Periodontiamedica.com

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.

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

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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.

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

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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

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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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