O assunto é desafiador e, mais uma vez, como fez no mestrado, Bruno emplacou seu material de pesquisa em revistas Quails A internacional.

Tese Doutorado

From the Center of Clinical Oral Siences, Department of

Periodontology and the Department of Medical Laboratory Sciences

and Technology, Division of Clinical Chemistry,

Karolinska Institutet, Stockholm, Sweden

Function of Neutrophilic Granulocytes in Periodontitis

A Pathogenetic Study of a Tissue-Destructive Inflammation

Anders Gustafsson

Stockholm 1995

Abstract

The presence of bacteria in gingival crevices causes an inflammatory response, that is tissue-destructive in about 5-10% of the population. This destructive inflammation seems to be patient associated, rather than to be caused by specific periopathogens -i.e., a host-specific response to an essentially normal bacterial colonization of the gingival sulcus. There are several reasons why neutrophils may be involved in tissue destruction in periodontitis: i) they release active proteases and oxygen radicals, ii) they are the predominant leukocytes in gingival pocket epithelium and iii) their numbers increase with inflammation.

This thesis is based on the concept of a host specific and tissue-destructive type of inflammatory reaction mediated by hyperreactive neutrophils. The aim was thus, to evaluate whether there is an association between neutrophil activity and attachment loss in periodontitis.

The fluid in the gingival crevices (GCF) showed a higher neutrophil elastase activity (chromogenic substrate) and a lower a-2-macroglobulin content per µL (ELISA) in sites with tissue destruction than in sites without destruction in patients with gingivitis alone (Papers I and II). The lower concentration of the protease inhibitor can be ascribed to greater consumption due to the release of a larger amount of active proteases -e.g., neutrophil elastase.

In Paper III, the elastase activity in GCF was related to the lactoferrin content (ELISA). Elastase was used as a marker of neutrophil activation and lactoferrin as a marker of the number of neutrophils in the area. A higher elastase/lactoferrin ratio in GCF from periodontitis patients was found even when the samples were taken from clinically similar sites -i.e., the release of elastase per cell was higher from neutrophils in periodontitis patients than that in patients with gingivitis alone. This accords with a neutrophil associated specific host response.

This conclusion was further supported by the finding of lower protein concentrations in GCF from patients with periodontitis regardless of the clinical status (Gingival Index and pocket depth) of the sampled site (Paper IV ).

The methodological evaluation in Paper V showed that it was not possible to recover pure elastase from the paper strips used for sampling in the previous papers. The elastase a-2-macroglobulin complex could be recovered. The elastase activity determined on low molecular substrates must therefore originate from the elastase a-2-macroglobulin complex.

In order to verify the conclusions in previous papers (I-III) concerning hyperreactive neutrophils in periodontitis, an in vitro activation of peripheral neutrophils was performed (Paper VI). Using luminol-enhanced chemiluminescence, a higher release of oxygen radicals from neutrophils activated after Fc-receptor stimulation was found in patients with periodontitis. The elastase release was also significantly higher when related to the simultaneous release of lactoferrin.

In conclusion, both in vivo and in vitro findings in this thesis show an association between neutrophil activity and tissue destruction in periodontitis. This corroborates the view that there is a tissue-destructive type of inflammatory reaction, mediated by hyperreactive neutrophils.

Key words: Periodontitis, inflammation, specific host response, tissue-destruction, neutrophils, free oxygen radicals elastase, GCF

ISBN  91-628-1510-5

CONTENTS

Preface 5

Introduction 6

Periodontitis – a host response disease                                                                      6

Neutrophilic granulocytes                                                                                          11

Neutrophil mediated tissue destruction in periodontitis                                           14

Aims 16

Methods 17

Clinical parameters                                                                                                    17

Sampling of gingival fluid                                                                                          17

Gingival crevicular fluid volume                                                                                   17

Elastase activity                                                                                                           17

Antigenic elastase                                                                                                       18

Lactoferrin and a-2-macroglobulin content                                                                  18

Total protein content                                                                                                       18

Chemiluminescence                                                                                                  19

Neutrophil preparation                                                                                                 19

Neutrophil activation                                                                                                    20

Flow cytometric analysis                                                                                              20

Investigations and results 21

Studies on the pathogenesis of periodontitis                                                             21

Paper I                                                                                                                         22

Paper II                                                                                                                    24

Paper III                                                                                                                     26

Paper IV                                                                                                                                28

Paper V                                                                                                                        30

Paper VI                                                                                                                  32

General discussion 34

Conclusions 40

Acknowledgements 41

References 42

Papers I-VI

PREFACE

The thesis is based on the following publications, which will be referred to in the text by their Roman numerals.

I                   Granulocyte elastase in gingival crevicular fluid

A possible discriminator between gingivitis and periodontitis

Gustafsson A., Åsman B., Bergström K. & Söder P.-Ö.

Journal of Clinical Periodontology 1992; 19: 535-540

II              Altered relation between granulocyte elastase and a-2-

macroglobulin in gingival crevicular fluid from sites with  periodontal destruction

Gustafsson A., Åsman B. & Bergström K.

Journal of Clinical Periodontology 1994; 21: 17-21

III          Elastase and lactoferrin in gingival crevicular fluid: possible  indicators of a granulocyte-associated specific host response

Gustafsson A., Åsman B. & Bergström K.

Journal of Periodontal Research 1994; 29: 276-282

IV            Lower protein concentration in GCF from patients with  periodontitis: an indicator of host-specific inflammatory reaction

Gustafsson A., Åsman B. & Bergström K.

Journal of Clinical Periodontology 1995; 22: 225-228

V                   Methodological considerations in GCF sampling with                paper strips: poor recovery of uncomplexed elastase

Gustafsson A. Submitted to Journal of Clinical Periodontology

VI                   Increased release of free oxygen radicals from peripheral                neutrophils in adult periodontitis after Fc-gamma                receptor stimulation.

Gustafsson A. & Åsman B.

Journal of Clinical Periodontology 1996; in print

INTRODUCTION

“This form of destructive pericementitis is as fatal in

its results as inflammation from deposits of calculus.

It usually runs a more rapid course, I think, and is less

amenable to treatment.”

G. V. Black 1882

Periodontitis – a host response disease

Periodontitis is a tissue-destructive inflammation that affects some sites in certain individuals (Lindhe et al. 1983, Socransky et al. 1984). The tooth supporting collagen fibres of the periodontal ligament may be broken down by neutrophils alone or in concert with other cells -e.g., monocytes/macrophages and fibroblasts, in the vicinity of the periodontal lesion.  The destruction is probably mediated by a specific host response that is a prerequisite for the inflammation to become destructive on bacterial challenge. The view that the tissue destruction is mediated by the host’s cells and not by bacteria is supported by the findings that anti-inflammatory drugs reduce attachment loss, but do not affect the microbiota (Williams et al. 1989) and that sterile granulation tissue degrades collagen in the absence of bacteria (Åsman et al. 1988).

The assumption of a specific host response is supported by epidemiological studies, that show that the frequency of moderate to severe periodontitis is in the order of 5-10% in most parts of the world, independently of oral hygiene habits (Yoneyama et al. 1986, Hugosson et al. 1992). In areas with little, if any oral hygiene, higher amounts of plaque and dental calculus, and higher frequencies of gingivitis have been reported, but the majority of the population still do not develop periodontitis (Bealum et al. 1986, 1988). Moreover, in families with a high frequency of early onset periodontitis, there is a genetic coupling, indicating that periodontitis is a patient associated specific host response (van der Velden 1990, Michalowicz 1991).

In conclusion, the idea of a specific host response expressed, as a tissue- destructive inflammation, is well supported while the involvement of specific pathogens is less clear.

Microbiology

Although numerous studies have been published that suggest a relationship between the microbiota and the clinical status of the gingival pocket, it is still difficult to point out a single specific periopathogen. Wolff et al. (1993) found, in agreement with others (e.g., Socransky et al. 1991, Beck et al. 1992), that the frequency and the levels of five Gram-negative anaerobic bacteria, previously known to increase with the severity of the disease, correlated with the probing depth of 6905 sites sampled. This could be either an indicator of disease or simply the result of a more favourable environment. Deep pockets favour anaerobic bacteria, such as P. gingivalis, P. intermedia, E. corrodens and F. nucleatum, but these bacteria can also be found in shallow pockets, even in subjects without deep pockets or periodontits (Dahlén et al. 1992, Chen et al. 1989). Wolff et al. (1993) conclude that the fact that the alleged periopathogens frequently inhabit sites that are not associated with advanced disease suggests that a susceptible host is required.

Since the environment influences the bacterial composition, it should be more interesting to compare the microbiological flora in clinically similar sites with and without progression. Studies done with this design have shown conflicting results. Dzink et al. (1988) studied 33 subjects with periodontal disease and compared the predominant cultivable flora in 100 active sites (attachment loss of at least 2 mm in two months) with that in 150 inactive sites with similar pocket depth and attachment level. They found a significantly increased frequency of F. nucleatum and W. recta in active sites and suggested that combinations of some species above certain threshold levels might initiate active periodontal disease.

Haffajee et al. (1991) studied six sites per tooth (excluding third molars) in 38 subjects with attachment loss. The total average viable count at each site in each subject was averaged in subjects with active disease, who exhibited one or more sites with an attachment loss of 3 mm or more, and in those with inactive disease. Those with active disease had higher frequencies of P. gingivalis and W. recta, while those with inactive disease had higher frequencies of C. ochracea and Capnocytophaga sp. These authors used the mean frequency of various microorganisms in the sampled sites to characterize each subject and they concluded that “ the present investigation suggested certain species which might be useful in identifying subjects at risk”.

Moore et al. (1991) compared the microbial flora in samples from sites with disease progression (-i.e., an attachment loss of 2 mm or more during the last two months) with the flora in clinically similar control sites with no disease progression, obtained at the same time from the same subject. No significant difference was found between the two types of sites. Similarly MacFarlane et al. (1988) and Liljenberg et al. (1994) were unable to detect differences between sites with or without progression in untreated patients with adult periodontitis. The latter authors ascribed the inability to find                        microbial differences to methodological reasons, such as smallness of the samples, or the delineation of only a few species,  or in fact no difference in the subgingival microbiota that could explain the differences in disease progression.

Although numerous microbiological studies have been performed, the association between specific periopathogens and periodontitis remains to be conclusively proved and it is not yet possible to state whether the microbiota found in deep pockets are the cause or an effect of periodontitis. This also applies to a relation between progression of the disease and certain bacteria. An intriguing question concerns the way in which the periodontal process starts. The theory that specific periopathogens may be involved in the causation of periodontitis does not explain the development of a tissue destructive inflammation in normal gingival cervices, in some sites and in certain individuals. It therefore seems important to study the normal supragingival microflora which contains considerable amounts of Gram-negative bacteria.

Aggravating factors

Three fairly common conditions, show an increased prevalence of periodontitis: diabetes, human immunodeficiency virus infections and Down’s syndrome. All three are associated with alterations in the immune system that significantly affect the inflammatory reaction and show dysfunctions in the neutrophils.

Diabetes has proved to be a risk factor for periodontitis. Recent epidemiological studies report an increased prevalence and increased incidence of periodontitis (Grossi et al. 1993, Haber et al. 1991, Yalda et al. 1994). Although the results of examinations of the periodontal microflora are conflicting, most studies indicate that there are no differences in the microbiota between periodontitis patients with or without diabetes. It therefore seems reasonable to assume that the high frequency of periodontitis is due to an aberrant host response. Diabetes may increase susceptibility to periodontitis by impairing neutrophil chemotaxis and phagocytosis (Manouchehr-pour et al. 1981, McMullen et al. 1981). Abnormal cross-linking and glycosylation of collagen and defective secretion of growth factors may also contribute to the increased susceptibility of patients with diabetes (Yalda et al. 1994). Monocytes from patients with diabetes secrete more prostaglandin E2 (PGE2) and interleukin 1b after stimulation with lipopolysaccharide (LPS). This accords with the finding of an  increased secretion of PGE2 from monocytes after LPS stimulation in patients with early-onset periodontitis (Shapira et al. 1994) and elevated levels of PGE2 in gingival crevicular fluid (GCF) from patients with periodontitis (Offenbacher et al. 1991).

Human immunodeficiency virus (HIV) is associated with distinguished forms of gingivitis and periodontitis. HIV-associated gingivitis is characterized by a distinctive type of marginal gingivitis accompanied by petechia-like or diffuse erythematous lesions of the attached gingiva and oral mucosa, which do not respond to conventional treatment and improved plaque control (Murray 1994). The clinical features of HIV-associated periodontitis include severe pain, spontaneous gingival bleeding, and exposure of  bone (Murray 1994). The microbiota of  HIV-associated periodontitis resembles that usually found in periodontitis patients without HIV. In contrast, the microbiota of HIV-associated gingivitis differs entirely from that seen in other subjects with gingivitis and the flora resembles that encountered in periodontitis lesions (Murray et al. 1989). HIV infection causes many changes in the immune system -e.g., neutrophils show decreased chemotaxis but increased phagocytosis and respiratory burst (Ryder et al. 1988).

Down’s syndrome. The prevalence of periodontitis in patients with Down’s syndrome (DS) is very high and exceeds 90% in some studies (Reuland-Bosma & van Dijk 1986, for review).  In a more recent Swedish study, alveolar bone loss was found in 39% of children (10-19 yrs) with Down’s syndrome (Modéer et al. 1990). The reason for this high prevalence is not clear, but DS is associated with many changes in the immune system -e.g., a decrease in the number of mature T-lymphocytes and functional defects in the neutrophils. Impaired chemotaxis and phagocytosis are present but the findings concerning respiratory burst are inconclusive (Kretschmer et al. 1974, Barkin et al. 1980 a, b). Preliminary results in our laboratory indicate that the extracellular release of free oxygen radicals from peripheral neutrophils in children with DS but without periodontitis is similar to that in healthy control subjects. However, in DS patients with periodontitis the release of oxygen radicals seems to be higher, which is in accordance with the findings in juvenile periodontitis (Åsman 1988). Reuland-Bosma & van Dijk (1986) suggest that the reason for the high prevalence in DS is a combination impaired collagen synthesis and abnormal capillary morphology as well as due to functional defects in neutrophils and monocytes.

Quantitative  and qualitative defects in the neutrophils have been implicated as the cause of the periodontal tissue destruction in a number of rare and more or less life-threatening diseases, such as the Chediak-Higashi syndrome, Papillon-Lefévre syndrome, acute myeloid and chronic leukaemia  (Wilton et al. 1988, for review). Another unusual and very serious condition associated with periodontitis is leukocyte adhesion deficiency (LAD) (Meyle 1994). Neutrophils from patients with LAD show defects in three adhesion molecules, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18) and gp 150,95 (CD11c/CD18), probably due to a primary defect in the common beta subunit (CD18). These three integrins mediate vital leukocyte functions, such as binding to endothelial cells, chemotaxis and phagocytosis. Considering the severity of this deficiency, the associated prepubertal periodontitis can be seen as a complication of LAD rather than a form of periodontitis.

Other conditions, such as physical and psychical stress (Green et al. 1986) and malnutrition (Enwonwu 1994) may impair protective responses, such as production of antioxidants and acute phase proteins, and can, as such, aggravate periodontitis but not cause destructive inflammation per se.

Smoking is now accepted as an additional risk factor for periodontal disease. Several epidemiological studies have demonstrated an increased prevalence of periodontitis among smokers (Haber & Kent 1992, Holm 1994, for review see, Bergström & Preber 1994). Periodontal treatment, both surgical (Preber & Bergström 1990) and non-surgical (Preber & Bergström 1985), is less effective in smokers. Smokers have also been shown to be highly over-represented in the groups of patients with refractory periodontitis studied by MacFarlane et al. (1992) and Magnusson et al. (1994).

