Role of bacterial debris in inflammatory diseases of the joint and eye

APMIS 98: 957 - 968,1990

Role of bacterial debris in innammatory diseases of the joint and eye

Review article

ALVIN FOX

Department of Microbiology and Immunology, University of South Carolina, School of Medicine, Columbia, South Carolina, U.S.A.

Fox, A. Role of bacterial debris in inflammatory diseases of the joint and eye. APMIS 98: 957-968,1990.

Several distinct rheumatic conditions (including Lyme arthritis, Reiter’s syndrome and rheumatic fever) as well as certain forms of the blinding disease, uveitis, may share a common etiology. In each instance specific bacterial pathogens may infect a distant site, which on interaction with the immune system, leads to a sterile inflammation in the joint or eye. These ”reactive” conditions may result, in some cases, from prior localization of non-viable bacterial remnants (including the cell wall or peptidoglycan) or alternatively ”dormant” fastidious bacteria in the affected joint or eye where they act as persisting antigens. Classical culture techniques, would not detect the presence of these putative microbial antigens. Alternative approaches for detection of ubiquitous components of bacteria in the host (using appropriate chemical, molecular and immu- nological techniques) are discussed.

Key words: Peptidoglycan; arthritis; uveitis; bacteria.

Alvin Fox, Department of Microbiology and Immunology, University of South Carolina, School of Medi- cine, Columbia, S.C. 29208, U.S.A.

A. INTRODUCTION

Reactive arthritis may be defined as a non-puru- lent disease developing after infection elsewhere in the body, in which viable microbes can not be isolated from the joint (Granfors et al. 1989). Uveitis is a sterile inflammatory disease of the eye, which is often associated with reactive arthritis and prior bacterial infection (Calin & Fries 1976). A key ques- tion, which has puzzled generations of researchers, is: ”How can a bacterial infection initiate these and other chronic inflammatory diseases without ever being culturable from the affected tissue”?

The major purpose of this review is to provide a framework that may help in examining this conun- drum. Particularly focused upon will be information, obtained in animal models, on the interaction of bacterial debris and the immune system during acute and chronic inflammation. A second area addressed will be biochemical pathway in-

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volved in the processing and handling of bacterial remnants generated during infection. Finally, information relevant to reactive arthritis from the use of new ”non-culture” based approaches for detecting bacteria and their remnants in mammalian tissues will be discussed.

Forms of reactive arthritis where bacteria have been shown to play a role include Reiter’s syndrome (Aho et al. 1974, Calin & Fries 1976), rheumatic fever (Unny & Middlebrooks ,1983) and Lyme disease (Steere et al. 1987). Reactive arthritis has in the past been felt to be less important clini- cally than rheumatoid arthritis. However, the newly recognized form of reactive arthritis, Lyme arthritis, is now realized to be the most common tick-borne illness in the United States and perhaps worldwide. Lyme arthritis is caused by the spirochete Borrelia burgdorferi, which is trans- mitted into the human bloodstream by a bite from ticks of the genus lodes (Burgdorfer et al. 1982,

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Steere et al. 1983). The discovery of a previously unrecognized bacterium causing one major form of inflammatory arthritis has focused renewed interest on the role of microbes in other forms of chronic arthritis.

In Scandinavia, reactive arthritis is a common post -infectious complication following dysentery mediated by Yersinia entercolitica and Yersinia pseudotuberculosis infection. Other bacteria associated with this form of reactive arthritis, often referred to as Reiter’s syndrome, include Salmo- nella, Shigella, Chlamydia and Campylobacter (Toivanen & Toivanen 1988). The blinding eye disease, uveitis, is a common feature of this syndrome (Calin & Fries 1976). There is a high association between Reiter’s syndrome and the histocompati- bility antigen (HLA) B27 (Brewerton et al. 1973b). Because most patients with ankylosing spondylitis are also HLA-B27 positive (Brewerton et al. 1973a), it is tempting to additionally speculate a bacterial origin for this disease ; although direct evidence is limited.

Rheumatic arthritis associated with rheumatic carditis occurs after pharyngeal infection mediated by Streptococcus pyogenes (the group A streptococcus). This disease is still highly important in many parts of the third world. It had been felt to be of almost historical interest in the US, having largely disappeared. However, there have been recent outbreaks of rheumatic fever suggesting that it might be a re-emerging disease (Kaplan et al. 1989).

