Periodontal Immunology

Dr. Ken Miyasaki

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• The Immune System Involvement in the Pathogenesis of Periodontal Disease
• Complement & Periodontal Disease
• Neutrophils & Periodontal Disease
• Lymphocytes-Monocytes and the Chronic Phase of Periodontal Diseases

The Immune System Involvement in
the Pathogenesis of Periodontal Disease

It is pretty much agreed that the immune system is involved in the pathogenesis of periodontal disease. This chapter will provide a paradigm for understanding how the immune system is involved. "Periodontal disease" is actually a process: involving first a local infection by indigenous plaque bacteria and second, a time-dependent immune response which includes the remodelling of the subjacent connective tissues (including bone). By definition, a local infection by indigenous bacteria is an opportunistic infection. An opportunistic infection usually reflects a failure of innate immune mechanisms. The "time-dependent" or "chronic" immune response represents the way in which the host responds to the repeated defeat of innate defense mechanisms. The chronic immune response involves the generation of specific defense (mainly, opsonic antibodies) and remodelling of tissues. The remodelling is a complex process itself, involving cycles of "destruction" and "reconstruction." Periodontal probes detect only the morbidity (incomplete reconstruction) of the remodelling process.

Histology: Temporal Sequence

In the early 1970's, Page and Schroeder published classical histological studies on the progressive "natural history" of experimental periodontal disease lesions using dogs which were first given meticulous oral hygiene and subsequently deprived (Figure 1). Their work described four types of lesions representative of stages of disease. Although numerous observations were made with respect to tissue changes, we will emphasize temporal changes which occurred with respect to the immune system.

Initial lesion. The initial lesion begins within 2-4 days after deprivation of oral hygiene. It exhibits a few neutrophils in the junctional epithelium, which are in the process of egressing into the gingival crevice. There are small numbers of macrophages and lymphocytes in the connective tissue, and the lymphocytes are primarily T-cells. A small fraction of junctional epithelium exhibits a pathologic conversion to "pocket epithelium."

Early lesion. The early lesion evolves from the initial lesion within 4-7 days and continues up to 14 days. Defining the early lesion is a marked increase in lymphocytes infiltrating the subjacent connective tissue; but also, the junctional and sulcular epithelia continue in conversion to pocket epithelium. Neutrophils increase in numbers in the periodontal pocket until about the 12th day. More coronal aspects of the junctional epithelium may resemble microabscesses, with severe leukocyte infiltration. Lymphoid cells dominate the subjacent connective tissue, comprising up to 75% of the infiltrate. Early in this period, the predominating lymphocytic infiltrate are T-cells, although B-cells become abundant and eventually dominate later in the lesion. T-cell blast transformation (ie., proliferation and functional activation) can be observed. Realize that the exact duration of each phase may vary, and that the phase during which the predominating infiltrating cell type are T-cells can be prolonged for up to three weeks (Seymour et al., 1983. J. Periodont. Res. 18: 375-385).

Established lesion. After 14 days, the neutrophil infiltration of the pocket and junctional epithelium is intense. B-cells undergo blast transformation and conversion to plasma cells (with local production of antibody) and monocytes undergo conversion to macrophage. Page and Schroeder suggested that the established lesion was extremely prevalent in man, and could be stable for many years.

Advanced lesion. The lesion is considered advanced when the destruction of bone is evident. At this late stage, the pocket epithelium shows ulceration, junctional epithelium shows great apical extension. Plasma cells continue to dominate the connective tissues, and neutrophils continue to dominate in the junctional epithelium and gingival crevice. Many of the plasma cells appeared to be "degenerating" (Page and Schroeder, 1976). Logically, one of the proposed mechanisms for the release of cytosolic molecules would involve degeneration. Importantly, the cytokine, interleukin-1b, (IL-1b) is actually a cytosolic molecule (it is synthesized without a leader sequence).

There are several important temporal aspects of the histology of periodontal disease that you should remember. First, that all of these lesions except the advanced lesion represent what we would call "gingivitis," from the clinical perspective (Table 1).