The pathogenetic mechanisms underlying this are not clear. Patients with periodontitis who smoke do not differ from those who do not, with respect to possible periopathogens (Preber et al. 1992). In subjects with gingivitis, there are no differences between smokers and non-smokers, either in the composition of the microbiota or in the plaque accumulation rate (Bergström & Preber 1994). Smoking causes several changes in the inflammatory response – for example, tobacco smoke and water soluble components of tobacco smoke impair the chemotaxis and phagocytosis of normal peripheral neutrophils (Kenney et al. 1977, Kraal & Kenney 1979). Cigarette smoking increases the number of circulating neutrophils in vivo and increases the release of reactive oxygen radicals from peripheral neutrophils  in vitro (Anderson et al. 1987, Anderson et al. 1991).

In conclusion, patients who have conditions associated with an elevated prevalence of periodontitis seem to have a compromised host response with alterations in neutrophil functions.

Neutrophilic granulocytes

In man, the neutrophilic granulocyte is the predominant leukocyte in blood comprising between 50% and 70 % of the circulating pool of leukocytes. The neutrophils are produced in the bone marrow, where the myeloid precursor cells mature to segmented neutrophils in about 9 days. The cells remain in the blood­stream for a relatively short time (T1/2 = 6-7h) and in the tissue for 1-4 days. The neutrophils are rapidly mobilized to the site of an injury or bacterial invasion (within 30 min) and constitute the first cellular host defence against invading bacteria.

Migration from the blood vessels (diapedesis) to the inflammatory lesion starts with binding between the selectins (P- and E-selectin on the endothelial wall, and L-selectin on the neutrophil) and their counter-receptors (ligands). This initiates the rolling of neutrophils along the wall and enables the cells to come into contact with chemoattractants along the endothelial lining of the vessels. The contact with chemoattractants triggers intracellular reactions which activate the integrin adhesiveness (Springer 1994). The neutrophils adhere to and penetrate the endothelial layer by binding integrins on their membranes (LFA-1, Mac-1, VLA-4) with the intercellular adhesion molecules on the endothelium (ICAM-1, ICAM-2, VCAM-1). Besides activating the integrins, the chemoattractants direct the migration of the cell once it is outside the blood vessel. The neutrophils can sense a chemoattractant concentration gradient of 1% across their diameter and move, using the integrins for traction, towards the inflammatory lesion.

In the gingival sulcus, the neutrophils form a “leukocyte wall” between the subgingival plaque and the junctional epithelium (Frank et al. 1980). The neutrophil recognizes and combats foreign substances by their receptors for several surface structures -for instance, the Fc-portion of attached antibodies, complement factors, capsule reactive proteins and the LPS-complex.

Neutrophils can deliver antimicrobial substances by four mechanisms:

1. Degranulation releases anti-bacterial substances extracellularly (Wright 1988), from  primary (azurophil) granule: defensins, lysozyme, myeloperoxidase and the neutral serine proteases cathepsin G and elastase, and from secondary (specific) granule: lactoferrin, collagenase and lysozyme. A tertiary granula containing gelatinase has been described (Dewald et al. 1982).

2) The respiratory burst releases highly reactive oxygen metabolites extracellularly.  Oxidative metabolism mainly generates hypochlorous acid (HOCl) because of the relatively high concentration of Cl- in plasma (Weiss 1989), which has both tissue-destructive and antimicrobial effects.

3) Phagocytosis, which isolates the organism intracellularly by creating a phagosome. Fusion between the phagosome and cytoplasmic granules result in the delivery of high concentrations of the antibacterial substances mentioned above into the vacuole.

4) Cytolysis or apoptosis (programmed cell death) is an ultimate mechanism for  antibacterial substances to reach the bacteria. Considering the high concentration of neutrophils in inflamed tissue, the release of antibacterial agents from dying cells may represent an important host defence mechanism (McNamara et al. 1988).

The neutrophil, however, is not only a terminally differentiated cell, incapable of protein synthesis, that participates in the inflammatory response only as an effector cell by releasing preformed substances. It can also produce several potent inflammatory mediators that may influence both cellular and humoral immunity (Table 1) (Lloyd & Oppenheim 1992) . T-cells, natural killer cells and monocytes/macrophages produce considerable amounts of cytokines, but the vast majority of cells infiltrating an inflamed tissue are neutrophils and they may thus be an important source of cytokines. This capacity enables the neutrophil to survive much longer in the inflamed lesion than was previously believed (Cassatella 1995).

In this scenario, neutrophils should be considered not only as active and central elements in the inflammatory response, but also as cells which, through cytokine secretion, may significantly influence the direction and development of the immune processes.

__________________________________________________________________________

Table 1. Proteins and other mediators produced and released by neutrophils to fulfil efferent (effector) and afferent (inductive) functions in inflammation, immunity and repair.

__________________________________________________________________________

Mediator Reference

Efferent

CR1, CR 3, FcR, class 1 MHC                           Jack & Fearon 1988

IFN-a                           Shirafuji et al. 1990

Platelet-activating factor                           Sisson et al. 1987

Leukotriene B4                           Sisson et al. 1987

Fibronectin                           La Fleur et al. 1987

PGE2                           Herrmann et al. 1990

Afferent

IL-1b                            Marucha et al. 1990

TNF-a                           Dubravec et al. 1990

G-CSF, M-CSF                           Lindemann et al. 1989

IL-6                           Cicco et al. 1990

IL-8                           Bazzoni  et al. 1991

IL-1 receptor antagonist                           Ulich et al. 1992

__________________________________________________________________________

Modified from Lloyd & Oppenheim 1992

Neutrophil mediated tissue destruction in periodontitis

Neutrophilic granulocytes are associated with tissue destruction in a number of chronic inflammations (Table 2). In periodontitis, neutrophils are the probable mediators of tissue destruction because: 1) their numbers increase with inflammation, 2) they lie close to the collagen fibres of the periodontal ligament and finally, 3) they are equipped with potent active tissue-destructive substances, which they may release in the inflammatory lesion.

Increasing numbers of neutrophils have been reported in several morphologic studies of the inflamed gingiva. Attström (1970) studied the presence of leukocytes in clinically healthy and chronically inflamed sites in humans and dogs. The leukocytes were found in healthy as well as in inflamed sites and differential counts showed 95-97% neutrophils, 1.2% lymphocytes and 2-3% moncytes. The proportions remained the same, although the number of leukocytes increased with inflammation. In another study by the same author (Attström 1971), leukopenia was induced by nitrogen mustard and heterologous anti-neutrophil serum in dogs with chronically inflamed gingiva. The leukopenia caused a reduction in the number of crevicular leukocytes, the enzymatic activity and the volume of gingival crevicular fluid. Kowashi et al. (1979) counted neutrophils in gingival washings during experimental gingivitis in man and found a doubling of the number of cells from day 0 to day 20.

The localization of neutrophils near Sharpey’s fibres and the formation of a leukocyte wall have been shown in microscopic studies. Christersson et al. (1987), using light and immunofluorescence microscopy, noted extensive subepithelial infiltration of neutrophils in gingival biopsies from patients with juvenile periodontitis. Frank et al. (1980), using transmission microscopy, found a wall of aggregated neutrophils between the subgingival plaque and the junctional epithelium.

The degranulation of the neutrophils, leads to the release of active and inactive proteases extracellularly. Elastase is a neutral serine protease that can degrade a number of important proteins in the extracellular matrix, such as elastin, fibronectin and collagen types I, II, III and IV (Starkey et al. 1977, Janoff 1985). Collagenase (matrix metallo-proteinase-8) is released in a latent form but can be activated extracellularly. Several authors have demonstrated the presence of neutrophil-derived collagenase, both functionally and immunohistochemically, in  gingival tissue biopsies and gingival crevicular fluid (GCF) (Gangbar et al. 1990, Overall et al. 1991, Ingman et al. 1994). The destructive capacity of released proteases is usually offset by the protease-inhibitors a-1-antitrypsin (A1AT) and a-2-macroglobulin (A2MG). The inhibitors are present in such abundance that all active proteases are inhibited within milliseconds, but the release of proteases in closed compartments and/or an oxidative inactivation of A1AT can give the proteases an opportunity to cause tissue damage.

The respiratory burst generates reactive oxygen radicals that are released extracellularly. Oxygen radicals are tissue destructive per se (Weiss 1989), but they can also act in concert with the simultaneous release of proteases. Extracellularly released oxygen radicals can oxidatively inhibit A1AT and thus allow the proteases to degrade matrix proteins in close proximity to the neutrophils (Weiss 1989). Neutrophil collagenase, released from the cell in an inactive form, may be activated by the simultaneous release of oxygen radicals (Saari et al. 1990).

Oxygen radicals, besides contributing to the tissue destruction, are also thought to inhibit phagocytosis (Stendahl et al. 1984). The mechanism for this is unclear: both the receptors on the neutrophils and their ligands may be affected.

In conclusion, neutrophils may play a major role in the tissue-destructive mechanisms causing degradation of the periodontal ligament in periodontitis.

_________________________________________________________________________

Table 2.  Noninfectious conditions in which symptoms and tissue injury may be partly mediated by neutrophils

__________________________________________________________________________

Disease Reference

Adult respiratory distress syndrome                           Rivkind et al. 1991

Cystic fibrosis                           Kharazmi et al. 1987

Emphysema                           Janoff 1983

Glomerulonephritis                           Holdsworth & Bellomo 1984

Inflammatory bowel disease                           Faden & Rossi 1985

Myocardial infarction                           Rowe et al. 1984

Rheumatoid arthritis                           Nurcombe et al. 1991

Secondary hyperparathyroidism                           Tuma et al. 1981

Trauma                           Tanaka et al. 1991

__________________________________________________________________________

AIMS

General aims

The investigations in this thesis were based on the concept that periodontitis is caused by a host specific tissue-destructive type of inflammatory reaction that is associated with hyperreactive neutrophils. The aims were thus to demonstrate an association between neutrophil activation and attachment loss.

Aims of studies included in thesis:

- to measure the enzymatic activity and the antigenic content of elastase in GCF in order to distinguish between periodontitis and gingivitis.

- to study the local balance between elastase activity and a-2-macroglobulin in GCF-samples from various types of gingival sites and to relate these components to tissue-destructive inflammation.

- to relate elastase activity to the amount of lactoferrin in order to assess the local reaction of neutrophils from patients with periodontitis and patients with gingivitis.

- to compare the protein concentration in GCF from 1) inflamed sites with or without tissue destruction from patients with periodontitis and 2) inflamed sites from patients with gingivitis alone.

- to assess the recovery of certain proteins from paper strips and evaluate the usefulness of various sampling methods for analyses of GCF.

- to measure the in vitro generation of oxygen radicals and degranulation from peripheral neutrophils in patients with adult periodontitis and pair matched healthy controls in order to evaluate the association between neutrophil reactivity and periodontal tissue destruction.

METHODS

Clinical parameters

Supragingival plaque (PLI) and gingival inflammation (GI) were recorded employing the criteria of Silness & Löe (1964) and Löe (1967) (Papers I-IV). Only sites with a GI value of 1 or 2 were used. Sites without inflammation (value 0) could not yield sufficient gingival crevicular fluid (GCF) volumes to permit measurement with the Periotron 6000® and very inflamed sites (GI value 3) usually could not be examined because it was difficult to avoid contamination of the GCF sample by blood.

Pocket depth and clinical attachment level, the distance from the enamel-cement junction to the bottom of the pocket, were measured with a calibrated periodontal probe. The marginal bone loss (MBL) was recorded in accordance with the recommendations of Lavstedt & Eklund (1975) (Paper I)and adjusted for age by subtracting of the means for the corresponding age group reported by Lavstedt et al. (1975).

Sampling of gingival crevicular fluid

The site to be sampled was isolated with cotton rolls, supragingival plaque removed and the site was gently dried with an air syringe (Papers I-IV). Thirty seconds later, GCF was collected with prefabricated paper strips (Periopaper ® GCF strips, IDE Interstate, Amityville, NY, USA). The strip was inserted into the pocket until mild resistance was felt and was kept there for 30 sec.

Gingival crevicular fluid volume

The volume of the GCF was measured with a Periotron 6000® GCF meter (IDE Interstate, Amityville, NY, USA) (Papers I-IV). The Periotron measures the elec­trical capacitance as the dielectric insulating properties of the filter paper vary with the quantity of fluid absorbed by the paper. Differences in the composition of the various GCF samples do not affect the measurements (Hinrichs et al. 1984). Before each study, the instrument was calibrated with saline, delivered with a Hamilton syringe.

Elastase activity

The amount of active elastase (Papers I-VI) was measured as proteolytic activity on a low molecular weight substrate, (445.5 Da) L-pyroglutamyl-L-propyl-L-valine-p-nitroanilide (S-2484, Pharmacia Diagnostica, Uppsala, Sweden). The substrate is highly specific for granulocyte elastase (Tanaka et al. 1990) but is also hydrolysed by the elastase-a-2-macroglobulin complex (Wewers 1988). No difference in activity was found between pure commercial elastase and the elastase a-2-macroglobulin complex.

Antigenic elastase

The antigenic elastase (Paper I) was assessed by a commercial method (PMN elastase IMAC MERCK # 11332), which measures both free elastase and the elastase-a-1-antitrypsin complex. The elastase is detected with a sheep antibody, against human neutrophil elastase, conjugated with inactive peroxidase. The enzyme is activated by the antigen antibody reaction.

Lactoferrin and a-2-macroglobulin

An Enzyme Linked Immunosorbent Assay (ELISA) was used to analyse the contents of lactoferrin and a-2-macroglobulin (Papers II, III, V, VI). The micro-well plate was coated with the antigen, samples or standard, diluted in carbonate buffer, pH 9.6, overnight at +4°C. After incubation, the plate was washed four times with PBS 7.4 + 0.05% Tween 20. The antigen was detected with a polyclonal rabbit antibody during incubation at +37°C for 1h. After another washing with PBS + Tween, the final antibody, a goat anti-rabbit IgG conjugated with alkaline phosphatase (ALP), was added. The plate was once again incubated at +37°C for 1h and washed as above. The ALP activity was measured with the substrate p-nitro-phenol-phosphate in a spectrophotometer at 405 nm.

In Paper VI, the amount of lactoferrin was analysed with a sandwich ELISA, using a monoclonal mouse anti-lactoferrin antibody as the first antibody. This antibody was coated on the plate overnight and the antigen was added after four washings. After incubation for 1h at +37°C, the procedure was the same as described above.

Each plate included a standard curve obtained by the serial dilution of a commercial standard.

Total protein content

The protein content of GCF was measured with a protein staining method described by Bradford (1976), using the Bio Rad protein assay (Bio Rad Laboratories GmbH, Munich, Germany) (Papers IV, V). The method is sensitive and reproducible, but stains vari­ous amino acids differently. This protein staining method seems to be particularly sensitive to arginine rich proteins (Compton & Jones 1985). To minimize the risk of differences in the staining of various proteins, a standard serum (Control serum, Behringwerke AG, Marburg, Germany), with a pro­tein composition similar to GCF, was used as standard.