All of the above diseases, which are initiated by bacterial infection, may share a common etiology since in each case bacteria are cultured with extreme rarity from inflamed joints. Although rheumatoid arthritis has not been shown to be associated with prior bacterial infection, since it is a disease of unknown etiology, it may be premature to rule out a role for bacteria at this time. The presence of rheumatoid factor (RF) is common in bacterial in- fections (Greenwood et al. 1971, Williams & Kunkel 1962). Indeed it has been suggested that RF might result from IgG being conformationally altered, as a result of binding to antigen on forming immune complexes, and play no part in maintaining chronic inflammation being merely a secondary phenomenon in the disease process (Glynn 1975).

Poorly degradable ”bacterial remnants” may persist indefinitely in certain forms of reactive arthritis, acting as a chronic antigenic stimulus. This debris would be disseminated to the joint after generation from partially degraded bacterial

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cells. Alternatively, reactive arthritis could result from the persistence of fastidious or dormant bacteria in the synovium. Neither debris nor dormant bacteria would be detected by traditional culture techniques.

Chlamydia1 antigens have been detected in synovial membranes of patients with Reiter’s syndrome during the first month of disease activity (Schumacher et al. 1988). Gram negative bacterial lipopolysaccharide has been found in human reactive arthritis triggered by Yersinia and Salmonella (Granfors et al. 1989, 1990). In joints of patients with Lyme arthritis spirochetes and globular antigen deposits can sometimes be seen in and around blood vessels in areas of lymphocytic infiltration (Steere et al. 1988). In none of these cases is it clear whether live bacteria or bacterial remnants are involved, since both will be detected equally well by antibody- based techniques such as immunofluorescence or Western blots. It may be somewhat easier to develop approaches to delineate the rela- tive role of dormant bacteria and bacterial debris in animal models.

A great deal of information on the mechanisms by which bacterial debris cause chronic inflammation has come from studies of animals. These studies have often involved the streptococcal cell wall polyarthritis, SCWP, (Cromartie et al. 1977) or adjuvant arthritis, AA, (Pearson 1956) rat models. In both cases the active bacterial components are non-viable fragments of cell wall peptidoglycan. Only recently have polyarthritis models, in which live fastidious bacteria are employed as the arthro- pathogenic agent, become available (Hill & Yu 1987, Barthold et al. 1988). Thus these models will not be discussed further, although it is anticipated they will prove important in shedding light on the etiology of reactive arthritides.

B. CHRONIC EXPERIMENTAL, ARTHRITIS, WITH EMPHASIS ON

THE STREPTOCOCCAL CELL WALL POLYARTHRITIS MODEL

I . Overview Sonicates of poorly biodegradable streptococ-

cal cell walls (peptidoglycan-polysaccharide complexes, PG-PS) in saline supension will cause polyarthritis in rats after systemic administration. Streptococcal PG consists of a backbone of N- acetyl muramic acid and N-acetyl glucosamine with tetra and pentapetide side chains made up

BACTERIAL DEBRIS AND INFLAMMATION

of alternating D- and L-amino acids (Munoz et al. 1967). The side chains are cross-linked by peptide bridges. Other eubacterial peptidoglycans are similar in composition and so it is not unexpected that group A streptococcal cell wall fragments are not unique in causing sterile joint inflammation after systemic administration (Lehman et al. 1983, Stimpson et al. 1986a). The third position of the side chain of PG, Glysine, is replaced by diamino- pimelic acid in mycobacteria and most Gram negative bacteria (Schleifer & Kandler 1972) , whilst the cross- bridges are quite variable among bacterial species. The group A streptococcal PS consists of a rhamnose backbone with side chains of N- acetylglucosamine (Fung et al. 1982).

Although adjuvant arthritis is most commonly elicited with a suspension of killed whole mycobacterial cells, the active component is also the bacterial peptidoglycan (Nagao & Tanaka 1980, Zidek et al. 1982). In AA, the mycobacteria are administered in mineral oil suspension. The oil itself is poorly biodegradable and probably more than just an inactive vehicle for bacterial debris in induction of polyarthritis (Kohashi et al. 1977). The mechanisms by which chronic inflammation is elicited in AA and SCWP may be similar. How- ever, results obtained with the SCWP model are simpler to interpret, because of the absence of the oil vehicle. The remainder of this section will thus be focused on the SCWP model.