Histology: Spatial Considerations

As alluded to above, the distribution of cell types within the periodontal arena is not uniform. The neutrophil infiltrates the junctional and gingival crevice (panel A, Fig. 2). Lymphocytes and other mononuclear cells are within the subjacent connective tissue (Page and Schroeder, 1976; Nisengard, 1977. J. Periodontol. 48: 505-516). Initially, neutrophils migrate through the coronal aspects of the junctional eptihelium, and may play a destructive role in the loss of contact made between the tooth and the junctional epithelium, thereby initiating the formation of pocket epithelium. Neutrophils are also stratified within the gingival crevice, and rather than forming a homogenous bacteria/leukocyte mixture in the gingival crevice, the neutrophils form a "leukocyte wall" interposed between the periodontal plaque mass and the junctional and sulcular (or pocket) epithelium (panel B, Fig. 2). The "leukocyte wall" is unique, no one in medicine has ever seen such a structure in which leukocytes overlay epithelium (the gingival crevice is unique). The leukocyte wall may function both as a secretory and as a digestive (phagocytic) organ. Crevicular neutrophils exhibit both partial degranulation and ingested bacteria.

Half the leukocytes infiltrating the junctional epithelium and 90% of the leukocytes isolated from crevicular fluid are neutrophils. Whereas neutrophils predominate the junctional epithelium and the gingival crevice, lymphocytes and monocytes/ macrophages predominate within the subjacent connective tissue. To put this in perspective, the concentration of neutrophils in the periodontal tissues exceeds the concentration of neutrophils in blood. In minimally-inflamed gingivitis, 2.5 x 107 neutrophils/cm3 infiltrate the gingival connective tissue and 1.7 x 108 neutrophils/cm3 are found in the junctional epithelium (blood levels range from 1 x 106 to 4 x 106 neutrophils/cm3). Although some lymphocytes, mainly B-cells (but no plasma cells), can be observed within the gingival crevice (just as neutrophils may be observed in the periodontal connective tissue) most mononuclear cells (lymphocytes and monocytes) can be found within the subjacent connective tissues.

A Paradigm for Pathogenesis Opportunistic Infection

Specific infections - the specific plaque hypothesis. It is widely accepted (though never rigorously proven) that certain periodontal diseases result from infection by specific microbes (as shown in Table 2); a belief which is referred to as "the specific plaque hypothesis."

The nastier of these bacteria also possess evasive strategies which can defeat aspects of both the acute and chronic immune systems. For example, A. actinomycetemcomitans has a well-known leukotoxin (kills phagocytes) and possesses a lymphosuppressive factor which can delay specific immunologic responses. Most of the oral Gram negative bacteria possess low molecular weight factors which seem to impair neutrophil chemotaxis. These observations lead to the concept that specific periodontal infections may result from the failure of a specific portion of the host immune system and not simply a general immune failure (Miyasaki. 1991. J. Periodontol. 62:761-774). These failures may be intrinsic or acquired. An intrinsic defect can lead to juvenile forms of periodontal disease, whereas acquired defects may lead to diseases with a later onset.

Natural History. The strategy of host defense against specific periodontal infections may be viewed as similar to that used in combatting any local infection (Fig. 3, from Chapter 1). In this extremely simplified scheme, potential periodontal pathogens first encounter plasma factors, such as complement, within the crevicular or extracellular fluids. If complement (± antibody) is not successful in controlling the pathogen, neutrophils are recruited into the gingival crevice (a hypoxic environment) and provide the first cellular host mechanism to control periodontal bacteria. Neutrophils are well-adapted to function in the hypoxic environments, in that virtually all of their energy is derived from fermentation of stored glycogen rather than oxidative phosphorylation. They also possess a large number of antimicrobial mechanisms which do not depend upon oxygen. The changes resulting from acute inflammatory activities are usually reversible.

In periodontal pathogenesis, chronic inflammation begins essentially a few hours after the appearance of neutrophils. However, a spatial separation remains between the two phases, as monocytes and lymphocytes are recruited into the connective tissue and seldom follow the neutrophil into the crevicular arena. Monocytic cells probably function more effectively in the oxygenated connective tissues than in the oxygen-deficient gingival crevice, since almost half of their energy is derived by mitochondrial respiration and oxidative phosphorylation.

The exact purpose of macrophages and lymphocytes in the gingival tissues has not been identified. However, we may speculate that macrophages ingest particulate and soluble microbial antigens, and either digest the antigen completely (and signal repair processes with cytokines such as transforming growth factor b) or present partially digested antigen in association with major-histocompatibility complex (MHC)-encoded class II molecules to lymphocytes. The lymphocytes, which predominate in the connective tissue infiltrate, may then generate specific immunologic responses. Both the monocytes and the lymphocytes elaborate cytokines which not only lead to differentiation and activation of the local immune cells, but also signal the surrounding tissues. Continued microbial insult will cause these cells to elaborate cytokines which lead to connective tissue remodelling. In continued microbial insult, remodelling will result in net loss of connective tissues. In the absence of microbial insult, anti-inflammatory signals will be elaborated and remodelling will result in healing.