Chemiluminescence

Chemiluminescence (CL) is a convenient and sensitive way to measure the respiratory burst. Native CL is the light produced by the neutrophil during its in­teraction with bacteria or other particles (Paper VI). The oxygen consumption, catalysed by the membrane enzyme NADPH-oxidase, during this interaction generates unstable products, such as superoxide, hydrogen peroxide, singlet oxygen  and hydroxyl radical. The light emitted results from a reaction between oxygen radicals and regions of high electron density in a wide range of substrates. Microorganisms in phago­cytic vacuoles provide a rich source of oxidizable substrates (for review, see Seymour et al. 1986). Cyclic hydrazine 5-amino-2, 3 dihydro-1,4 phthalazine­dione (luminol) is used as a substrate for the oxygen radicals to enhance the amount of light, thus making it possible to use smaller numbers of neutrophils.

CL correlates well with the killing of Staphylococcus aureus initially but, after a prolonged incubation, the CL decreases, while the killing rate remains stable (Ewetz et al. 1981).

Neutrophil preparation

The neutrophils (Papers III, VI) were prepared on a two-layer discontinuous Percoll® gradient (1.098 kg / L ± 1 g and 1.079 kg / L ± 1 g). Nine mL of venous blood were layered on the gradient and centrifuged. After an initial centrifugation at 108g for 8 min, the supernatant containing plasma and platelets, could be removed and after a second centrifugation at 1200g for 10 min, the neutrophils could be separated from the mononuclear cells. The erythrocytes in the pellet were lysed with 0.83% NH4Cl, supplemented with 0.25% human serum albumin (HSA). The cells were finally washed twice with phosphate-buffered saline (PBS) + 0.25% HSA. The cell preparation contained 96% neutrophils and 4 % mononuclear cells. The remaining erythrocytes were about as numerous as the neutrophils and the platelets were no more than 50% of the neutrophils. The recovery of neutrophils was more than 75% and 98% of the cells were viable (Bergström & Åsman 1993).

Neutrophil activation

In order to obtain a clear-cut Fcg-receptor stimulation, the neutrophils (Papers III, VI) were activated with bacteria, opsonized with human gamma-globulin. Opsonized Staphylococcus aureus (S.a.), Actinobacillus actinomycetemcomitans (A.a.) and Porphyromonas gingivalis (P.g.) all induce chemiluminescence (CL) from peripheral neutrophils in vitro. Åsman & Bergström (1992) found that the CL after activation with any of these three bacteria was higher in patients with juvenile periodontitis than in healthy controls and that neither unopsonized bacteria nor monomeric IgG induced measurable CL. This indicates that the bacteria merely serve as carriers for the polymeric IgG. In our studies, S.a. was chosen for practical reasons.

The bacteria were harvested from agar plates and counted in a Bürker chamber. Opsonization was done with IgG at +20°C during agitation for 2h. The opsonized bacteria were frozen in aliquots at -70°C.

Flow cytometric analysis

Flow cytometry was performed on 0.5×106 neutrophils (Paper VI), premixed either with an antibody or with the corresponding immunoglobulin as the negative control for 20 min at ±0°C and washed twice in PBS before suspension in 500 µL of the same buffer. Using unconjugated antibodies, a second FITC-conjugated antibody against mouse-IgG was added before performing the same procedures of incubation, washing and final suspension. FITC-conjugated monoclonal antibodies were used against CD15, CD 16, CD 62L (Mel-14) and CD 11b. Unconjugated antibodies were used against CD 11a and CD 35. Immunofluorescence was measured on an EpicsR -profile II Flow Cytometer (Coulter Electronics, Inc., Hialeah, FL, USA). The instrument was calibrated on every experimental occasion with standard beads for alignment of sheet flow and adjustment of the photo-multiplier. After gating of the granulocyte population on the scattergram, the % of antigen positive cells and their median immunofluorescence were registered.

INVESTIGATIONS AND RESULTS

Studies on the pathogenesis of periodontitis

Pathogenetic studies of periodontitis have been done for more than a century. Black (1882) divided his cases into two types: i) inflammation of the gums occurring from deposits of salivary calculus and ii) destructive inflammation of the peridental membranes not caused by calcareous deposits. Black observed that the destructive inflammation was not related to the general health of the patient and that it was more difficult to treat.

Histological studies seem to have been based mainly on examinations of biopsy specimens. Despite technical and ethical sampling difficulties, this method can give a good picture of the inflammatory processes. Histological changes in the gingival tissues from the initial lesion to the advanced periodontal lesion are described in a comprehensive review by Page & Schroeder (1976).

Many studies are based on analyses of plasma components (Schenkein & Genco 1977a, b), lymphocytes (Lehner 1972) and neutrophils (Van Dyke et al. 1983, Miller 1984) in the blood.

A convenient and noninvasive way to study the pathological processes in the gingival lesion is to analyse samples of gingival crevicular fluid (GCF). GCF is primarily a filtrate of plasma, but it also contains leukocytes and leukocyte derived products, tissue degradation products and microorganisms and their products. All of these components may be representative of the inflammatory lesion and therefore of interest to analyse (see Cimasoni 1983 for review). The possibility of identifying subjects susceptible to periodontitis and of predicting the progress of the disease have been investigated extensively during recent decades (see Curtis et al. 1989, Page 1992, Lamster 1992, for review).

In this thesis the tissue-destructive type of inflammation in periodontitis has been studied in vivo by analyses of GCF in Papers I-IV and in vitro by functional analyses of peripheral neutrophils in  Paper VI. A summary of the investigations and results of the studies are presented paper by paper on the following pages.

Paper I

Granulocyte elastase in gingival crevicular fluid.

A possible discriminator between gingivitis and periodontitis

Elastase is released from the azurophil granula of the neutrophils during activation and can therefore be used as a marker of neutrophil activation in inflammatory lesions. The amount of elastase, both functional and antigenic, was therefore measured in samples of gingival crevicular fluid (GCF) from inflamed sites in patients with adult periodontitis (n=16) and in patients with gingivitis alone (n=10). Two deep gingival pockets were sampled in each periodontitis patient (mean pocket depth 6.9 mm) and two shallow pockets (2.8 mm) in the gingivitis group. The two groups of sites had the same gingival index (Löe 1967). The GCF was collected with paper strips, the volume of the samples was measured with a Periotron®, and eluted in PBS with 0.1% Tween for 1h and centrifuged at 3000g for 10 min.  Elastase activity was measured with a substrate (S-2484, L-pyroglutamyl-L-propyl-L-valine-p-nitroanilide) shown to be highly specific for neutrophil elastase (Tanaka et al. 1990). The antigenic content of elastase was determined with a specific antibody conjugated with peroxidase (PMN elastase IMAC Merck # 11332).

The study showed a correlation between pocket depth and GCF volume, and between elastase activity per site and GCF volume. In order to minimize the influence of the pocket depth, the amount of elastase was calculated per µL.  The amount of antigenic elastase showed no difference between the two groups, calculated either per site or per µL. In contrast, the elastase activity, per site and per µL, was significantly higher in the periodontitis group  (Fig. 1).

The elastase activity per µL increased with pocket depth and attachment loss, in spite of increasing GCF volume. The higher elastase activity in the periodontitis sites was  a consequence not only of larger fluid volumes in the deep pockets but also of a higher release of elastase per neutrophil and/or of higher numbers of enzyme releasing neutrophils in the inflamed tissue.

Fig. 1

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Fig. 1. Paper I. Mean antigenic elastase per µL and elastase activity per µL in GCF samples from patients with periodontitis (n=16) and gingivitis (n=10). *  = p < 0.05 calculated with the Mann-Whitney U-test. Bars indicate + 1SD

Paper II

Altered relation between granulocyte elastase and a-2-macroglobulin in gingival crevicular fluid from sites with periodontal destruction

The elastase substrate used in Paper I is hydrolysed not only by free elastase but also by the elastase bound to a-2-macroglobulin (A2MG), which, together with a-1-antitrypsin, is the principal inhibitor of elastase. These facts made it interesting to relate the elastase activity to the amount of A2MG in the same GCF sample.

Neutrophil elastase activity and A2MG were studied in GCF from three categories of sites, in six patients with gingivitis alone and six patients with periodontitis. Six inflamed sites were sampled in each gingivitis patient and 12 sites, six with and six without attachment loss and periodontal pockets in each periodontitis patient. These three types of sites were chosen in order to study the influence of GI, pocket depth and attachment loss on these two parameters, as well as to compare patients with periodontitis to those with gingivitis alone. The GCF was collected with paper strips and eluted in 500 µL PBS for 1h in room temperature, and centrifuged at 3000g for 10 min. The volume of the samples was measured with a Periotron 6000®. To avoid the influence of increased GCF volume from deep pockets (Paper I), the elastase activity and the A2MG content were calculated per µL of GCF. The elastase activity was measured with a specific substrate (S-2484) and the A2MG with ELISA. The study showed an increase in elastase activity and a decrease in A2MG  in the sites with attachment loss, but no difference between the two types of sites without attachment loss (Fig. 2). The lower concentration of A2MG in the samples from sites with attachment loss might be the result of consumption of the inhibitor due to increased release of elastase, since the protease-inhibitor complex is cleared faster than the native A2MG (Debanne et al. 1975).

The study showed an increased protease activity in sites with tissue destruction, indicating an association between neutrophil activity and periodontitis, compatible with the results of Paper I.

Fig. 2

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Fig. 2. Paper II. Clinical attachment loss measured from the enamel-cement junction to the bottom of the pocket, elastase activity per µL and a-2-macroglobulin concentration in three types of sites: GG – inflamed sites without attachment loss in subjects with gingivitis alone, GP – inflamed sites without attachment loss and PP – inflamed sites with attachment loss in subjects with periodontitis (n= 6 subjects). The findings in six sites were averaged in

each subject and then the average in all the subjects calculated.

Paper III

Elastase and lactoferrin in gingival crevicular fluid: possible indicators of a granulocyte-associated specific host response

The previous studies (Papers I and II) showed increased elastase activity in GCF from sites with attachment loss, but no difference between clinically similar sites in patients with periodontitis and gingivitis alone (Paper II). This may have been due to difficulties in assessing the number of neutrophils in the lesion with the gingival index (Adonogianaki et al. 1993). In this study lactoferrin, proposed as a marker of the number of neutrophils (Wright & Gallin 1979, Adonogianaki et al. 1993), was related to the elastase activity in the same sample. Initial in vitro experiments with Fcg-receptor mediated activation of peripheral neutrophils from healthy volunteers showed that the release of elastase and lactoferrin was controlled independently. It was therefore pertinent to relate the two substances in the same GCF sample to obtain an estimate of the release of elastase activity per neutrophil.

The samples of GCF were collected and eluted in the same way and from the same three types of sites as in Paper II. Both the periodontitis and the gingivitis groups consisted of seven subjects. The elastase activity was measured with a specific substrate (S-2484) and the lactoferrin with ELISA.

Higher elastase activity per µL was found in sites with attachment loss and deep pockets, in agreement with the findings in Papers I and II. Furthermore there was a significantly higher activity in the sites without attachment loss from the periodontitis patients than in the sites from the gingivitis patients, although they had similar clinical status, assessed by pocket depth and gingival index. Unlike elastase, lactoferrin showed no differences between the three types of sites, suggesting that the amounts of neutrophils were comparable (Fig. 3). This means that the ratio of elastase to lactoferrin was higher in the GCF samples from the patients with periodontitis, regardless of the clinical status of the sampled site.

A higher ratio of elastase to lactoferrin indicates the occurrence of a higher release of elastase from the neutrophils in periodontitis per se, which strongly suggests a neutrophil associated specific host response.

Fig. 3

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Fig. 3. Paper III. Elastase activity per µL, lactoferrin concentration and ratio of elastase / lactoferrin (mean ± SE) in three types of sites: GG – inflamed sites without attachment loss or deep pockets in subjects with gingivitis alone, GP – inflamed sites without attachment loss or deep pockets and PP – inflamed sites with attachment loss and deep pockets in subjects with periodontitis (n= seven subjects).  Six sites of each type were averaged in each subject and then the average in the seven  subjects was calculated.

Paper IV

Lower protein concentration in GCF from patients with periodontitis: an indicator of host-specific inflammatory reaction

The previous paper showed a decreased a-2-macroglobulin concentration in GCF from sites with attachment loss, probably due to consumption of the inhibitor because of an increased release of proteases. However, an alternative explanation might be dilution of the GCF. In order to test this possibility, the protein concentration in GCF was analysed with the same experimental design as in Papers II and III.

The GCF samples were collected with paper strips from six inflamed sites in each of 14 subjects with gingivitis alone (mean pocket depth 2.0 mm) and from six inflamed sites with (mean pocket depth 4.7 mm) and six sites without attachment loss (mean pocket depth 2.0 mm) in 13 patients with periodontitis. The volume of the GCF samples was measured with a Periotron 6000®. The GCF samples were eluted in 500 µL PBS and centrifuged at 3000g for 10 min. The protein content in the GCF samples was measured by the Bradford method (Bradford 1976), using the Bio-Rad protein assay.

The study showed a lower protein concentration in GCF samples from patients with periodontitis, in sites with or without attachment loss, than in GCF samples from patients with gingivitis alone (Fig. 4). A weak negative correlation was found between the protein concentration and the GCF volume, suggesting an increased dilution of the GCF samples from the patients with periodontitis. This may result from contributions by the interstitial fluid (Alfano 1974), which has a protein concentration around 2 g/L compared to that of 76 g/L in plasma.

A low protein concentration seems to be characteristic of patients with periodontitis and independent on the clinical findings at the sampled site. This would accord with a specific host response which distinguishes patients with periodontitis from those with gingivitis alone.

Fig. 4

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Fig. 4. Paper IV. Protein concentration (µg/ µL) in three types of sites: GG – inflamed sites without attachment loss in subjects with gingivitis alone (14 subjects), GP – inflamed sites without attachment loss or deep pockets and PP – inflamed sites with attachment loss and deep pockets in subjects with periodontitis (13 subjects).  Six sites of each type were averaged in each subject and then the average was calculated in all the subjects with a given type of site.

Paper V

Methodological considerations in GCF sampling with paper strips: poor recovery of uncomplexed elastase

The purpose of this study was 1) to measure the recovery of proteins, as regards tissue destruction and with a wide range of molecular weights, from the paper strips, and 2) to compare the usefulness of paper-strip sampling with other sampling methods.

Various proteins were applied to the strips with a Hamilton syringe and eluted  in isotonic PBS, pH 7.4, without detergent for 1h and centrifuged at 3000g for 10 min. This eluent does not lyse the neutrophils applied to the strips, that  may otherwise release intracellular substances -e.g., neutrophil elastase – to the eluate of the GCF samples. The recovery of these proteins was satisfactory, about 90% (Table 1), which is in agreement with earlier studies (Griffins et al. 1988). However, commercial pure neutrophil-elastase, unlike the same elastase premixed with a-2-macroglobulin, could not be recovered from the paper strips. The reason for this is not clear, but it was not dependent on the molecular weight in the range tested -i.e., 12-725 kD (Table 1).

The recovery of elastase from Durapore strips, which are also used for GCF sampling (Giannopoulou et al 1992), was investigated. About 30% of pure elastase applied to these strips was recovered.

However, a comparison of measurements made with a Periotron 6000®  GCF meter (IDE Interstate, Amityville, NY, USA) on the Durapore and the Periopaper strips, showed that Durapore gave lower readings making it difficult to measure small volumes of GCF collected with this material.

This study showed a satisfactory and reproducible recovery of proteins from paper strips, when the elution was made in isotonic buffers at neutral pH and without detergents to minimize lysis of the neutrophils. However, there was one important exception: the recovery of uncomplexed elastase was very low, showing that there may be differences between various proteins.