The clinical pattern of polyarthritis elicited by systemic administration of streptococcal cell wall fragments is related to particle size. Soluble PG- PS fragments elicit acute edema in the joints of rats (Cherry et al. 1982). Small insoluble particles cause an acute arthritis that develops within a few days. Intermediate size particles cause a moderate early inflammation and severe chronic erosive joint lesions. The largest particles induce a polyarthritis that can take weeks to develop (Fox et al. 1982).

Certain bacterial debris (including group A streptococcal peptidoglycan- polysaccharide) induce chronic arthritis in animal models, because of two related properties; activity as an inflammatory agent and poor biodegradability, with persistence in the host for extensive periods, (Cromartie et al. 1977, Eisenberg et al. 1982, Gilbart & Fox 1987). As noted above the cell wall skeleton of all eubacteria (including S . pyogenes and B. burgdorferi) is similar chemically (Schleifer & Kandler 1972, Munoz et al. 1967, Beck & Habichr 1988). Thus studies on the streptococcal cell wall polyarthritis model are

not just relevant to rheumatic fever, but to other reactive arthritides including Lyme arthritis.

How arthritogenic bacterial debris is processed and handled is fundamental to an understanding of reactive arthritis in man. First, although streptococcal, and certain other cell walls, are resistant to biodegradation they are slowly eliminated from the joint. This is a complex process that includes catabolism, excretion in the urine and perhaps egestion in the gut. There may also be some redistribution from deposits in the joint to depots in the reticuloendothelial system.

The physical form of cell wall debris and its distribution within cellular and non-cellular elements in the bloodstream and lymphatics may also be important in understanding the etiology of reactive arthritis. Larger cell wall particles are probably transported within phagocytes in the blood and lymphatics (Daldorf et al. 1980). How- ever, during processing by phagocytes soluble cell wall debris is egested (Vermuelen & Gray 1984). Small cell wall particles may be carried in a soluble form, and readily penetrate into the joint. There they elicit mast cell degranulation in the joint, which in turn affects vacular permeability which may allow the bulk of the larger cell wall particles to then localize (Daldorf et al. 1988). It may be necessary in vivo for some processing of large cell wall fragments, in order to generate permeability eliciting soluble cell wall oligomers, before their penetration into the synovium can occur. Although small cell wall fragments may readily cause acute joint inflammation (Cherty et al. 1982), because of rapid elimination (Parant et al. 1979, Ladesic et al. 1981, Fox & Fox 1990) they do not cause chronic inflammation.

II . The role of the immune system Intermediate size particles of PG-PS, as noted

above, elicit a biphasic disease in which arthritis develops within several days, decreases in severity within one-two weeks and subsequently develops into the chronic erosive granulomatous phase during the next several months. Thus SCWP has an acute and chronic phase (Cromartie et al. 1977). Decomplementation with cobra venom factor re- duces the severity of the acute, but not the chronic phase of SCWP. Thus, complement activation is involved in the acute phase of SCWP (Schwab et al. 1982). Cell walls do not induce chronic arthritis in nude rats (Ridge etal. 1985). Furthermore, cyclosporin, which is cytotoxic for T cells, significantly inhibits chronic arthritis induced by cell walls

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(Yocum et al. 1986). Thus T cells appear to be involved in the chronic phase of SCWP. Purified T cells can adoptively transfer SCWP (Quin Dejoy et al. 1989), however, the cells may contain residual undegraded cell wall debris.

Monocytes are the predominant cell in chroni- cally inflamed joints. Streptococcal cell walls activate macrophages to become cytotoxic and cyto- static for fibroblasts in vitro; this might also occur in vivo (Smialowicz & Schwab 1977). This has suggested a central role of the macrophage in mediating the chronicity of SCWP. However, since epi- sodes of reactivation occur in arthritic tissues in which neutrophils collect even months into the disease process, polymorphonuclear cells may also be important (Daldorf et al. 1980).