Perspective

In summary, neutrophils control the periodontal microecology within the gingival crevice. Although we may view neutrophils as antimicrobial cells, it is possible that they may protect the host by enhancing the growth of non-pathogens within the complex ecology of the gingival crevice. There is also good evidence that neutrophils are in constant communication with the chronic immune cells, and may exert anti-inflammatory effects initially and pro-inflammatory later. Chronic inflammatory cells, monocytes and lymphocytes, do all that it takes to prevent a local infection from becoming systemic and life-threatening. A destructive tissue response is a mechanism of amputation and isolating the infection. Please realize that there is much overlap in the actual local lesion: acute and chronic phase elements actually participate throughout the lesion, both spatially and temporally, and that the above paradigm only focusses on the predominant reactions. A more even consideration of the role of various elements in host defense will be provided, but the student should first understand the general strategy before attempting to go on.

In this paradigm, the student should note that the neutrophil is protective with respect to the periodontium. The chronic cells are destructive. We will explore the validity of these assertions in the subsequent sections. Also, note that several notions are discarded: (a) that bacteria directly produce periodontal disease; (b) that immunopathologic reactions are involved; and (c) that immunoregulatory disorders are involved. Periodontal disease is a well-regulated, normal immune response to chronic bacterial infection (Ranney, 1991. J. Periodont. Res. 26:243-254; Genco. 1992. J. Periodontol. 63:338-355).

Conflicting Opinions to The Paradigm

So far, I've discussed my own opinions. Read this following section (thoughtfully provided in fine print) to judge for yourself how dogmatic I am, because someday, you will meet people with other points of view.

Can't bacteria directly produce periodontal disease? That is, can't we equate "periodontal infection" with "periodontal disease" and use the terms interchangeably? One would think so listening to the microbiologists. Microbial virulence factors from certain periodontal bacteria may directly cause the connective tissue destruction that we refer to as periodontal disease (Socransky and Haffajee. 1991. J. Periodontal Res. 26: 195-212). Although the direct destruction of tissues by bacterial toxins is possible and well-reviewed in the citation above, to date there are no studies confirming a direct pathogenic role for microbial toxins in periodontal disease. This is despite the fact that many potential toxins have been identified (Including bacterial metalloproteases and thiol-dependent proteinases). No microbiologist has designed a study involving the induction of periodontal disease in an animal model by a mutated putative pathogen (either reduced pathogenicity in a mutant or increased pathogenicity by a nonpathogen possessing a virulence factor from a putative pathogen). As such, there is no study which proves that microbes possess substances which directly produce periodontal pathology. Although this does not disprove the contention that bacteria possess toxins which directly causes the tissue destruction of periodontitis, it seems implausible that microbial toxins would (or could) orchestrate an organized destruction of the periodontal tissues, being so careful as to destroy bone prior to producing soft tissue erosion.

Can't immunopathology be involved? Nisengard (1977. J. Periodontol. 48: 505-516) pointed out that classical immunopathologic mechanisms may be involved in periodontal disease destruction. Classical immunopathologic reactions include (a) Type I - immediate hypersensitivity mediated by IgE and mast cells, (b) Type II - cytotoxic hypersensitivity involving the interaction of antibodies to antigens bound to target tissues and subsequent destruction of tissues by complement lysis, antibody neutralization, or phagocyte degranulation (c) Type III - immune complex interactions involving the formation of circulating antibody-antigen- complement aggregates ("immune complexes," IC) and the deposition of these IC on membranes and subsequent destruction by phagocytes, and (d) Type IV - delayed-type hypersensitivity involving specific CD4+ T-cells and monocytes (Summarized in Chapter 16).