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Table 3. Paper V. Recovery of proteins applied to Periopaper strips. n= number of determinations, IP = isoelectric point

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Paper VI

Increased release of free oxygen radicals from peripheral neutrophils in adult periodontitis after Fc-gamma receptor stimulation

Neutrophils may participate in the tissue destruction in periodontitis by releasing oxygen radicals and proteases. Increased release of these agents has been demonstrated in juvenile periodontitis. Extracellular release of oxygen radicals and degranulation during in vitro activation of peripheral neutrophils was studied in 14 patients with adult periodontitis, nine men and five women (mean age 49.9 yrs.) and a group of age- and sex-matched healthy controls.

The peripheral neutrophils were purified, using a two-layer discontinuous Percoll gradient, washed and resuspended in PBS, pH 7.4, with 0.25% HSA. The peripheral neutrophils were activated with FcgR-stimulation using IgG- opsonized Staphylococcus aureus . The release of radicals during activation was measured with luminol-enhanced chemiluminescence. The light reaction was followed until a maximal light intensity was reached and was registered as mV(peak). Degranulation was assessed as the release of elastase (primary granula) and lactoferrin (secondary granula) after similar activation for 1h at +35°C. Elastase was measured with a specific substrate and lactoferrin with ELISA. The study showed a 113% greater release of oxygen radicals from neutrophils in the patients than in the healthy controls. The release of elastase was also elevated in the patients -the mean difference was 25%. However, the release of lactoferrin did not differ between the two groups (Table 4). The ratio of released elastase to lactoferrin was higher in the periodontitis group. This is in accordance with our in vivo findings in Paper III.

The present study therefore demonstrated that neutrophils from patients with adult periodontitis had a more marked reaction to Fc-receptor mediated activation even before they entered the inflammatory lesion. Whether this difference was due to a constitutional difference in the neutrophils per se or to a priming of the circulating cells was not evaluated, but it indicates a neutrophil-associated specific host response.

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Table 2. Paper VI. Release of free oxygen radicals measured with luminol-enhanced chemiluminescence (CL) (mean maximal light intensity ± SD), and cumulated release of elastase and lactoferrin during one hour (mean ± SD) after FcgR-stimulation. The release was calculated in 106 neutrophil granulocytes from 14 healthy controls (HC) and 14 patients with adult periodontitis (AP). The stimulation was performed with a bacteria-to-cell ratio of 300:1 and 75:1 for radical generation and degranulation, respectively.

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The difference (presented as the mean of 14 pairs) was calculated in each pair as follows: AP x 100 – 100 = %

HC

GENERAL DISCUSSION

This thesis deals with some characteristics involved in the pathogenesis of the tissue-destructive inflammation in the gingiva -i.e., periodontitis – that leads to degradation of the collagen fibres of the periodontal ligament. The accumulation of bacteria in the gingival crevice causes an inflammatory response which is tissue-destructive in about 5-10% of the population. This type of inflammation only seems to develop in some patients and not to be caused by specific periopathogens. Thus periodontitis is a host-specific response to a comparatively normal bacterial colonization of the gingival sulcus. The tissue destruction appears to be primarily mediated by neutrophils.

In these studies, elastase has been used as a marker of neutrophil activation and degranulation of primary granule. Elastase is also able to cause tissue destruction (Cergneux et al. 1982). Elastase is released as active protease and degrades most extracellular matrix proteins, including collagen type I (Starkey et al. 1977) -e.g., in Sharpey’s fibres. In Paper I the GCF analyses showed increased elastase activity, per site and per µL, in sites with attachment loss and deep pockets of patients with periodontitis (Fig. 1).

Elevated levels of neutrophil derived substances, such as b-glucuronidase and elastase, have been reported by a number of investigators (see Page 1992 and Lamster 1992, for review). Lamster recommends that substances found in GCF -e.g. elastase – should be calculated per site because of the risk of contamination with saliva, which can change the volume and subsequently the concentration. However, the effect of such contamination can be markedly reduced by careful sampling techniques and by discarding the strips that may be contaminated (Griffiths et al. 1990). Deep gingival pockets contain larger volumes of GCF and probably have higher amounts of various substances. In Paper I a correlation was found between elastase activity per site and pocket depth. The measurements of GCF content should therefore be calculated per µL in order to avoid the influence of larger volumes in deep pockets. The elastase activity per µL was higher in GCF from periodontitis sites than in GCF from gingivitis sites in subjects with gingivitis alone although the clinical signs of inflammation (GI) were the same.  In contrast, there was no difference in the amount of antigenic elastase per µL. This could be caused by a combination of higher release of elastase and an oxidative inactivation of a-1-antitrypsin (A1AT) followed by a compensatory increased inhibition by a-2-macroglobulin (A2MG). This  indicates that the tissue destruction in periodontitis is associated with hyperactive neutrophils.

Release of oxygen radicals from neutrophils may oxidatively inactivate and thus, reduce the inhibitory capacity of the otherwise sufficient amounts of A1AT (Weiss 1989).  The impaired inhibitory capacity of A1AT may, to some extent, be compensated for by the equally efficient and less oxidatively sensitive A2MG. This makes it important to study the relation of A2MG to increases in elastase activity in connection with inflammatory destruction.

In Paper II, the local balance between A2MG and elastase was analysed in the same GCF sample from three types of sites (cf. Paper II in Investigations and Results). The amounts of this inhibitor per µL were significantly lower from sites with tissue destruction, while the elastase activity per µL was higher (Fig. 2). The findings of lower concentrations are in agreement with those of Skaleric et al. (1986). The most likely mechanism for reduced concentrations at the sites with destruction seems to be consumption of A2MG by free proteolytic activity  due to  the faster clearance of the A2MG -protease complex than that of the native A2MG. This kinetic difference in elimination of A2MG has been demonstrated by Debanne (1975). The lower concentration of A2MG is therefore a secondary sign of proteolytic activity and, together with the higher elastase activity, strongly indicates the presence of a tissue-destructive inflammatory reaction, in agreement with Paper I.

However, in Paper II, the two variables elastase and A2MG could not distinguish between the two types of inflamed sites without attachment loss and without deep pockets in patients with periodontitis and patients with gingivitis alone. One possible explanation is a difference in the number of neutrophils accumulated in the lesion, although they had the same clinical signs of inflammation, as assessed by GI. However, GI is a rather insensitive measure of the degree of inflammation, expressed as the number of neutrophils (Kowashi et al. 1980, Adonogianaki et al. 1993). It is possible that the inflamed sites in the patients with gingivitis alone, in agreement with their higher GCF volumes (Paper II), in fact were somewhat more inflamed than the clinically similar sites in patients with periodontitis. In consequence, the lesions of the gingivitis patients may have harboured more neutrophils, which increased the elastase activity to the levels of the sites in the periodontitis patients. However, if it were possible to establish the degree of inflammation, as regards the amount of neutrophils in the lesion, in the two types of gingivitis sites rather than by using the GI, the relation between proteolytic activity and the varying degrees of inflammation might reveal differences.

An increased release of elastase into the tissue and into the GCF must be caused either by an increase in the number of neutrophils and/or by an increase in the release of this substance from the neutrophils per se.

In Paper III,  the elastase activity was related to the lactoferrin content in the same GCF sample in order to assess the release of elastase in relation to the number of neutrophils in the lesion.  Lactoferrin was chosen as a marker of neutrophils because it is specific for these cells (Benett & Kokocinski 1978) and is only found in trace amounts in plasma (Hetherington et al. 1983). In vitro, lactoferrin is released following chemotactic stimulation (Bentwood & Henson 1980) and it can be assumed that this mainly occurs during the migration from the blood vessel to the inflamed gingival site. Samples were obtained from the same three types of sites as in Paper II. The study showed a higher elastase activity per µL in the two types of sites in patients with periodontitis than in the sites in subjects with gingivitis alone.

In contrast, no difference was noted in the lactoferrin concentrations in the three types of sites (Fig. 3). If lactoferrin can be regarded as a marker of the number of neutrophils, this means that the “periodontitis neutrophils” released more elastase per cell, regardless of the pocket depth (PD, gingival inflammation (GI), and attachment level (CAL) at the sampled site. However, since the substrate used in this study is hydrolysed by the elastase-A2MG complex, an alternative explanation is that a larger proportion of the released elastase is inhibited by A2MG instead of by A1AT. In any case, both interpretations indicate a neutrophil associated specific host response in periodontitis.

The analysis of the protein concentrations  (Paper IV) in the same three types of sites as in the previous two studies (Papers II and III) showed a lower concentration in GCF samples from patients with periodontitis than in samples from gingivitis patients, which was not related to the clinical findings (PD, GI, CAL) in the sampled site (Fig. 4). This study indicates that dilution of GCF may contribute to the lower concentration of A2MG in sites with attachment loss (Paper II). However, other mechanisms must be involved, since the low A2MG concentration was  associated with attachment loss (Fig. 2) and not as the low protein concentration, found in all sites in patients with periodontitis. The physiological mechanism and the pathogenetic importance of this finding are not clear, but the result strongly supports the theory of a host specific response in periodontitis.

Papers I-III dealt with measurements of elastase in GCF that were made with paper strips. For this reason the sampling of GCF was evaluated in Paper V. Unlike elastase complexed with A2MG, pure elastase could not be recovered from the strips (Table 3). This means that the elastase measured in the previous GCF studies (Papers I-III) originates from this complex. Moreover, it is unlikely that there is any free active elastase in GCF, since the calculated in vivo half-life of active elastase is 0.6 msec and, consequently, all activity  should be inhibited after 3 msec (Travis & Salvesen 1983). It is not apparent why uncomplexed elastase cannot be eluated in isotonic phosphate-buffered saline (pH 7.4). The molecular weight had no influence on the recovery, since the other proteins tested had a recovery of about 90 %, regardless of their molecular weight (12-725 kDa). The fact that elastase has a very high isoelectric point (IP) may provide an explanation, but an increase in the pH of the eluent did not improve the recovery.

In conclusion, most proteins could be satisfactorily and reproducibly recovered when the elution was made in isotonic buffer at neutral pH and without detergents to minimize lysis of the neutrophils. In contrast, the recovery of uncomplexed elastase was poor, showing that the recoveries of various proteins may differ. According to these findings, the previous investigations of GCF in this thesis, and in earlier studies made with paper strips and low-molecular elastase substrates, probably did not measure free elastase activity, but rather the activity of the elastase-A2MG complex.

In Paper III it was deducted that increased elastase activity in GCF from periodontitis patients could be due to an oxidative inactivation of A1AT with a subsequent increase in the need for inhibitory activity of A2MG or to an increased activity of the neutrophils per se. Both explanations indicate an increase in the  neutrophil activity. It was therefore of interest to study the in vitro activation of peripheral neutrophils. An increased release of oxygen radicals (e.g., Åsman et al. 1984,  Leino et al. 1994) and of elastase (Åsman 1988) has been reported in juvenile periodontitis.

Fcg-receptor-mediated activation of peripheral neutrophils (Paper VI) showed a doubling of the release of oxygen radicals from the neutrophils in a group of adult patients with periodontitis as compared to a group of healthy controls (Fig. 5). The release of oxygen radicals was measured with luminol-enhanced chemiluminescence. In juvenile periodontitis, the findings concerning in vitro release of oxygen radicals are inconclusive. Disagreement in the results are probably due to different activation mechanisms, such as complement factor activation (-e.g., opsonized zymosan), protein kinase C (phorbol myristate acetate) and activation by specific receptors (-e.g., Fc-gR). However, investigators involving Fcg-receptor activation with opsonized bacteria have consistently shown increased CL (Whyte et al. 1989, Shapira et al. 1991) in agreement with five separate studies in our laboratory (Åsman et al. 1984, 1986, 1988, Åsman 1988,  Åsman & Bergström 1992). We also found a  higher release of elastase, while the release of lactoferrin did not differ within the pairs. The ratio of elastase to lactoferrin release, however, was significantly higher in the patients with periodontitis, which accords with the finding in Paper III of a higher elastase/lactoferrin ratio in GCF from this type of patients.

Since most studies using receptor mediated activation of the neutrophils have shown an increased release of oxygen radicals from neutrophils in periodontitis patients, while studies using direct PKC activation with PMA have failed, it was reasonable to assume, that the difference is to be found at the level of the  receptors. However, the higher activity was not associated with increased number of FcgIII-receptors (CD16) on the cell membranes in Paper VI. This is in agreement with earlier findings in juvenile periodontitis (Åsman & Bergström 1992, Leino et al. 1994, Mouynet et al. 1995). Nor did the neutrophil populations in the same study differ, regarding the expression of the adhesion molecules CD11a, b (LFA-1, Mac-1), CD15 (Lewis X), CD35 (CR1) and CD62L (Mel-14/LECAM-1), between patients with periodontitis and gingivitis.

Taken together, the studies in this thesis support the theory of a specific host response associated with hyperreactive neutrophils. Whether this hyperreactivity of the neutrophils is a constitutional trait or is due to priming (an enhancement of the neutrophils’ ability to respond to various stimuli) of the circulating cells is not known. Åsman et al. (1988) found that the increase in the release of oxygen radicals from neutrophils in patients with juvenile periodontitis persisted even after “successful” treatment. The view of a constitutional trait is supported by a recent study by Pippin et al. (1995), that found higher intracellular content of b-glucuronidase in neutrophils from patients with rapidly progressing periodontitis. Other investigators report that the neutrophil hyperreactivity in juvenile periodontitis is serum-associated (Agarwal et al. 1994, Shapira et al. 1994), which would indicate a priming of the cells. Cytokines, such as tumor necrosis factor a (TNFa) could be important primers of circulating neutrophils (Steinbeck & Roth 1989). A priming by TNFa has been suggested in association with adult respiratory distress syndrome (ARDS). ARDS is a very serious condition, usually seen in connection with severe injuries. It is  characterized by rapid destruction of lung tissue, probably resulting from an excessive release of proteases from neutrophils in the lungs. Chollet-Martin et al. (1993) found higher plasma concentrations of TNFa in patients suffering from ARDS than in patients with similar injuries but without ARDS. Neutrophils from patients with certain types of obstructive jaundice are less responsive to in vitro priming with TNFa (Jiang et al. 1994). This could be due to an in vivo priming that makes the cells less responsive to a second priming in vitro .

Some studies show impaired or altered chemotaxis of neutrophils in patients with localized juvenile periodontitis (-e.g., Clark et al. 1977, Van Dyke et al. 1982, 1990) and in patients with refractory periodontitis (Oshrain et al. 1987). This is not necessarily in conflict with a higher extracellular release of free oxygen radicals, since a priming of the peripheral cells, besides enhancing the oxidative metabolism, might also increase the adherence of the cells, and thereby impair their motility. In fact, there are several conditions with aberrant neutrophil functions that exhibit the combination of impaired chemotaxis and increased respiratory burst, such as Down’s syndrome, HIV and diabetes (cf. Introduction). Priming of human neutrophils by tumor necrosis factor in vitro has shown an enhanced release of oxygen radicals and degranulation, and a simultaneous down-regulation of chemotaxis (Bajaj et al. 1992, Agarwal et al. 1994). It seems reasonable that future pathogenetic studies of periodontitis should include evaluation of a possible influence of priming on the tissue-destructive activity of the neutrophils.

Conclusions

The GCF studies (Papers I-III) show that patients with periodontitis have an increased neutrophil elastase activity, which is independent of pocket depth, fluid volume and numbers of neutrophils. This would suggest a relation between neutrophils and a host specific tissue-destructive type of inflammation. The in vitro study (Paper VI) shows an increased release of oxygen radicals and elastase from the neutrophils in periodontitis patients. This would imply that hyperactive neutrophils participate as effector cells in the tissue-destructive mechanism. The findings in this thesis consistently support the view that neutrophils play a major role in the specific host response in periodontitis.