Peptidoglycan activates a variety of cell types, including fibroblasts to produce interleukin 1 ( I L 1) (Dinarello 1984, Takahashi et al. 1988). Tumor necrosis factor (TNF) should also be considered for a role in PG mediated inflammation (Cybul- sky et al. 1988). These cytokines activate endothelial cells, which in turn causes attraction and dia- pedesis of polymorphonuclear cells into tissues (Streiter et al. 1989). I L 1 and TNF also increase the adhesion of blood leukocytes to vascular endothelium by causing the expression of surface re- ceptors (endothelial leukocoyte adhesion molecules) (Bevilacqua et al. 1989).

Streptococcal cell wall particles elicit severe, chronic leukocytosis (Wells et al. 1989). All three leukocyte elements are effected, but leukocytosis correlates most well with degree of joint inflammation. In chronic tissue lesions, it is common to see periods of acute inflammation with neutrophil infiltration and fibrin deposition superimposed on the characteristic mononuclear cell infiltrate (Daldorf et al. 1980). However, it is rare to find elevated neutrophil counts in the systemic circulation at chronic time periods. The PG-PS induced leukocytosis may provide a source of polymorphonuclear cells (and monocytes) which may be recruited to aid in the clearance of bacterial components and tissue debris in chronic lesions of joints. Once recruited such cells could be activated to produce inflammatory mediators. It has been noted that PG-PS induce chronic hemopoiesis (Geratz et al. 1988). Thus cell wall debris interacting with systemic leukocytes or their progenitors is important in terms of what happens in the joint.

One possible scenario for the neutrophilia and monocytosis induced by streptococcal cell walls is as follows: 11-1 may be produced by macrophages

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exposed to cell walls. 11-1 may then induce fixed bone morrow indothelial cells and fibroblasts to produce colony stimulating factor for granulocytes and monocytes (CSF-GM). CSF-GM, in turn, may stimulate the formation of granulocyte/mo- nocyte colonies which produce large numbers of neutrophils and monocytes that are released from the bone marrow into the general circulation. These circulating cells may participate in the chronic phase of SCWP.

III. Elimination and degradation of bacterial debris There is a dearth of biochemical information on

the events involved in the processing, dissemina- tion and elimination of bacterial peptidoglycan in the mammalian host. PG polymers, oligomers and monomers are all potent inflammogens. Major factors in determining the severity and chronicity of inflammation elicited by PG include: how rapidly it is eliminated in the urine and by other routes and how readily it is degraded by mammalian tissue enzymes.

Streptococcal cell walls are highly resistant to degradation by mammalian enzymes, and thus provide a persisting inflammatory stimulus (Oha- nian & Schwab 1967). They are not unique in this regard, for example, gonococcal peptidoglycan can be quite resistant to lysozyme, and may be involved in causing reactive arthritis associated with gonorrhoea (Fleming ef al. 1986). Both the composition of peptidoglycan itself and of associated polysaccharide affect the inflammatory properties of streptococcal cell walls. Extensive O-acetylation of the glycan backbone of PG decreases the susceptibility of peptidoglycan to lysozyme (Gallis et al. 1976, Rosenthal et al. 1982). Extensive N-acetylation has the opposite effect (Amano et al. 1980). The presence of the group A polysacc- harid also inhibits the action of lysozyme on PG (Schwab & Ohanian 1967).

However, PG-PS is slowly eliminated from the host; antigenic determinants (the terminal D-alanine dipeptide of PG, and the rhamnose backbone and N-acetyl glucosamine side chains of PS) (Eisenberg et al. 1982) and chemical components (rhamnose, muramic acid and D-alanine) of streptococcal cell walls all slowly decrease in amount in vivo (Gilbart & Fox 1987, Ueda et al. 1989). Rham- nose, muramic acid and D-alanine are not syn- thesized by normal mammalian tissues. Over a 60 day period, the levels of PG-PS in the liver and joint of rats only decreases four-fold. This indicates that after an initial streptococcal infection in man,


there might be debris present almost indefinitely. Similarly, after an initial invasion of the joint, debris derived from spirochetes might also persist for extensive periods in Lyme arthritis.