In immune complex (Type III) immunopathology, IC form deposits in association with basement membranes or vascular walls. Neutrophils attack the immune complexes, forming relatively tight seals (via FcR, CR3, or CR4) with the tissue containing the IC deposits. Neutrophils damage the tissue by "frustrated phagocytosis," resulting in the secretion of damaging substances in the area of the immune complex. Exaggerated tissue destruction occurs for two reasons: (1) because the phagocyte can never completely engulf the target and continues to secrete toxic substances, and (2) because protective factors (such as the plasma antiproteases) are excluded from the space between the neutrophil and the tissue. This is the mechanism of immune complex glomerulonephritis, serum sickness, and certain destructive phases in rheumatoid arthritis. Type III immunopathologic reactions have been proposed as a potentially important mechanism in periodontal diseases (Nisengard, 1977). In these types of immunopathologic reactions, the neutrophil is usually an important factor in the destruction of tissues, although antibody and complement alone may also cause the immunopathologic reactions. At present, there is conflicting evidence for IC formation in adult periodontitis (AP) in man and dogs (Clagett and Page. 1978. Arch. Oral Biol. 23:153-165; Yamashita et al., 1987. J. Periodont. Res. 22: 353-358). It is probably safe to conclude that immune complexes form within the periodontium in adult periodontitis but in a rather limited manner. Therefore, one mechanism whereby neutrophils would be directly immunopathogenic may be via immune complex immunopathogenesis, but this does not seem to be a hallmark of the disease. That is, IC are an inconsistent finding in a rather consistent disease.

Can't periodontal disease be an immunopathologic autoimmune reaction? Periodontal disease may result from antibody directed against tissue antigens ("Type II"). Tissue destruction occurs as a result of antibody neutralization of tissue functions (Eg., cell-to-cell cohesion in pemphigus) or the activation of complement and neutrophils. To date, there appears to be no evidence for such autoimmune reactions against the gingival tissues (Anusaksathien et al., 1992. J. Periodontol. 63:194-199). One caveat is that there has been no truly systematic examination of autoimmunity and certain forms of periodontal disease, and no one has asked whether maturity onset forms of periodontal disease may represent an autoimmune disease in which the neutrophil (not the periodontal tissues) is the target. Most forms of RAP exhibit serum factors which impair leukocyte function, and although some of these are immunoglobulins, they appear to function as cell-directed inhibitors (CDI) of chemotaxis rather than as autoantibodies (Lavine et al., 1979. J. Periodont. Res. 14:10-19). CDI function via their Fc regions rather than their Fab regions. However, CDI have not been described for all cases of RAP. Furthermore, autoantibodies against neutrophils which inhibit chemotaxis have been described, and although the periodontal disease status of that individual was not recorded, a history of recurrent pyogenic infection was noted (Kramer et al., 1980. N. Engl. J. Med. 303: 1253-1258).

Ranney (1991) has concluded that the clinical and histopathologic signs of periodontal disease do not fit with any immunopathologic mechanism and that periodontal disease does not show many of the exaggerated characteristics important in immunopathology (eg., an autoimmune nature or abundant immune complexes). The idea of immunopathology in garden-variety AP has been abandoned (but I wouldn't be so sure whether it is not important in unusual forms of periodontal disease, especially those with an adult onset).

Can't the neutrophil be destructive? Certain immunologists would suggest that the relationship of the neutrophil to the periodontium is the "proverbial double-edged sword," capable of producing periodontal disease as well as protecting against such disease (Lamster and Novak. 1992. Crit. Rev. Oral Biol. Med. 3:31-60; Miller et al., 1984. J. Clin. Periodontol. 11: 1-15). They suggest that both hyporeactivity and hyperreactivity of the neutrophil can produce disease. This is a very even-handed and fair point of view, but is this fair point of view substantiated by experimental evidence?

The evidence presented by these individuals has not made a strong case. For example, they suggest that the neutrophil can cause periodontal destruction by the release granule proteases, but admit that many of those proteases can be neutralized by serum antiproteases. In nature, effector cells are usually required make direct contact (or sequester the target in a phagolysosome) with the target in order to damage that target. The neutrophil must also form a seal with the tissue it damages. One mechanism neutrophils may use to adhere to host tissues involves the formation of large antigen-antibody-complement aggregates (immune complexes); which, as mentioned above, have not been observed with any consistency.

It has been suggested that perhaps neutrophils do not need to form tight seals with the tissue they damaged. Neutrophil-derived myeloperoxidase and H2O2 may inactivate serum antiproteases. There is no evidence for or against such an occurrence within the gingival tissues. It is clear that neutralization can occur in vitro in the absence of other proteins. In the presence of other proteins in high concentrations, myeloperoxidase and its product, hypochlorous acid, are not specific for antiproteases and therefore, may be much less efficient.