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to all of you who helped me with this study at the Centre for Clinical Oral Science, Department of Periodontology and the  Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge Hospital, Huddinge, Sweden. In particular, I wish to thank:

Dr. Björn Åsman, tutor and friend, for never-failing enthusiasm and sincere interest in the daily life of the neutrophil. Without his encouragement, optimism and expertise there would have been no thesis.

Docent Kurt Bergström, my tutor and supervisor, for his constant support and for sharing with me his vast medical knowledge and scientific experience.

Professor Per-Östen Söder for introducing me to the field of scientific research and to Bergström and Åsman.

Docent Jan Bergström for valuable support and inspiring discussions about my research.

Dr Eva Åkerlöf for helping me with all my ELISA problems.

Laboratory engineer Göran Nilsson for continuous assistance in my laboratory work.

Gunvor Nilsson & Gunnel Zeisig for their optimism, help and consideration.

Laboratory engineer Hildegard Sablica who gave me technical assistance and good advice.

Gun Nygren for her excellent assistance in the laboratory.

Leyla Gümüsay for patiently helping me with all those samples.

Francis and Zoe Walsh for making my English more readable, especially by introducing the comma.

My wife, Anita and my boys, Erik and Oskar, for their great patience, love and support.

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I– Protease activity in gingival crevicular fluid. Presence of three protease. Figueredo CMS & Gustafsson A. Journal of clinical Periodontology 1998:25; 306 –310.

II– Activity and inhibition of elastase in GCF.
Figueredo CMS & Gustafsson A. Journal of clinical Periodontology 1998:25; 531 –535.

III– Increased release of elastase from in vitro activated peripheral neutrophilis in patients with adult periodontitis. Figueredo CMS, & Gustafsson A, Asman B and Bergström K. Journal of clinical Periodontology 1999:26; 206 –211.

IV– Increased interleukin–1ß concentration in GCF as a characteristic tarit of patients with periodontitis. Figueredo CMS, Ribeiro MSM, Fischer RG, and Gustafsson A. Journal of Periodontology; resubmitted for publication in February 1999.

V– Increased amounts of laminin in GCF from untreated patients with periodontitis.
Figueredo CMS, & Gustafsson A. Journal of Clinical Periodontology; submitted for publication in March 1999.

II– Introduction 1

Periodontitis

A crevicular accumulation of microbes, follewed by inflammation and immune reaction, are the main features of gingivitis and periodontitis. A small but definite infiltrate of inflammatory cells can be detected in the coronal portion of the connective tissue, although the clinical findings are not typical of inflammation. Thus, a subclinical inflammatory reaction is generally present in clinically healthy gingiva (Page & Schroeder 1976).

Plaque accumulation in the crevice leads to an increase in the inflammatory reaction that can be seeen clinically and microscopically. Until a gengival lesion becomes “established” (Page & Schroeder 1976), this pattern persists, with inflammation following plaque accumulation. At a certain stage in this process, marked irreversible tissue destruction begins. Degradation of the connective tissue is followed by junctional epithelial migration and bone resorption. This stage is at the border line between gingivitis and periodontitis. It is not clear why some lesions remain localized on the marginal part of the gingival tissues, while others go on to loss of connective tissue attachment and supporting alveolar bone. Moreover the severity of periodontal tissue damage often varies from tooth to tooth and even from surface on the same tooth in the same subject.

Epidemiological studies show that the frequency of the severe form of adult periodontitis is very low in the population. The percentage of subjects with the disease tends to increase with age, reaching a peak at 50–60 years. Brown et al. (1989) reported that only 3.4% of the United States population between 19–44 years had 1 or more pockets deeper than 6 mm. This percentage increased to 16.5% in the population between 45–64 yeras, and decreased to 14 % in those 65 years or more.

Baelum et al. (1986) studied adult tanzanians aged 30–69 years, having large amounts of plaque and caculus, and found that only 19% showed pockets deeper than 3 mm and attachment loss > 6mm. Seventy–five percent of the tooth sites with attachment loss 7 mm were found in 31% of the subjects, showing that advanced periodontal disease does not correlate with supragingival plaque levels. The same investigators (Baelum et al.1988) observed, in a 15 to 65 year old population in Kenya where poor oral hygiene was reflected by plaque, calculus and gingivitis, that pockets 4 mm deep were found on less than 20% of the surfaces, and the proportion oof sites per subject with deep pockets and advancedloss of attachment showed a markedly skewed distribuition.

The current view of the role of micro–organisms as the principal etiologic factor in periodontal diseases can be summarized by saying that the periodontopathic bacterial flora is “necessary, but not sufficient to cause disease” or that periodontal disease are” specific mixed infections which cause periodontal destruction in an appropriately host” (Offenbacher 1996). This renewed emphasis on the host–response in pathoogenesis is based on three factors: (1) the inflammataryresponse varies greatly from one subject to another (Movig et al. 1988, Pociot et al. 1992, Shapira et al. 1994). (2) epidemiological population studies show that microbial parameters account for only some of the incidence and prevalence of the disease. Other factors, such as smoking, stress, systemic diseases, genetic and biochemical markers of inflammation, also contributesignificantly to multivariate models of disease expression, independently of the microbial infections (Beck at al. 1990; Beck et al. 1992; Christersson et al. 1992; Eheeler et al. 1994). (3)Epidemiologic studies in twins have suggested that, overall, about half the variability of periodontal disease expression is controlledby genetic, not microbial, factors (Michalowicz 1993; 1994).

Wolff et al. (1993) found that the frequency and levels of five gram–negative anaerobic bacteria, P. gingivalis, A. actinomycetemcomitans, P. intermidia, E. corrodens and F. nucleatum, correlated with the probing depth of 6905 sites sampled. This could be either an indicator of disease or simply the result of a more favourable environment. Deep pockets favour anaerobic bacteria, such as those mentioned above but they can also be found in shallow pockets, even in subjects without periodontitis (chen et al. 1989; Dahén et al. 1992). According to Wolff et al. (1993) the finding that periopathogens frequently inhabit sites not associated with advanced disease suggests that a susceptible host is necessary.

Although reports on the presence of still unidentified etiologic organisms or pathogenic clonal types cannot be disregarded, it is not correct to say that specific organisms are the cause of severe disease. According to Kornman & di Giovine (1998), the response to chronic inflammation may be modified by factors that do not directly cause the disease, but modify some aspects of it to aggravate the clinical conditions. The chronic reaction of an inflammation may be disproportionated to the pathogenic challenge, resulting in a response detrimental to the host. Smoking (Harber 1994), diabetes (Salvi et al. 1997c) and genetic influences (Kornman et al. 1997) may place some subjects at relatively high risk for an increase in the severity of periodontitis.

1.1– Risk factors

A risk factor is part of the causal chain of a particular disease or can lead to exposure of the host toa a disease (Beck 1994). The presence of a risk factor implies a direct increase in the probability of that a disease will occur, and if a risk factor is absent or removed, a reduction will probably ensue. This differs from markers, which generally result from a disease, vary over time and contribute to the natural history of disease progression (Salvi et al. 1997b)

1.1.1 Diabetes

Diabetes entails a risk of periodontitis, with an odds ratio of 2 to 3 for diabetics, as compared to non–diabetics (Salvi et al. 1997b). The increase in susceptibility seems to be associated more with an aberrant host–response (Salvi et al. 1997a;c) than with differences in the microbiota (Mandell et al. 1992). Diabetics may increase susceptibilityto periodontitis by impairing neutrophil chemotaxis and phagocytosis (McMullen et al. 1981). Moreover, monocytes from patients with diabetes secrete more prostaglandin E, TNF– and interleukin–1 after stimutalion with lipopolysaccharide (Salvi et al. 1997a; Salvi et al. 1998).

1.1.2– Smoking

Smoking is a well–accepted risk factor in periodontitis. In a meta–analysis, Papapanou (1996) confirmed that smoking is a risk in periodontitis, with an odds ratio of 2.8. The pathogenic mechanisms underlying this are not clear. regarding subjects with gingivitis, no differences were found between smokers and non–smokers in the composition of microbiota or the plaque accumulation rates (Bergström & Preber 1994). Preber et al. (1992) reported that patients with periodontitid who smoke do not differ from those who do not, with respect to possible periopathogens. However, Zambon et al. (1996) reported that smokers had ssignificantly higher levels of B. forsythus and were at significantly greater risk of infection with this periodontal pathogen than non–smokers. Smoking causes several changes in the response to inflammation – e.g., it can impair the chemotaxis and phagocytosis of normal peripheral neutrophils (Kraal & Kenney 1979) and alter their oxidative bursst (Ryder et al.1998). Locally, Numabe et al. (1998) showed that salivary neutrophilis intensify their phagocytic activity after smoking.

1.1.3– Genetic

the clearest evidence of genetic factors in adult periodontitis ccomes from periodontal findings in adult twins. More than 50% of the variance in several clinical and radiographic measures appears to be explained by genetic factors (Michaliwicz et al. 1991a; Michaliwicz et al. 1991b; Corey et al. 1993).

Kornman et al. (1997) showed that a composite genotype including 2 polymorphisms in the cluster of IL–1 genes (allele 2 of IL–1B +3953 plus allele 2 of IL–1A –889) was significantly overrepresented in the severe periodontitis group. Gore et al. (1998) found that the frequency of IL–1 genotypes, including the IL–1 + 3953 allele 2 (IL– 1 + 3953 1/2 and IL–1 + 3953 2/2), previously correlated with increased IL–1 production, was significantly higher in patients with advanced than in those with early–to–moderate adult periodontitis.

2. Neutrophils

Neutrophils are produced in the same bone mmarrow, where the myeloid precursor cells mature to segmented neutrophils in about 9 days, and are then released into the circulation. The cells remain in the bloodstream for a relatively short time (half–life = 6–7h) and in the tissue for 1–4 days . They can be rapidly mobilized within 30 mim to the site of an bacteria. In man, neutrophils are the predoominant leukocyte in blood, comprising between 50% and 70% of the circulation pool of leukocytes.

Neutrophils exist in three states: quiescent, primed and actiivated. Neutrophil activation seems to be mediated by a rise in cytosolic free Ca 2+, which correlates with activation of the oxidase system. “Primed” means that the cells are “ready to go”, but await a further stimulus before the oxidase response is elicited . A resting cell can receive a priming or an activation stimulus. Only the activated cells show oxidase activity but, if the primed cell also receives an activating stimulus, the ensuing oxidase activity will be higher than that in unprimed, activated cells (Hallet & Lloyds 1995). Greater chemotaxis and degranulation have also been found when neutrophils are primed with cytokines, such as TNF– (bajaj et al. 1992) and interleukin–1 (Brandolini et al. 1997).

Cytokines are ssolube protein mediators of immunity. They produce their effects on target cells via specific membrane receptors. Cytokines are generally rather small polypeptides (17 – 20 kDa), which are divided into four major groups: interferons, TNF, interleukins (IL) and growth factors. Most of them are not stored in secretory granules, but are synthesized when needed (Leffell 1997). Although T–cells, natural killer cells and monocytes/macrophages produce large amounts of cytokines, nearly all cells infiltrating an inflamed tissue are neutrophils, which may therefore be a large source of cytokines. At least two interleukins, IL–1 and IL–8, have a strong association with neutrophils and periodontal diseases.

A relation between interleukin–1 (IL–1) and perioddontal disease has been reported in many studies. IL–1 is a potent proinflammatory cytokine that can influence the host–response in the periodontal lession. It is produced in and forms, wich are different gene productos, but utilize the same cellular receptor. Both are produced mainly by macrophages, but alsso partly by other cells, including neutrophils (Tokoro et al. 1996; Galbraith et al. 1997). IL–1 is the major inflammatory cytokine in gingival tissue associated with periodontitis (Tokoro et al. 1996). It is formed and released in response to several immunostimlatory agents – e.g., lipopolysaccharide (LPS) and tissue degradation products (Hanazawa et al. 1985) – and can activate endothelial cells to upregulate ICAM–1 and E–selectin expression (Bevilacqua et al. 1985; Carlos & Harlan 1994), wich may increase the diapedesis of leukocytes. IL–1 has also been found to activate osteoclasts (Dewhirst et al. 1985). All these IL–1effects increase the inflammatory response and can subsequently cause degradation of the periodontal ligament and alveolar bone.

Interleukin–8 (IL–8) is a small cytokine that exhibits chemotactic activy against neutrophils, but not against nmonocytes 8Harada et al. 1996). It can be produced in vitro by a wide variety of cell types, including monocytes, lymphocytes, neutrophils and keratinocytes (mukaida et al. 1995). Il–8 is not constitutively produced, but occurs in response to an inflammatory stimulus and has a strong effect on the migration and activation of neutrophils – e.g., it can induce the degranulation of neutrophils (Brandolini et al. 1997).

2.1 Antimicrobial functions

The neutrophil recognizes and combats foreing substances by their receptors for several surface structures – for instance, the fc–portion of attached antibodies, complement factors, capsule reactive proteins and the LPS–complex. Antibodies(especially IgG) and complement proteins like C3b can opsonize and are therefore referred to as “opsonins”. Opsonization is the process whereby particles such as micro–organisms become coated with molecules, which allow them to bind to receptors on phagocytes.IgG antibodies bind to the antigens in the Fab region,leaving the Fc region sticking out. phagocytes have Fc gamma receptors(FcR) and they can therefore bind to the coated molecules and internalize them.

Three classes of FcR are currently distinguished: (1) FcRI (CD64) – a 72 kDa high–affinity receptor for IgG, constitutively expressed on monocytes, macrophages,myeloid progrenitor cells and dendritic cells; (2) FcRII (CD 32) –a broadly distributed 40 kDa molecule, found in most types of blood leukocytes, “Langerhans” cells, various populations of endothelial cells, dendritic cells, macrophages and platelets; and (3) FcRIII (CD16) – a glycoprotein having a molecular weight ranging from 50 – 80 kDa with two gene expressions. The FcRIIIA gene product is a transmembrane receptor named FcRIIIa, found on natural killer cells, macrophages, subpopulations of T–cells and fresh isolated blood monocytes, immature thymocytes and placental trophoblasts. The FcRIIIB gene product is FcRIIIb, which is celectively expressed on neutrophils (for a review, see Rascu et al. 1997).

Activated neutrophils csn destroy foreign substances by at least three mechanisms: 1) respiratory burst, 2) cytolysis and 3) phagocytosis and degranulation.

2.1.1 Respiratory burst

The respiratory burst realeases highly reactive oxygen metabolites extracellularly. Oxidative metabolism mainly generates hypochlorous acid (HOCI), because of the relatively high concentration of CL in plasma (Weiss 1989), which has both tissue–destructive and antimicrobial effects.

2.1.2 Cytolysis

This is a mechanism by which antibacterial substances reach the bacteria. Considering the high concentration of neutrophils in inflamed tissue, the release of antibacterial agents from dying cells may be an important host–defense mechanism(McNamara et al. 1988).

2.1.3 Phagosytosis and degranulation

Phagocytosis is a process in which the neutrophil isolates the organism intracellulary by creating a phagosome . Fusion between the phagosome and cytoplasmic granules deliveres high concentrations of antibacterial substances into the vacuole.

Degranulation leads to the release of anti–bacterial substances extracellularly(Wright 1988) from the primary (azurophil) granule(defensins, lysozyme, myeloperoxidase, cathepsin G and elastase) and the secondary 8specific) granule 8lactoferrin, collagenase and lysozyme). A tertiary granule containing gelatinase has also been described (Dewald et al. 1982). The degranulation of the primary and secondary granules is of special interest in this study. Bentwood & Henson(1980) reported that human neutrophils respondednto soluble stimuli withh sequential release of, first, the secondary(specific) granule constituent, followed by the constituents of the primary granule.