The slow elimination of PG-PS from tissues could occur by several mechanisms: PG-PS might be egested or excreted from the host in an intact or partially degraded form in the feces or urine. There may also be some redistribution from the joint to depots in the RES. In some instances complete catabolic breakdown of PG-PS to monomers and conversion of unique cell wall constituents into related mammalian substances might occur. For example one could speculate that rhamnose (once enzymatically released from PG- PS) might be converted into its isomer fucose. Although rhamnose is not a component of normal mammalian tissues, fucose is a component of mammalian glycoproteins. D-alanine if formed as a breakdown product of PG-PS might be converted into Lalanine and then converted into mammalian protein. D-alanine is not found in normal mammalian tissues, but Lalanine is a component of proteins. Muramic might be converted into glucosamine by loss of its lactyl group. The catabolic pathways for in vivo degradation of bacterial debris have not been widely investigated.

There is some evidence that group A streptococcal cell walls are physically broken down in vivo. After injection of "'1 PG-PS into rats, tissues were removed at various time periods and homogenates fractionated on the basis of molecular weight by size exclusion chromatography. Initially, all the 1251 label was associated in a high molecular form, but some was solubilized after in vivo processing (Stimpson et al. 1986b).

The enzymatic processes in degradation of PG- PS in vivo are complex and involve a battery of different enzymes. Lysozyme, N-acetyl muramyl-Lalanine amidase, peptidases, glucosaminidase, and other enzymes may all play a role in the degradation of PG-PS. Lysozyme will partially degrade streptococcal peptidoglycan under some circumstances (Gallis et al. 1976). In vivo there appears to be glucosaminidase action on streptococcal cell walls since A-variant antigenic activity is ex- pressed, presumably because of removal of the N- acetyl glucosamine moiety from the group carbohydrate (Scwab & Ohanian 1967). Degradation of gonococcal peptidoglycan by glucosaminidase occurs in vitro (Striker et al. 1987). Small cell wall monomers and muramyl dipeptide (MDP, N-ace- tylmuramyl-L-alanine-D-isoglutamine, the small-

est biologically active sub-unit of PG) are degraded by serum N-acetyl muramyl Lalanine amidase (Tomasic et al. 1980, Mollner & Braun 1984, Harri- son & Fox 1985). This enzyme breaks down MDP into N-acetyl muramic acid, Lalanine and D-glu- tamic acid. Serum also has peptidase activity on MDP (Harrison & Fox 1985).

MDP and other small PG subunits cause a variety of short-lived effects in vivo. These effects include induction of slow wave sleep (Johannsen et al. 1989, Cookson etal. 1989) and inflammatory uveitis in the rabbit (Kufoy et al. 1990). A now classic study (Parunt et al. 1979) demonstrated that, within a few hours of intravenous administration the major part of radiolabelled MDP was present in an intact form in the urine. Other small cell wall monomers are also extremely rapidly passed through the kidney (Ladesic et al. 1981). Any residual PG residues would be degraded into inactive by-pro- ducts by serum amidase and peptidase. Thus there are unlikely to be persisting depots of muramyl peptides in normal mammalian tissues (Fox & Fox 1990).

N: Summary of the etiology of streptococcal cell wall polyarthritis

The process by which streptococcal cell walls cause polyarthritis is a multi-step process. These steps may include differential transport and deposition of different forms of debris from blood and lymphatics to the joint, interaction with inflammatory elements within the synovium, reticuloendothelial and hemopoietic system and finally processing and/or removal of bacterial debris from the host. Bacterial debris is initially deposited in the reticuloendothelial system, as well as the joint. Some small particles may be carried free in the bloodstream and lymphatics and readily localize in joints. These might cause the release of immune mediators (e.g. from mast cells) which allow larger particles within phagocytes to localize in the joint. Once large cell wall particles are deposited chronic arthritis results perhaps by continued stimulation of production of IL-1 and TNF. Neutrophils and monocytes from pools in the bloodstream (generated in the bone marrow and elsewhere in response to stimulation by deposited debris) will be attracted to the affected joint. Thus not only are local immune elements important in joint inflammation, but events in the bloodstream, hemopoietic system and reticuloendothelial system. After deposition in the joint, streptococcal debris is only slowly eliminated and thus provides a perpetuat-

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ing stimulus for inflammation. The rat model may have a similar etiology to

some forms of human reactive arthritis, including rheumatic fever and Lyme disease. As noted above, there have been reports that bacterial debris is found in joints of patients with reactive arthritis. There may, however, be instances where bacterial cell walls (as components of "dormant" bacteria) are involved in chronic inflammation.