Lamster and Novak (1992) cite the use of gold sodium thiomalate, an agent known to interfere with oxygen radical formation by phagocytes by blocking the activation of protein kinase C. They have shown that gold sodium thiomalate decreases periodontal destruction in squirrel monkeys induced to have periodontal disease by wrapping wire ligatures around the teeth (The ligatures serve as plaque traps). Fairly, they admit that gold sodium thiomalate is not that specific, and that it blocks many phagocyte functions as well as the function of other cells.

Others have argued that in certain animals (minks, marmosets, rodents) there is very little evidence that lymphoid cells (and B-cells in particular) are at all important in periodontopathogenesis. In fact, in these animal models, all destructive phases appears to relate to the influx of neutrophils and acute phase inflammation. Inactive phases are characterized by the decrease in neutrophils. Although this histologic evidence suggests that neutrophils may play some role in periodontal disease destruction, it should be pointed out that neutrophils from different species can differ to a great extent in the biochemicals packaged within their granules, and further, molecules from one species may not have the same biologic effect as analogous molecules from another species. The histologic data may simply reflect that in certain small animals with a brief lifespan, the task of inducing periodontal tissue amputation has been assigned to the neutrophil rather than the more intelligent lymphocyte-monocyte arm. Therefore, we cannot rationally extrapolate findings from small, brief-lived animals to humans.

A limited pathogenic role of neutrophils. It has been demonstrated in gnotobiotic animals and conventionally-reared dogs that in the absence of plaque, there is no gingival sulcus (Schroeder, 1976). The sulcus appears after plaque and inflammatory cells (neutrophils) develop. The attraction of the neutrophil is chemotactic, but not complement dependent. Neutrophils are capable of inducing damage to gingival epithelium in vitro (Altman et al., 1992. J. Periodontal Res. 27:70-79) and therefore, neutrophils may exert a pathologic effect in forming the gingival sulcus at this point. This is not a rigorous scientific association (one could argue that plaque or the gingival keratinocytes themselves were responsible for the tissue changes). Others may suggest to you that neutrophils are associated with changes in the perivascular connective tissue. Realize that such connective tissue changes are reversible and that they are not necessarily a sign of pathogenesis. Neither alteration of gingival epithelium nor changes in the perivascular connective tissue are the histological manifestation of what we call periodontitis.

Dysregulation of the immune system and periodontal disease. Seymour and colleagues (Seymour, 1987. J. Dent. Res. 66: 2-9; Taubman et al., 1988. Adv. Dent. Res. 2: 328-333; Seymour, 1991. J. Clin. Periodontol. 18: 421-426) advanced the proposition that a local or systemic dysfunction in "immune regulation" may be important in the immunopathogenesis of periodontal disease. Moreover, philosophically, they suggested that periodontal disease may be a failure of T-cell regulation, and rather than the proper functioning of immunoregulatory pathways as has been outlined in the 'paradigm' above. The evidence they present is mainly histologic, although biochemical information (regarding cytokine and MHC molecule expression) is emerging which may support this view. The concept of an immunoregulatory disorder has not been proven or disproven; however it is virtually certain that a primary, systemic immune dysregulation is usually not the cause of the periodontal destruction resulting from periodontal infection in most cases. What about a local, secondary immune dysregulation? To me, that fits the definition of normal, ie., the immune system is supposed to be responsive to local challenge.

Back to the paradigm. Because you are future doctors, you should decide what you would like to believe with respect to the role the immune system plays in periodontal pathogenesis. Be aware that there is no need to invoke direct bacterial toxicity, destruction of periodontal tissues by neutrophils, immunopathology or autoimmunity against the gingival tissues, or immune dysregulation in the pathogenesis of periodontal disease. With the rapid advances in our understanding of cytokines, periodontal disease may now be viewed as a properly functioning, orchestrated mechanism of connective tissue amputation designed to prevent systemic and bone infection (Ranney, 1991; Genco, 1992). To put things in perspective, the preservation of life is more important than the preservation of teeth. The net loss of bone and connective tissue is relatively slow and well-designed for the maintenance of the human dentition until death (which used to occur by the age of 35 until about 250 years ago). It is nonetheless important to understand the immune system and the role it plays in periodontal pathogenesis. Future diagnosis, methods of determining disease activity and susceptibility, and treatment will be based upon a firm understanding of why certain individuals are susceptible to periodontal diseases.

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