2.2 Neutrophils and tissue destruction

Neutrophils are associated with tissue destruction in chronic inflammation – e.g., adult respiratory ditress syndrome (Lee et al. 1981) and emphysema(Janoff 1985). During periodontal inflammation, increased numbers of neutrophils have been found in sevarel morphologic studies of the inflamed gingiva. Attström & Egelberg (1970) examined leukocytes in clinically healthy and chronically inflamed sites in humans and dogs. The leukocytes were present in both healthy and inflamed sites. Differential counts showed 95–97% neutrophils, 1.2% lymphocytes and 1–3% monocytes.The proportions remained the same, although the number of leukocytes increased with inflammation. In another study by the same author (Attström 1971), leukopenia was induced with nitrogen mustard and with heterologous anti–neutrophil serum in dogs having chronically inflamed gingiva. The leukopeina rediuced the number of crevicular leukocytes, the enzymatic activity and the volume of gengival crevicular fluid. Kowashi et al.(1979), who counted neutrophils in gingival washings during experimental gingivitis in man, found a doubling of the number of cells from day 0 to day 20. Although the local number of neutrophils are associated with the degree of inflammation, it is not conclusively shown that a marginal chronic gingivitis evolves into a perodontitis lesion. Gustafsson et al. (1994) showed that, in chronic gingivitis and in periodontitis sites, the numbers of neutrophils seem to be very similar, supporting the concept that hyperactivity per cell, rather tham differences in the local cell numbers, is important for initiation of tissue destruction.

The term “hyperactive” was chosen to describe higher activity of neutrophils in an inflamed site, while “hyperreactive” was chosen to describe higher reactivity of peripheral after in vitro activation. Studies of peripheral neutrophils from patients with juvenile periodontitis, uisng the production of oxygen radicals as a marker for neutrophil activity, have shown hyperreactivity after Fc–receptor stimulation (Asman 1988; whyte et al.1989; Shapiran et al.1991). The same phenomenon has recently een seen in adults with periodontitis(Gustafsson & Asman 1996; Fredriksson et al. 1998). Chollet–Martin et al.(1992) called “hyperresponsive” a subpopulation of neuttrophils, isolated from patients withacute respiratory distress syndrome(ARDS), which showed an increased capacity to generate hydrogen peroxide (H2O2). Although the hyperreactivity of neutrophils from periodontitis patients might be related to the generation of oxygen species other than HO (Fredriksson et al.1998), the accumulating neutrophils may produce extensive tissue damage by generating reactive oxygen species in both diseases.

Besides reactive oxugen species, there are strong indications that neutrophil proteases can injure the lung and periodontal tissues. Neutrophil proteases can degrade lung and arterial–wall elastin(Janoff & Zeligs 1968), cause emphysema in animal(Weinbaum et al. 1974) and collagen degradation(Janoff 1983). Increased amounts of neutrophil–derived substances, such as (beta)–glucuronidase(Lamster et al. 19949, neutrophil collagenase(Overall et al. 1987) and elastase(Gustaffsson et al. 1992), have been found in GCF in deen pockets in periodontitis patients.

The release of elastase from in vitro–activated peripheral neutrophil has been shown to differ significantly in patients with juvenile periodomtitis, as compared to healthy controls(Asman 1988), but it has not been shown in patients with adult periodontitis.

2.2.1 Neutrophil elastase

One of major end–products of neutrophil activity is the extracellular release of active and inactive proteases. Elastase is a neutral serine protease(33kDa), synthesized primarily in promylocytes and stored in the cytoplasmatic azurophil granules of maturing neutrophils in amounts ranging up to 3 picogramas per cell(Janoff 1985). Although neutrophils contain large amounts of elastase, they have no elastase mRNA transcripts, indicating that matureneutrophils cannot produce this enzyme. The expression of the elastase gene is limited to a very short period in leukocytes differentiation(Fouret et al. 1989).

Elastase can degrade many important proteins the extracellular matrix, such as elastin (Janoff & Zeligs 1968), laminim (Heck et al. 1990), fibronectin and collagen (Janoff 1985, Owen & Campbell 1995). The destructive capacity of released elastase is usually offset by the protease inhibitors –1–antitrypsin(A1AT) and –2–macroglobulin (A2NG). A1AT (52 kDa) seems to be the main regulator of elastase. This is a glycoprotein synthesized in the liver, and it can also be produced locally by macrophages and neutrophils(Bois et al. 1991; Pääkkö et al.1996). Normally, A1AT is present in such abundance that all active proteases are inhibited within milliseconds. However, elastase can avoid inhibition by three mechanisms: 1) when the enzyme is released in closed compartments in concentrations sufficient to overwhelm the available inhibitor; 2) release of enzyme in close proximity to its substrates, which then successffully compete with A1AT ti bind elastase; 3) enzyme released in sites where local A1AT molecules have been inactivated by oxidation.

The respiratory burts generates reactive oxygen radicals that are released extracellularly. Oxigen radicals are tissue–destructive per se (Weiss 1989), but they can also act in concert with simultaneously released proteases. Extracellularly–released oxigen radicals can oxidatively inhibit A1AT and thus allow the proteases to degrade matrix proteins close to the neutrophils (Weiss 1989). The facilitation of neutrophil elastase activity by oxidative inactivation of an inflammatory reaction. It can degrade tissue inhibitors of metalloproteinases (Weiss 1989) and, at the same time, may be involved in the extracellular activation of latent metalloproteinase (Ferry et al. 1997). Elastase is able to activate pro–geletinase B, which is one of the majo factors in neutrophil migration across the basement membrane (Delclaux et al. 1996) while causing extensive damage to the basement membrane by degrading laminin (Heck et al. 1990).

Several studies have shown increased elastase activity against specific low molecular weight substrates in periodontitis (e.g., Gustafsson et al. 1994; Ingman et al.1994) and increased levels of elastase activity have also been suggested as a perdictor of periodontal disease progression8Palcnis et al. 1992; Armitage et al. 1994). Such substrates are hydrolyzed by free elastase and bound to A2NG (Travis & Salvesen 1983). This means that measurements of elastase activity with these substrates cannot distinguish between free elastase and the elastase–A2MG complex. Moreover, to our knowledge, the presence of free elastase activity in GCF from patients with adult periodontitis has not been convincingly shown.

In summary, elastase is very important for normal tissue turnover and for combating infection, but an excessive release in the active form together with a higher production of oxigen radicals, might cause partial inactivatiion of the major inhibitor, A1AT, and thus lead to extensive tissue destruction.

III – AIMS

The main goals in the thesis were: (1) to detect protease activity that might explain differences in site–specific tissue destruction; (2) to find in vitro evidence of peripheral neutrophil hyperreactivity related to the release of elastase; (3) to determine whether increased levels of interleukin–1(beta) could explain the local neutrophil hyperreactivity; (4) to show that inflamed sites from periodontits patients have more activated neutrophils tham inflamed sites from healthy controls, by measuring basement membrane degradation; (5) and to determine whether there is a relation between levels of IL–8 and site–specific tissue destruction.

1. Specific aims

Paper I – To determine whether free protease activity is present in GCF.

Paper II – To determine whether free elastase is present in GCF.

Paper III – To determine whether there free differences in the release of first and secundary granule between peripheral neutrophils from periodontitis patients and healthy controls.

Paper IV – To test the hyputhesis high levels of IL–1(beta) in GCF are characteristicof patients with periodontitis.

Paper V – To test the hypothesis that the presence of hyperactive neutrophils generates more basement membrane degradation in the inflamed sites of periodontitis patients.

IV MATERIAL AND METHODS

1– Clinical parameters

The presence of supraginginval plaque (PI) and gingival inflammation(GI) were recorded using the criteria of Silness & Löe (1964) (Papers I, II, IV and V). Only sites with a GI value of 1 or 2 were included. In sites without inflammation (value 0), the volume of gingival crevicular fluid (GCF) was insufficient to permit measurement. It was usually impossible to examine very infalmed sites (GI=3), because of difficulty in avoiding contamination with bllod. Pocket depht was measured with calibrated periodontal probe.

2– Gingival crevicular fluid samples

In Papers I and II, GCF samples ewrw collected with repeated intracrevicular washings, as described by Salonen & Paunio (1991), to avoid retention of free elastase in the more commonly used paper strips (Gustafsson 1996). Each pocket was washed five times with 5l of PBS during continuous aspiration . All samples from sites in the same category in each person were pooled and diluted with PBS up to 1 ml. Since one can not measure the volume of GCF with the intracrevicular washing method, we related protease activity to the concentration of transferrin in the wash–fluid, to obtain a semiquantitative estimate (Asma et al.1981). The amount of transferrin in GCF increases with fluid volume, probably because of leakage from plasma(Adonogianaki et al. 1994) and can therefore, be viewed as a marker of GCF volume.

In Papers IV and V, the sites were sampled with paper strips. The strip was inserted into the pocket until mild resistance was felt and kept there for 30sec. The volume of GCF was measured with a Periotron 8000 ®GCF meter: Before each study, the unstrument was calibrated, using saline delivered with a Hamilton syringe.

3– Protease activity

Free preotease activity was measured with FITC–conjugated casein. The mixture of casein and sample and incubated for 20h at +37°C, during agitation. After incubation, trichloroacetic acid 0.6M/l was added to stop the reaction and percipitate the intact casein molecules. After 30 minutes, the samples were centrifuged at 3000g for 10 minutes. The amountesof FITC–conjugated cassein fragments in the aupernant corresponds to the amount of protease activity in the samples. Fluorescence was analysed in a fluorescence spectrophotometer, at an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

4– Elastase activity

The levels of elastase activity were measured with the low molecular weight substrate(445.5 Da) L–pyroglutamy–L–propyl–L–valine–p–nitroanilide. The substrate is highly specific for granulocytes elastase(Tanaka et al. 1990) but it is also hydrolyzed by the elastase––2–macroglobulin complex (Wewers 1988). To distinguish between activity derived from free elastase and elastase bound to A2NG, an excess amount of A1At was added to the samples. A1AT is a very effective inhibitor of free elastase activity, but it can innhibit activity from the elastase bound to A2MG (Travis & Salvesen 1983). The activity inhibited by A1AT could thus be seen as derived from free elastase and the remaining activity from the elastase–A2MG complex.

5– ELISA assays

5.1 –Elastase – A1AT complex

A polyclonal antibody against A1AT was coated on a 96–well microtitre plate overnight at + 40°C. The wells were washed five times with PBS + 0.05% Tween ®20. Samples or standards were added to each well and incubated for one hour at +37°C. The wells were washed again, as above. and an alkanline phosphate–conjugated polyclonal sheep antibody aginst elastase was added and incubated for one hour at + 37°C. After a final washing, the substrate p–nitrophenol–phosphate was added to each well and the absorbance was read after 10 min at 405 nm in a spectrophotometer.

5.2– Antigenic elastase

A monoclonal antibody against elastase was coated on a 96–well microtitre plate overnightnat +4¼C. The wells were washed four times with PBS + 0.05% Tween®. Diluted samples or standards were added to each well and incubated for 1h at +37¼C. The wells were washed again, as above, and a polyclonal sheep antibody against elastase was added to them and incubated for one hour at + 37¼C. After another wash, the third antibody, an alkaline phosphatase–conjugated rabbit ant–goat IgG, was added and incubated, as above. After a final washing, the substrate p–nitrophenol–phosphate was added to each well. The absordance at 405nm was read in a spectrophotometer after 10 min. The antigenic elastase assay does not detect elastase bound to A2MG.

5.3– Lactoferrin

A microtitre plate was coated with a monoclonal mouse antibody against lactoferrin and incubated overnight at + 4¼C. The wells were washed five times with PBS + 0.05% Tween¨. Samples and standards were added to the wells and incubated for one hour, washed once again and the second antibody, a polyclonal rabbit anti–lactoferrin, was added. The plates were incubated for one hour at +37¼C and washed five times before adding of the third antibody, an alkaline phosphatase–cconjugated polyclonal goat antibody against rabbit IgG, and incubating for one hour. The phosphatase activity was measured after the additing of the substrate p–nitrophenol– phosphate and the absorbance at 405 nm was read in a spectrophotometer after 10 minutes.

5.4– –1–antitrypsin (A1AT) and –2macroglobulin (A2MG)

Antigenic A1AT and A2MG were measured with a one –layer ELISA. The microtitre plate was coated with samples and standard, diluted in carbonatee buffer, pH 9.6, and icubated overnight at +4¼C. After incubation, the plate was washed four times with PBS + 0.05% Tween¨. The antigen was detected with a polyclonal rabbit antibody against A1AT or A2MG during incubation at +37¼C for one hour. After another washing, as described above, the final antibody, a goat anti–rabbit IgG conjugated with alkaline phosphatase, was added. The plate once again incubated at +37¼C for one hour and washed, as above. The phosphatase activity was measured after the addition of the substrate, p– spectrophotometer after 10 minutes.

5.5– Interleukin–1 andInterleukin–8

A monoclonal antibody to IL–1 or IL–8, diluted in carbonate buffer, was coated onto the microtitre plates overnight at +4¼C. After the coating, the plates were washed four times with PBS+0.05% Tween, blocked with 1% HSA, and incubated for 1h at room temperature. After washing, as above, a standard curve and samples were coated onto the plates. The plates were incubated at +37¼C and shaken for 45 min and washed as above. The detection antibody, a biotinylatedpolyclonal goat anti– IL–1 or IL–8, was incubated at +37¼C and shaken for 45min. After washing, as above, the horse radish peroxidase (HRP)–conjugated streptavidin was added to the plates and incubated in the same way as the detection antibody. The plates were washed once again, as above, before the HRP–substrate, 3,3«, 5,5«–tetramethyl–benzidine, was added. The reaction was stopped with 1M H2SO4 after approximately 10min and the absorbance at 450 nm was read in a spectrophotometer.

5.6– Laminin

the standard curve and diluted samples were added to the microtitre plates and incubated overnight at +4¼C. After washing the plates, a polyclonal rabbit antibody against laminin, diluted in PBS + 0.1%HSA, was used as a primary antibody. The plates were then incubated during shaking 45 min at 37¼C. After another washing, the second antibody, a biotinylated polyclonal goat anti–rabbit IgG diluted in PBS+0.1% HSA, was added and incubated during shaking at +37¼C fro 45 min. After a third wash, as above, HRP–conjugated streptavidin was added to the plates and incubated in the same way as the detection antibody. The plates were once again washed, as above, before the HRP–substrate, 3,3« , 5,5«–tetramethyl–benzidine, was added. The reaction was stopped with 1M H2SO4

5.6– Laminin

the standard curve and diluted samples were added to the microtitre plates and incubated overnight at +4¼C. After washing the plates, a polyclonal rabbit antibody against laminin, diluted in PBS + 0.1%HSA, was used as a primary antibody. The plates were then incubated during shaking 45 min at 37¼C. After another washing, the second antibody, a biotinylated polyclonal goat anti–rabbit IgG diluted in PBS+0.1% HSA, was added and incubated during shaking at +37¼C for 45 min. After a third wash, as above, HRP–conjugated streptavidin was added to the plates and incubated in the same way as the detection antibody. The plates were once again washed, as above, before the HRP–substrate, 3,3« , 5,5«–tetramethyl–benzidine, was added. The reaction was stopped with 1M H2SO4 after approximately 40 min, and the absorbance at 450nm was read in a spectrophotometer.