C. ACUTE OCULAR INFLAMMATION ELICITED BY MURAMYL DIPEPTIDE

Bacterial remnants may be involved in mediating anterior uveitis in man, based on studies in animal models. This work has primarily emphasized a role for Gram negative lipopolysaccharide (LPS) in modulating the blood-aqueous barrier and induc- ing infiltration of cellular elements of the immune system (Howes etal. 1970, Rosenbaum etal. 1980).

More recently it has been demonstrated that systemic injection of MDP in the rabbit also elicits modulation of the blood-aqueous barrier (Waters et al. 1986, Fox et al. 1990). Since MDP is found in Gram positive (as well as Gram negative) bacterial cell walls it has been hypothesized that Gram positive bacteria might also play a role in causing uveitis in man. Alternatively PG and LPS might act synergistically in Gram negative bacterial in- fections in causing inflammation in the eye.

After systemic administration of MDP in the rabbit, severe breakdown of the blood-aqueous barrier occurs within several hours with increased flow of protein into the eye from the blood and increase in absolute endogenous protein levels in the aqueous (Kufoy et al. 1990). The magnitude of the response to MDP is dramatic and polypeptide profiles of normal and inflamed aqueous are quite distinct, with the latter resembling serum (Fox et al. in press 1990). There are also overt clinical changes in blood vessels in the iris, limbus and conjunctiva. Eyes essentially return to normal within 24 h. Even after direct injection of MDP into the rabbit eye only acute inflammation is elicited: however, ocular administration of PG-PS does elicit chronic inflammation (Fox et al. 1984).

In contrast, after systemic administration of MDP the rat eye is totally insensitive. Although rats readily develop polyarthritis after injection of PG-PS, ocular effects have not been reproducibly observed. A uveitis observed in rats, initially at- tributed to the systemic administration of PG-PS

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(Wells et al. 1986), was later established to be a secondary feature of keratoconjunctivitis sicca, a previously undescribed side-effect of xylazine- mediated anesthesia (Kufoy et al. 1989).

The observations of differences in uveal response of rats and rabbits to MDP are consistent with the ocular response to LPS (Cousins et al. 1982,1984). In both cases, the rat is quite insensitive when compared to the rabbit. Although LPS and lipid A cause cellular infiltration of the uvea of the rat, but not the rabbit (Howes et al. 1970, Rosenbaum et al. 1980).

Unlike the joint, the eye is isolated from the circulation with the blood- aqueous barrier function- ing as a molecular sieve to inhibit penetration of large molecules (Dernouchamps & Hermans 1975, Burns-Bellhorn et al. 1978). There is poor penetration of both large streptococcal cell wall complexes (Wells et al. 1986) and lipopolysaccharides (Rosenbaum et al. 1983, Howes et al. 1984).

Anterior segment capillaries supply both the ciliary body and the iris. The endothelium of ciliary vessels is leaky in contrast to those of the iris. Pro- tein can thus pass into the stroma of the iris and ciliary body from the ciliary vessels, and remain there due to tight junctions of the ciliary and iris epithelium (Freddo 1983, Freddo 1986). LPS appears to affect vascular permeability by acting on both iridial vessels (Howes et al. 1971) and the tight junctions of the ciliary epithelium (Freddo 1984, Freddo 1987). MDP appears to act in a similar fashion.

Prostaglandins are well known to cause vasodila- tion and breakdown of the blood-aqueous barrier (Whitelocke et al. 1973). For exayple, LPS stimul- ates production of prostaglandins within the eye after systemic administration (Battacherjee & Phylactos 1977, Herbort et al. 1988). Although it has not been directly demonstrated that MDP will stimulate synthesis of intra-ocular prostaglandins, pharmacologic inhibitors of prostaglandin synthetase will inhibit MDP-mediated uveitis (Waters et al. 1986).

MDP activates a variety of cell types including macrophages to produce IL1 (Dinarello 1984). When injected intra-vitreally, both IL1 and TNF cause many features of uveitis (Rosenbaum et al. 1988, Rosenbaum et al. 1987). Thus prostaglandins and cytokines appear to play a role in MDP mediated ocluar inflammation.

MDP might be locally involved in causing uveitis, since it is a small inflammatory molecule which could enter the eye from the bloodstream, perhaps


unimpeded by anatomical ocular barriers as are larger molecules. Although it is possible that ocular inflammation might be caused by immune mediators elicited in the bloodstream. Regardless of the mechanism of initiation of ocular inflammation, uveitis caused by MDP may be transient, since as noted above this molecule is readily eliminated from the host. Although certain large cell wall particles can persist indefinitely in reservoirs of the reticuloendothelial system and joint they do not readily penetrate blood-ocular barriers (Wells et al. 1986). This may explain the difficulty in experimental induction of chronic uveitis in animals with bacteria or large bacterial cell wall constituents.

D. APPROACHES TO DETECTION OF

BACTERIAL DEBRIS IN MAMMALIAN TISSUES AND BODY FLUIDS

DORMANT BACTERIA OR NON-VIABLE

Our interest in the role of bacteria and arthritis be- gan with the Dumonde-Glynn experimental aller- gic monoarthritis model. The disease is elicited by sub-cutaneous injection of an oil emulsion of killed Mycobacterium tuberculosis and an unrelated protein, often ovalbumin. An intra-articular injection of ovalbumin then causes a self-perpetuating mono-arthritis. The last intra-articular injection may cause cell mediated immunity in the joint with influx of macrophages carrying mycobacterial debris. This debris would be released in the joint and cause chronic monoarthritis. The persistence of mycobacterial debris in tissues has been studied using radioiodinated killed M. tuberculosis as the mycobacterial component of the adjuvant. The protein portion of the mycobacteria was labelled. Bacterial debris was demonstrated to persist in granulomas at the initial S.C. injection site and radioactivity was found in the limb. However in vivo degradation would be expected to produce free 1251 or 1251 labelled peptides and unlabelled cell wall debris. Thus the levels of radioactivity might not reflect levels of cell wall debris in tissues (Doble et al. 1975).

In other experiments the dead M . tuberculosis component of the adjuvant was replaced with live ( M . avium). Ziehl-Neelsen staining and culture demonstrated the persistence of viable mycobacteria in the adjuvant granuloma, but no mycobacteria were detected in the arthritic joint. However, it is conceivable that only debris and not viable mycobacterial cells are carried to the joint in these

circumstances. Such debris would not be readily visible in the light microscope and not detectable by culture. Thus these experiments proved in- conclusive (Doble et al. 1975).

Similar experiments using radioactive and fluor- escent-labelled M. tuberculosis have been perform- ed in the adjuvant arthritis model and apparently mycobacteria were detected in arthritic limbs. However, as argued above, dissociation of label from relevant mycobacterial structures due to in vivo degradation might obscure interpretation of these experiments (Vernon-Roberts et al. 1975, Jones & Ward 1962). The in vivo dissociation of la- bels attached to bacteria or bacterial components (particularly in chronic studies of inflammation) is a general problem in studies of this type.

It was thus vital to develop more specific ways for quantitation of levels of bacterial debris in mammalian tissues. Schwab and co-workers per- formed pioneering studies on qualitative detection of cell wall debris in mammalian tissues using immunofluorescence after injection of well-defined streptococcal cell wall particles (Ohanian & Schwab 1967). The first semi-quantitative immunoassay for the detection of streptococcal cell wall debris in mammalian tissues was develop more recently (Eisenberg et al. 1982). Affinity -purified antibodies against rhamnose, D - alanine - D - alanine and N-acetylglucosamine immunodetermi- nants were used to detect streptococcal cell walls in arthritic joints. A direct correlation between arthritic index and the amount of antigen in the joint was established. It was also demonstrated that the three portions of PG-PS studies were all slowly degraded.

Immunoassay only provides semi-quantitation of the levels of cell wall particles in mammalian tissues, since streptococcal cell wall sonicates are polydisperse in size. After in vivo processing cell wall antigens are degraded and become even more physically heterogeneous. The basis of im- munochemical quantitation of cell wall antigens in tissues is the antigen-antibody interaction. Binding affinity is dramatically affected by the number of antigenic determinants exposed on a molecule. This does increase with the size of a particle, but there is also some steric hindrance with some buried determinants not available for antibody binding (Janusz et al. 1984).