5.7– Transferrin

Samples and standards were coated on a 96 micro–well plate overnight at +4¼C. After incubation, the plate was washed four times with PBS +0.05% Tween. One hundred l of a polyclonal rabbit antibody against trasferrin was added to the plate and incubated for onw hour +37¼C. The plate was washed again and the second antibody, an alkaline phosphatase–conjugated swine anti–rabbit immunoglobbulin, was added. After one more hour if incubation and still another wash, 100 l of the substrate p–nitrophenol–phosphate was added. The absorbency at 450nm was read after 30 min in a spectrophotometer.

6. Flow cytometric analysis

To confirm the presence of intrcellular stores of A1At, 250 000 leukocytes were fixed and permeabilized. After permeabilization, 2l of a rabbit polyclonal antibody against A1AT was added to the cells and incubated for 20 min during aditation, followed by the addition of 2l of a FITC–conjugated polyclonal swine antibody against rabbit IgG. A rabbit IgG fraction was used as the negative control. Flow cytometric meassurements were mada after gating the subpopulations on a histogram with foward– and side– scatter. The mean fluorescence intensity and the staining in percentage were recorded.

7– Cell Preparation

Venous blood was collected into vacuum tubes containing EDTA and allowed to rest for 1 hour at room temperature. A leukocyte–rich preparation from 7ml of venous was made by lysing the red blood cell in whole blood at 4¼C for 10 min. with 0.83% NH4Cl solution (NH4 Cl 8.3g, KHCO3 1.0g, Na2 EDTA 2H2O 0.04 g and distilled water to 100 ml) suplemented with 0.25% HSA, ratio of blood to reagent = 1:7. After centrifugation (350 g, 5 min), the harvested leukocyte pellet was washed twice with PBS/HSA and stored at + – 0¼C in the same buffers for one hour.

8– Degranulation

The leukocytes (1.0 x 106 netrophils) stored in PBS + 0.25% HSA, were mixed with IgG–opsonized Staphylococcus aureus (75 bacteria per neutrophil) (Bergström & Asman 1993). Tubes without bacterial stimulation were used as controls for each subject. The mixture was further diluted with Hankks«Balanced Salt Solution up to a final reaction volume of 1 ml, and incubated for one hour during horizontal agitation. The reaction was stopped by centrifuging at 1000 g for 5 minutes. The supernatant was removed and stored at –70¼C, pending analysis. the pellet containing the leukocytes was diluted up to 1ml with distilled watter and the cells were homogenized by repeated freezing and thawing (four times), centrifuged again (1000g for 5 min.) to remove the cell fragments and stored at –70¼C, pending analysis.

9– Statistical analysis

The significances of differences between patients and controls were calculated with the Mann–Whitney U–test and of differences between the categories of sites in periodontitis patients with the Wilcoxon signed–rank test. The correlations were calculated with the Spearman rank correlation coefficient or the Kendall rank correlation coefficient.

V– RESULTS

In Paper I, we measured non–specific protease activity in GCF from inflamed sites with or without tissue ddistruction. The clinical findings are shown in Table 11. The free protease activity was higher in samples from inflamed sites with tissue destruction in subjects having periodontitis (PP), than in samples form inflamed sites without such destruction in the same subjects (GP) and samples from subjects with gingivitis alone (GG). The difference between PP and GP was significant (p=0.0019). There was a large variation in protease activity, but the highest activities were found mostly in samples from the periodontitis patients. PP showed a mean protease activity, corresponding to 230 ng trypsin (SD± 481.6), 52 ng in GP (SD±174.7) and 25 ng in GG (SD± 33.6). Median values are presented in Table 2. The residual protease activity, i.e. the activity measured after the addition of excess A1AT, was usually low in the GP samples than in GG and PP samples. The addition of A1AT inhibited a larger proportion of the protease activity in the GP sample than in the GG and PP samples (Table 2). The transferrin concentration was significantly lower in the GP samples than in the other two categories . We found no difference between GG and PP samples. The ratio of protease activity to trasnferrin was higher in PP than in GP. A significant difference was observed between GP and PP (p=0.0001) (Table 2).

Table 1. Comparison of mean values of clinical findings (± standard deviation) between GG (gingivitis in gingivitis patients), GP (gingivitis in periodontitiss patients) and PP (periodontitis in periodontitis patients). GI– gingival index; PI plaque index; PPD–probing pocket depth.
GG (n12)     GP (n19)     PP (n:19)
GI     1.0 (±0.4)     1.1 (±0.3)     1.3 (±0.4)
PI     0.9 (±0.5)     1.3 (±0.6)     1.5 (±0.5)
PPD (mm)     1.7 (±0.7)     2.4 (±0.6)     6.3 (±0.9)

Table 2. Median of protease activity (after inhibition with A1AT) expressed in ng, median of the amounts of transferrin expressed in ng, the ratio between protease activity and transferrin and mean of protease inhibition expressed as a %.
Test / Site     GG     P1     GP     P2     PP     P3
Protease activity     15.7     NS     5.0     p<0.002     56.9     NS
Residual activity     2.1     NS     0.3     p=0.02

2.2     NS
Transferrin     355.1     p<0.04     194.6     p=0.003     345     NS
protease/transf     0.038     NS     0.023     p<0.001     0.140     NS
Mean inhibition     86     NS     94     p=0.041     88     NS

P1: Differences between GG and GP, calculated with Mann – Whitney U–Test. P2: Differences between GP and PP, calculated with Wilcoxon signed–rank test. P3:Differences between GG and PP, calculated with Mann–Whitney U–teste.

In paper II, elastase activity was measured with a low molecular weight substrate and elastase bound to A1AT was measured with with an ELISA. To distinguish between free elastase and elastase bound to A2MG, we added an excess of A1AT to the samples. The activity inhibited by A1AT was considered as free elastase and the uninhibited activity as derived from the elastase–A2MG complex (c.f. materials and methods, page 19). The amounts of free elastase, total elastase and elastase bound to A2MG, were significantly higher in PP than in GP and GG (Table 3; Fig. 1). There were no significant differences in the amounts of ealstase bound to A1AT betwenn the groups. Mean values are shown in Table 3.

The amounts of transferrin in the wash fluid were similar in the PP and GG sites while they low in the GP sites. Since the distribution was skewed, the median concentration of transferrin is shown in Table 3 .

The ratio of total elastase to tranferrin was significantly higher in PP samples than in GP and GG samples. The ratio between free elastase and transferrin was also significantly higher in PP sites than in Gp and GG sites. It was almost twice as high in GP sites as in clinically similar GG sites(table 3). The was a strong negative correlation between the percentage of free elastase and the percentage of A1AT complex (R= – 0.93) calculated in all samples, n= 50 (Fig.2). We found a positive correlation between free elastase and the non–specific protease activity analyzed previously(Paper I), R= 0.41 in GG and 0.71 in PP (date not shown).

Table 3. Mean values of total elastase, free elastase, elastase inhibited by –1–antitrypsin (A1AT) and by –2–macroglobulin (A2MG), exprressed as cell equivalents x 10(3). The ratios of total elastase to tranferrin and of free elastase to transferrin are expressed as mean the amounts of transferrin are expressed as median.
Test / Site     GG (n12)     P1     GP (n 19)     P2     PP (n19)     P3
Total elastase     67.5     NS     64.3     <0.001     162.9     0.005
Free elastase     17.7     NS     28.8     <0.001     79.9

0.007
Elastase – A1AT     42.8     NS     27.6     NS     61.8     NS
Elastase A2MG     8.4     NS     9.6

0.001     21.1

0.014
Free elastase/transf     59.7     NS     104.5     0.025     339.0     0.012
Total elastase/transf     148.6     NS     263.4     0.05     572.5

<0.001
Transferrin – ng     355     0.036     195     0.003     345     NS

p1: Differences between GG and GP, calculated with the mann–Whitney u ––test. p2: Differences between GP and PP, calculated with the Wilcoxon signend–rank test. p3: Significance of differences between GG and PP, calculated with the Mann–whitney u –test. NS: not significant.

Fig.1. Mean values of total elastase, free elastase, and of elastase bound to A2MG and to A1AT, expressed in cell equivalents x 10(3).

Fig.2. Correlation between the percentage of a free elastase and the percentage of elastase –– 1–atitrypsin complex (A1AT) in all samples (n = 50).

In Paper III, we found that the release of elastase from peripheral neutrophils after stimulation with opsonized bacteria was significantly higher in patients with periodontitis than in healthy controls (p= 0.01) (Table 4, Fig.3). There was also a significant difference between the groups in the unstimulated sample s(p= 0.024)(Table 4). The total amount of elastase, i.e. the sum of the elastase activity released and the remaining activity in the cells, were similar in the unstimulated samples from the two groups. During bacterial stimulation, however, the total amount of elastase increased in samples from patients (p= 0.013). In contrast, no corresponding increase could be seen in the control group(Fig. 4). The total amount of A1AT and the amount of elastase –A1AT complex in the supernatant from the stimulated cells were higher than from the unstimulated cells in both patients and controls, but the differences between the groups were not significant (Table 4). The flow cytometric analysis showed that the granulocytes contained intracellular stores of A1AT and that only negligible amounts of the inhibitor were present in monocytes and lymphocytes. The release of lactoferrin, used as a marker of the release of secondary granula, was the same in both groups.

Table 4. Significances of differences in elastase released, total elastase–A1AT complex (E–A1AT), total A1AT and lactoferrin. The degranulation was analysed in four groups: CO – Control subjects without bacterial stimulation; CB – Control subjects after bacterial stimulation; PO – Patients without bacterial stimulation and PB – Patients after bacterial stimulation.
CO – CB*     PO – PB*     CO – PO**     CB – PB**     pairs
Elastase released     0.002     0.001     0.024     0.010     15
Total elastase     NS     0.013     NS     0.044     15
Total A1AT     0.041     0.002     NS     NS     13
E–A1AT complex     0.007     0.002     NS     NS     13
Lactoferrin     0.012     0.008     NS     NS     10

* Significance of difference calculated with the Wilcoxon signed–rank test. ** Significance of difference calculated with the Mann–Whitney U – test. NS not significant.

Fig.3. Elastase activity, expressed as cell equivalent x 10(3), released from peripheral neutrophyls during Fc R–mediated simulation for 60 min in 15 healhy controls and 15 patients with adult periodontitis. Horizontal bars indicate mean.

Fig.4. Mean values of elastase activity released extracellularly and elastase extracted from the pellet (expressed as cell equivalents x 10(3). Samples from 15 control subjects incubated in Hank’s buffer with opsonized bacteria (CB) or in Hank’s buffer alone (C0) and from 15 patients with (PB) or without simulation by bacteria (P0).

In Paper IV, we measured the concentration of IL–1 and the elastase activity in GCF samples from untreated subjects with periodontitis and gingivitis. The plaque index (PI), gingival index (GI) and the volume of GCF collectec were similar in the periodontitis sites from patients and in the gingivitis sites fromhealthy controls, but the values were lower in inflamed shallow pockets from periodontitis patients (Table 5). No significant correlations, were found between the clinical data at various sites and the IL–1 concentrations, with the exception of GCF volume, were a significant positive correlation was found (p<0.001) (data not shown).

Both the concentration and the total amount of IL–1 were significantly higher in shallow (GP) and deep pockets (PP) in patients with periodontitis than in gingivitis sites(GG) in healthy sobjects. No signgficant difference was noted between GP and PP. PP also showed higher elastase activity, althogh the difference was not signgficant (Tables 6a and b). A weak positive correlation was present between elastase activity and IL–1 (p= 0.06). This correlation was most pronounced in the GG samples (p= 0.03).

The amount of elastase–A1AT complex was significantly higher in PP tahn in GG and GP(Table 6b). The concentrations of E–A1AT, A1AT and A2MG were similar in the three types of sites (Table 6a).

Table 5. Comparison of mean values at various sites in 13 subjects with gingivitis and 18 patients with periodontitis. GG– gingivitis sites in gingivitis in patients.GP– gingivitis sites in periodontitis patients,PP– periodontitis sites in periodontitis patients. PI– plaque index; GI– gingival index; PPD– probing pocket depth expressed in mm and GCF– gingival crevicular fluid expressed in l.
Site     GG     p1     GP     p2     PP     p3
PI     1.7     NS     1.2     0.001     1.7     NS
GI     1.7     0.05     1.4     0.003     1.9     0.021
PPD     1.9     NS     2.1     0.001     6.1     0.001
GCF     1.8     0.013     1.3     0.006     1.9     NS

p 1 : Differences between GG and GP, calculated with the Mann–Whitney U –test. p 2: Differences betwenn GP and PP, calculated with the Wilcoxon signed–rank test. p3: Significance of differnces between GG and PP, calculated with the Mann–Whitney U–test. NS: nit significant.

Tables 6a and b. Mean concentrations (6a) and total amounts (6b) of elastase activity (E activity), elastase––1–antitrypsin complex(EA1AT), –1–antitrypsin(A1AT), –2–macroglobulin (A2MG) and interleukin–1 (IL–1) in GG (gingivitis sites in gingivitis patients), GP (gingivitis sites in periodontitis patients) and PP(periodontitis sites in periodontitis patients), n= 13 control subjects with gingivitis and 18 periodontitis patients.

Table 6a
Site     GG     p1     GP     p2     PP     p3
E activity ( cell equiv/l )     26600     NS     22400     NS     30000     NS
EA1AT (nmol/l)     0.26     NS     0.23     NS     0.44     NS
A1AT (nmol/l)     0.24     NS     0.17     NS     0.24     NS
A2MG (ng/l)     458     NS     738     NS     621     NS
IL–1 (pg/l)     2.0     0.009     5.0     NS     5.4     0.0036

Table 6b
Site     GG     p1     GP     p2     PP     p3
E activity ( cell equiv)     46500     0.04     27700     0.03     68500     NS
EA1AT (nmol)     0.42     NS     0.29     0.001     0.75     0.04
A1AT (nmol)     0.38     0.04     0.20     NS     0.40     NS
A2MG (ng)     656     NS     823     0.002     1164     NS
IL–1 (pg)     2.9     0.02     5.5     0.002     9.0     0.001

p1: Differences between GG and GP, calculated with the Mann–Whitney U –test. p2: Differences between Gp and PP, calculated with the Wilcoxon signied–rank test. p3: Significance of differences between GG and PP, calculated with the Mann–Whitney U –test. NS: not significant.

In paper V, we studied laminin and IL–8 in GCF samples from untreated subjects with periodontitis and gingivitis. The clinical findings are shown in table 7. The amounts of laminin were significantly higher in PP, as compared to GG, but no significant differences was noted between shallow and deep pockets in the patients group (table 8). The distribution of the amounts of laminin in the samples is shown in Figure 5. The concentrations of laminin tended to be higher in the GP samples (table 8). The amounts of IL–8 and lactoferrin were very similar in the three groups (table 8). Strong negative correlations were found when the GCF volume was correlated with the concentrations of laminin (Fig. 6) lactoferrin (Fig. 7), while no correlation with the IL–8 concentration was observed.

A strong positive correlation was observed between the concentrations of laminin and lactoferrin (R= 0.72,p= 0.001). This correlation was more pronouced in patients with preiodontitis (R= 0.81, p=0.003) than in control subjects (R= 0.69, p= 0.02).