An alternative quantitative chemical approach has also been developed for detection of muramic acid as a ”chemical marker” for the PG portion of streptococcal cell walls in mammalian tissues (Fox

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et al. 1980). This technique combines the exquisite separating power of gas chromatography with the extreme selectivity of mass spectometric detection. Muramic acid can be detected in mammalian tissues, after injection of streptococcal cell wall fragments, and can also be used as a generic marker for bacterial peptidoglycan. Subsequently, the other unique carbohydrate component of streptococcal cell walls, rhamnose, was shown to be detectable in mammalian tissues (Gilbart er al. 1986). Rhamnose and muramic acid levels have been measured over a 60 day period in livers and joints of arthritic rats, after systemic injection of streptococcal cell walls (Gilbart & Fox 1987). Dur- ing this period levels of both muramic acid and rhamnose only decrease four fold; a surprisingly slow biodegradation rate. Levels of muramic acid in inflamed joints at 60 days after injection were less than lng/mg wet weight of tissue.

More recently another chemical method for detection of D-alanine as a marker for the peptide portion of streptococcal cell walls in mammalian tissues has been developed (Ueda et al. 1989). This method used a state-of-the-art mass spectrometric approach for trace analysis ”negative ion chemical ionization”. The D-alanine portion of the cell wall was also demonstrated to be eliminated extremely slowly in vivo. Unfortunately, amino acids undergo racemization during analysis, thus Lamino acids (derived from mammalian tissue proteins) produce background D-amino acids. This limits the sensitivity of the approach. Diami- nopimelic acid, which is found in most Gram negative peptidoglycans, but not mammalian tissues, is potentially a better amino acid marker (Sones- son et al. 1989).

There have been reports of bacterial antigens in joints of patients with reactive arthritis, including Lyme disease (Schumacher et al. 1988, Steere et al. 1988, Grunfors et al. 1989, 1990). Whether these remnants represent bacterial debris or ”dormant” microbes remains to be determined. The poten- tial for muramic acid as a ”chemical marker” for bacterial peptidoglycan has been demonstrated in a study of human septic arthritis (Christensson et al. 1989). This approach can detect both bacterial debris and viable bacteria, but can not differenti- ate on from the other. It might be predicted that in chronic inflammation, after prolonged digestion within phagocytes, bacterial remnants, in contrast to live bacteria, would not contain appreciable amounts of undegraded nucleic acids. Thus if pro- karyotic genes were detected in chronic Lyme dis-

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ease, or other reactive arthritides, this might sug- gest the presence of viable organisms.

It has been demonstrated that use of enzymatic amplification of DNA with a thermostable DNA polymerase (the polymerase chain reaction, PCR) dramatically increases the sensitivity of hybridiza- tion or sequencing (Saiki et al. 1988). Using PCR, the outer membrane protein genes (OSP A and OSB B) of B. burgdorferi have been detected in the tick vector, but not so far in synovial fluids or tissues of patients with Lyme disease (Persing et ul. 1990). There appears to be variation in these proteins among strains of B. burgdorferi and more conserv- ed genes (such as 16s rRNA) might have greater utility as a probe (Persing et al. 1990, Giovannoni et al. 1988). In Yersinia triggered reactive arthritis in synovial cells shown to contain Yersinia antigens, PCR was unable to detect bacterial genes (Viitanen et al. submitted 1990). This might indicate that bacterial debris can be present in the absence of live bacteria. Further work in this area is clearly needed.

E. CONCLUSIONS

The ability of cell wall components to cause acute or chronic inflammation results from the interaction between the bacterial inflammogen and various elements of the immune system. Peptidoglycan oligomers or monomers can cause acute inflammation within a few hours of administration. How- ever, for maintenance of perpetuating inflammation bacterial cell wall polymers must persist in host tissues. There is a dearth of information on the processing and handling of bacterial debris in relation to reactive arthritides, (including Reiter’s syndrome, rheumatic fever and Lyme disease) and uveitis. Direct correlations of the presence of fastidious/dormant bacteria with chronic immuno- pathology also remain limited. Approaches that have addressed these issues have been described. Further extension of these concepts with the ex- panded utilization of improved techniques for the quantitation of defined bacterial antigens, cell wall ”chemical markers” and genes in animal and human tissues may prove useful in elucidating a role for persisting bacterial debris and/or fastidious bacteria in a variety of acute and chronic inflammatory diseases.

This work was supported by a grant from the National Institutes of Health EY 04715.


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