Table 7. Mean values for PI– plaque indexm GI–gingival index, PPD– probing pocket depth expressed in mm and GCF– gingival crevicular fluid expressed in ml, in 12 subjects with gingivitis and 13 with periodontitis. GG– gingivitis sites in subjecs; GO– gingivitis sites in periodontitis patients; PP– periodontitis sites in periodontitis patients.
Site     GG     p1     GP     p2     PP     p3
PI     1.4     NS     1.6     NS     1.6     NS
GI     1.3     NS     1.7     NS     1.9     0.01
PPD     2.4     NS     2.6     0.0001     5.5     0.0001
GCF     1.8     NS     1.6     0.02     2.4     0.03

p1: Differences between GG and Gp, calculated with the Mann–Whitney U–test. p2: Differences between GP and PP, calculated with the Wilcoxon signed–rank test. p3: Significance of differences between GG and OO, calculated with the Mann–Whitney U –test. NS: not significant.

Table 8. Mean values of the total amounts and concentrations of laminin, interleukin–8(IL–8), expressed in picogramas(pg) and picogramas per microlitre(pg/ml), and lactoferrin expressed in arbritary units(a.u) and a.u/ml, in GG(gingivitis sites in gingivitis subjects) GP(gingivitis sites in periodontitid patients) and PP(periodontitis sites in periodontitis patients). N= 12 gingivitis and 13 periodontitis patients.
Site     GG     GP     PP
Laminin (pg)     287     397     385*
IL–8 (pg)     7.4     7.1     9.2
Lactoferrin (a.u.)     526.6     423.6     546.8
Laminin concentration (pg/l)     182     397     179
IL–8 concentration (pg/l)     4.4     8.9     3.8
Lactoferrin concentration (a.u./l)     352.8     378     237.1

* Difference between GG and PP, calculated with the Mann–Whitney U –test.

Fig 5. Boxplot showing the total amounts of laminin in 12 GG (gingivitis sites in control subjects), 13 GP (gingivitis sites in patients with periodontitis) and 13 PP (periodontitis sites in patients with periodontitis). Median, 10th , 25th , 75th and 90th percentiles in boxplot.

Fig 6. Correlation between laminin concentration and GCF volume in all samples (n= 38).

Fig 7. Correlation between lactoferrin and GCF volume in all samples (n= 38)

VI – DISCUSSION

The studies in this thesis show that neutrophil hyperreactivity is probaby one of the mechanisms causing tissue destruction in patients with adult periodontitis. This view is supported by the following findings: 1) sites with tissue destruction showed a higher free elastase activity than sites without; 2) neutrophils from periodontitis patients reacted more strongly against Fcy–receptor–mediated stimulation in vitro; and 3) basement membrane degradation tenteded to be greater in these patients. Moreover, such patients also have increased concentrations of interleukin–1b in gingival crevicular fluid(GCF), regardless of the degree of tissue destruction.

For he past few years, several authors have studied protease activity in GCF from patients with periodontitis(Ohlsson et al. 1973; Kowashi et al. 1979). Here we assessed protease activity by evaluating as the degradation of FITC–conjugated casein. Casein is degraded by most protease but, in contrast to low molecular weight substrates, not by protease complexed to A2MG. We showed that free protease activity can be detected in GCF in inflamed sites with or without tissue destruction, and that it is higher in sites with tissue destruction. The addition of A1AT to the GCF samples inhibited almost all activity(approx. 90%), suggesting that the protease activity was due to an imbalance between protease and antiprotease rather than to activity of proteases not sensitive to inhibition by A1At.

Elastase activity in GCF was measured with a low molecular weight substrate, highly specific for granulocyte elastase(Kramps et al. 1983). Such a substrate is hydrolyzed by free elastase and by elastase bound to A2MG (Trevis & Salvesen 1983). This means that measurements of elastase activity with this substrate cannot distinguish between free elastase and the elastase–A2MG complex. To distinguish between activity derived from free elastase and elastase bound to A2MG, an excess amount of A1AT was to the samples. A1AT is a very effective inhibitor of free elastase activity, but it cannot inhibit activity from the elastase – A2MG complex(Teavis & Salvesen 1983). The activity inhibited by A1AT can thus be seen as derived from free elastase and the remaining activity from the elastase–A2MG complex.

The total amount of neutrophil elastase was significantly higher in sites with tissue destruction and a large proportion of this elastase was free – i.e., still active against important proteins such as collagen and elastin . The free neutrophil elastase activity was stronbly correlated with the free protase activity, and showed the highest R–value in sites with tissue destructio (R= 0.71). Incontrast to our results, Giannopoulou et al.(1992) found no free elastase in GCF in patients with periodontitis. The reason for this discrepancy is not clear, but one explanation might be the sampling technique. Giannopoulou collected samples with microcapillaries, a method that may disturb gingival vessels ans cause a leakage of plasma. A large influx of plasmacould innhibit all elastase activity in GCF.

Higher elastase activity in sites with tissue destruction may be due to both a higher relase of elastase per all and/or an increased number of neutrophils. Gustafsson et al.(1994) showed that a higher elastase activity in GCF from periodontitis patients was due to increased release from each cell rather than to differences in the number of local cells, which mght be due to local hyperactivity of neutrophils. We studied the association between peripheral neutrophil reactivity and destructive periodontal disease by means of Fcy–receptor–stimulated elastase release. Significantly more elastase was released by in vitro–activated peripheral neutrophils from adult patients having periodontitis than by those from healthy controls. This was also true of juvenile periodontitis, using tha same type of stimulation(Asman 1988), but it has not been shown before in adult periodontitis. On the other hand , a similar study showed no differences in the release of elastase between subjects with various degrees of periodontal disease when peripheral neutrophils were activated with formyl–methionyl–leucyl–phenylalanime(fMLP) (Giannopoulou et al.1994). However, the increased degranulation of neutrophils in such patients may depend on the kind of stimuli used. Corresponding studies of oxygen radical prodiction, using Fcy–receptor–mediated stimulation, have consistently shown a difference between periodontitis patients and healthy controls(Asman 1988; Whyte et al. 1989; Fredriksson et al.1998), while studies using other methods of activation have shown conflicting results(Henry et al. 1984; Mouynet et al. 1994).

The higher release of elastase by peripheral neutrophils from patients with adult periodontitis might be due to a priming/activation of neutrophils already in the circulation, which could influence the intracellular activity of elastase. Elastase is present in both an active(Gardim et al. 1991) and a proenzyme form inside the azurophilic granules(Cavarra et al. 1997), but the mechanism controlling the transition from one to another is not well understood. According to Tamura et al. (1998), primed neutrophils activated with fMLP release almost twice as much elastase as fMLP activation does alone. Priming is a process that enhances the cellÕs ability to respond to a second stimulus. An increased priming response may reflect an increased susceptibility to priminig or increased amounts substances with a priming effect. Such substances may be derived from the host, e.g., proinflammatory cytokines and degradation products(Wachtfogel et al. 1988; Steinbeck & Roth 1989), or from bacteria, e.g., formyl–methionyl–leucyl–phenylalanime(Allred & hill 1978) and LPS(Forehand at al. 1991) . We recently found that unstimulated neutrophils from periodontitis patients expressed higher intracellular elastase activity than in those from healthy controls. Unlike elastase activity, the total amounts of antigenic elastase were similar in patients and controls(Figueredo et al. Unpublished data). This might indicated greater intracellular activation of latent elastasein perioheral neutrophils from patients with adult periodontitis.

Although elastase may have a salutary effect on the normal turnover of tissues and on combating infection, excessive release, especially under conditions that compromise the function of regulatory innhibitors – for instance, a–1–antitrypsin (A1AT) and a–2–macroglobulin(A2MG) – can damage tissue(for a review, see Janoff 1985). A1AT inactivates virtually all mammalian serine proteases. However, the rate of inhibition shows that elastase activity is inactivated by A1AT at a rate tenfold higher tahn of other proteases, indicating that the primary function of A1AT is to contorl neutrophil elastase activity(Travis & Savesen 1983). Most A1AT is produced in the liver, but cells like macrophages and neutrophils can contribute to local production of this inhibitor. In neutrophils, A1AT is stored in the same granula as elastase, but it does not seem to bind elastase intracellularly. Elastase tends to be present in the peripheral of the granule(Gramer et al. 1989), while A1At is usually found in the granule matrix(Mason et al. 1991). One reason why elastase activity was higher in the supernatant from the patientsÕ cells could be release of A1AT. However, we found no differences in the release of A1AT that explained the increase in elastase activity. Another possibility could be differences in inhibitory capacity of the A1AT released, but this possibility was ruled out by determining the levels of the elastase–A1AT complex in the supernatantm which were also similar in patients and controls.

We found a strong negative correletion between the percentage of free elastase and the percentage of elastase–A1AT complex in GCF. Since the concentrations of A1AT are high in plasma and in GCF, local inactivation rather than a shortage of A1AT probably occurred, in combination with increased release of elastase. Systemic inactivation of A1AT has also been suggested in a recentstudy by our group, in whuch plasma levels of A1AT were analysed in patients with adult periodontitis and compared to those in healthy controls. We found higherconcentrations of antigenic A1AT in patients with periodontitis, but a similar A1AT protease inhibitor capacity. Moreover, when this capacity was related to the antigenic A1AT, the levels of functional A1AT had a tendency to be lower in patients with periodontitis(Fredriksson et al. Unpublished data).

A1AT is sensitive to oxidative and proteolytic inactivation. Thus, an excessive realease of active protease in combination with simultaneous release of oxigen radicals might cause an inactivation of A1AT and subsequent tissue destruction(Weiss 1989). A similar process has been described in patients with lung emphysema, where high levels of oxidants, mainly hydrogen peroxide, can produce a situation in which the amounts of A1AT are normal, but the functional periodontal are near zero(Travis 1988). The situation may be even worse in periodontal disease, due to the activity of microbial protease that can degrade and inactivate A1AT(Carlsson et al.1984; Travis et al. 1994).

Apart from A1AT; –2–macroglobulin(A2MG) can inhibit elastase and almost all other proteases in periodontitis sites. A2MG is a rapid and efficient clearing for free proteases in the circualtion (Travis & Savesen 1983). We found that a higher percentage of elastase was inhibited by A2MG in samples from sites with tissue destruction, than in those without, an observation that accords with studies of GCF samples collected with paper strips (Gustafsson et al. 1994; Meyer et al. 1997). This may because impaired inhibition by A1AT is patly compensated by increased inhibition by A2MG. However, the simultaneous realease of oxigen species and proteases can also inactivate A2MG (Abbink et al.1991), which could explain the presence of free elastase in our samples. Moreover, the large molecular weight (725 kDa) of A2MG makes it difficult to compensate an extravascular deficiency/inactivation of A1AT.

Having assessed local peripheral differnces in elastase activity, we also wished to find a pro–inflammatory substance that might be characteristic of a given patient. IL–1 has been reported to be much higher in periodontitis sites than in healthy ones (Stashenko et al. 1991; Tsai et al. 1995; Ishihara et al.1997). This could be the result of severer inflammation and/or constitutional differences in Il–1b production. Kornman et al. (1997) and Gore et al. (1998) showed that patients with severe periodontitis have a significantly higher frequency of an IL–1 genotype associed with increased production of IL–1 in vitro (Pociot et al. 1992). We compared the levels of interleukin–1b in GCF from inflamed shallow and deep pockets in sujects without attachment loss. We found that the concentration of IL –1b in GCF is higher in periodontitis patients, even when inflamed shallow pockets in patients are compared to those in subjects with gingivitis alone. There were no significant differences in IL–1b concentration between shallow and deep pockets in the same patient. Since the GCF volumes and concentrations of elastase–A1AT, A1AT and A2MG were similar, the local degree of inflammation could be assumed to be the same in the three categories of sites. This supports the hypothesis that IL–1 is more a characteristic of a given patients and less a result of tissue destrution in the sampled site. Wilton et al. (1992; 1993) studied IL–1 levels in GCF from patients with adult periodontitis and found no correlation with plaque index, bleeding or pocket depth. Reinhardt et al. (1993) likewise found no differnce in IL–1 levels between shallow and deep pockets in patients with periodontitis. Salvi et al. (1997c) reported significantly higher IL–1 levels in GCF from patients with moderated/severe periodontitis than in those with gingivitis/mild periodontitis. However, no comparisom was made between shallow and deep pockets within the same patient.

Our findings differ to some extent from those of others, who have reported increasing levels of IL–1 with increasing inflammation and pocket depht (Ishihara et al. 1997; Hou et al. 1995; Yavuzylmaz et al. 1995). This may be because our subjects had not been treated and had similar degrees of inflammation in shallow and deep pockets. It seems likely that even if the level of IL–1 is characteristic of a given patient, it is also influenced by the degrees of inflammation since IL–1 is not constitutively produced, but needs external stimuli.

In our last investigation (Paper V), we found higher amounts of laminin in GCF from patients with adult periodontitis , suggesting the presence of hyperactivated leukocytes during the process of transmigration through the basement membranes. This difference was clearest when comparing PP and GG sites (p=0.03), but there was also a tendency towards a difference between GP and GG. However, due to the wide range of values, it did not reach significance (p=0.16). This finding indicates that these patients have more activated leukocytes that cause greater destruction of the basal membrane during their migration through the endothelium /epithelium. The high prevalence of neutrophils in the inflammatory lesions the periodontitis (Attström & Egelberg 1970). Together with previous evidence of local and peripheral neutrophil hyperreactivity (e.g., Gustafsson et al. 1994; Fredriksson et al.1998), suggest that these cells act the primary mediator of local basement membrane degradation in patients with adult periodontitis . The similar amounts of lactofferrin indicated that the differences in basal membrane degradation are not due to differences in the number of neutrophils in the lesion.

Activated neutrophils are said to express membrane–bound neutrophil elastase om the cell membrane (Owen et al. 1995). It is an important bioactive form of enzyme, where elastase is catalytically active against extracellular matrix macromolecules but resistant to inhibition by naturally occurring protease inhibitors (Campbell & Campbell 1988). Owen et al. (1995) showed that N–formyl–methiony–leucyl–phenylalanine (fMLP), complement component 5a (C5a) and phorbol myristate acetate (PMA) stimulate a dose–dependent increase in membrane–bound elastase expression. They also showed that priming neutrophils with bacterial LPS and then incubating them with fMLP induces a threefold increase in the cell surface expression of elastase, as compared to cells incubated with fMLP alone. We speculated that membrane–bound elastase activity might be the machanism by which activated neutrophils cause basement membrane degradation.

Even if neutrophil hyperreactivy occurs in periodontitis patients, sites–specific differences in tissue destruction still remain to be explained. We hypothesized that local differences in IL–8 could be one of the factors involved in these differences, but we found no association between hogher Il–8 levels and pocket depht. Chung et al.(1997) reported a lower concentration of IL–8 in patients with periodontal disease prior to tratament, but similar total amounts, which accords with our findings. Our results suggest that the amounts of IL–8 in GCF are influenced by the severity gengival inflammation rather than by constitutional differences in the local production that could explain site–specific tissue destruction.

VII – CONCLUSION

The main conclusion of this thesis are: (1) Increased levels of free protease activity are associated with tissue destruction in periodontitis patients, and part of this activity is derived from neutrophil elastase. Free elastase activity is associated with a local impairement in the majo inhibitor of elastase, A1AT. (2) Peripheral neutrophil hyperreactivity in patients having adult periodontitis is also associated with increased release of elastase after Fcy–receptor stimulation. (3) Increase concentration of interleukin–1 is a characteristic trait of a given patiens. (4) The tendency towards higher basal membrane degradation in patients having periodontitis seems to confirm the presence of local hyperactivityof neutrophils. (5) No evidence was found of a correaltion between IL–8 and site–specific differences in tissue destruction.

VII – ACKNOWLEDGEMENTS

IX – REFERENCES

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