tmmunologicai Reviews t99l. No. 121 Published by Munksgaard, Copenhagen. Denmark No part may be reproduced by any process without written permission from the author(s)

Role of hsp60 during Autoimmune and Bacterial Inflammation ROLF KIESSLING*, ALVAR GR6NBERG^ JURAJ IVANYI^ KALLE SODERSTROM*', MATS FERM*, SANDRA KLEINAU*, ETHEL NILSSON* & LARS KLARESKOG*

INTRODUCTION Heat-shock proteins (hsp) are a group of proteins with a highly conserved structure in evolution, which originally attracted the interest of molecular biologists studying gene regulation, but now are challenging also for the immunologist with a number of fundamental questions in relation to infection and autoimmunity. The immunogenicity of several hsp constituents of bacteria and parasites for the host T-cell system makes them of central interest for those who are trying to manipulate the protective immune responses against microorganisms. The hsp60 protein, originally characterized in E. coli as the groEL protein, and subsequently shown to have homologues in several other species, including humans (Jindal et al. 1989), is probably among the most studied in this regard, partly because recombinant myeobacterial hsp60 was made easily available for experimental immunologists. An almost bewildering number of observations suggest that T cells specific for hsp60 are involved in phenomena ranging from organ-specific inflammatory diseases like arthritis (van Eden et al. 1988) or diabetes (Elias et al. 1990) to tolerance induction (van den Broek et al. 1989), suggesting that many of the T cells recognizing bacterial hsp60 also crossreact with endogenous hsp60. Starting with our original interests in T-cell responsiveness against mycobacteria and local immunity in arthritis, respectively, we considered that at least two basic questions needed to be addressed from the findings related above; one question being when, where and how mammalian hsp60 is expressed; particularly, when do the determinants of this protein become available for recognition by immune cells? 'Department of Immunology, Karolinska Institute, Stockholm, Sweden. 'MRC Tuberculosis and Related Infection Unit, Royal Postgraduate Medical School, London, U.K., ^Department of Inflammation Research, Kabi-Pharmacia Therapeutics AB, Uppsala, 'Department of Medical and Physiological Chemistry, University of Uppsala, *Department of Rheumatology, Karolinska Hospital, Stockholm, Sweden. Corresponding author: Rolf Kiessling, Department of Immunology, Karolinska Institute, Box 60400, S-10401 Stockholm, Sweden.

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This would be of interest both for an understanding of why anti-hsp60-reactive T cells exist in the mature T-cell repertoire, and whether local variations of hsp60 expression might account for focusing anti-hsp60 immunity to particular organs. A second question relates to the T-cell subtypes that recognize hsp60, and whether their specificity or localization may explain the seemingly paradoxical finding of autoreactivity to this ubiquitous protein. The present review addressed these questions by studies of local expression of hsp60 in different tissues, and in different forms of inflammation, together with studies of regulation of its expre.ssion by established cell lines. There was also an obvious need for information concerning the receptor usage, specificity and requirements for activation of hsp60-reactive T cells. I

TISSUE AND CELLULAR DISTRIBUTION OF THE HUMAN AND RAT hsp60 HOMOLOGUE Evidence implicating a role for hsp60 in autoimmunity was first provided by the observation that T cells recognizing epitopes of bacterial hsp60, derived from rats with adjuvant arthritis (AA) after immunization with M. tuberculosis, could transfer arthritis to naive rats (van Eden et al. 1988). Further evidence was provided from the claims of specific prevention of arthritis in rats after pretreatment with bacterial hsp60 (van Eden et al. 1988, van denBroek et al. 1989). In association with these findings, and following the observation that synovial T cells show an enhanced capacity to recognize hsp60 (Res et al. 1988), it has been hypothesized that T cells reactive with hsp60 obtained from RA patients might also contribute to joint inflammation. T-cell cross-reactivity between endogenous and bacterial hsp60 might account for the observed influence ofthe intestinal bacterial flora on experimental arthritis (Kohashi et al. 1986). No explanation, however, was available on the question as to why T cells recognizing conserved epitopes of hsp60 focus on particular organs, such as the joint. These observations obviously motivated an investigation of the distribution of hsp60 in rat and human tissues. Thus, although the hsp60 protein was reported to be a mitochondrial protein present in most mammalian cells (McMullin & Hallberg 1988, Evans et al. 1990), quantitative differences in tbe expression of epitopes of this protein, or the possible expression of "new" bsp60 molecules at intracellular locations other than the originally described mitochondrial one, may explain the tissue-restricted inflammatory disease in mammals. We have therefore analyzed the expression of hsp60 in human and rat tissue, both by immunohistochemical methods on tissue sections and by analysis of the hsp60 protein/mRNA at the cellular level.

Immunohistochemical studies of hsp60 expression in human and rat tissues To the above end, we have used a murine monoclonal antibody (mAb) (ML30) produced against M. leprae (Ivanyi et al. 1983). This mAb has been found to

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react with a sequential epitope of the M. leprae hsp60 within residues 311-322 (Anderson et al. 1988) of which the tetrapeptide DMAI (315-318), shared with the human hsp60 sequence but distinct from the non-cross-reactive E. coli hsp60, is a possible core for the antigenic determinant site (Fig. 1). Its cross-reactivity with the human hsp60 is apparently due to binding to an epitope with restricted homology rather than to one which is highly conserved between many different species (Fig. 1). This view is based on the restriction of pronounced ML30 binding to only about 25% of strains (mostly human pathogens within the mycobacterial genus (Ivanyi et al. 1985, Buchanan et al. 1987)) and the paucity of reactions with non-mycobacterial genera (Ivanyi et al. 1988), as summarized in Table I. In contrast, another anti-human hsp60-reactive monoclonal antibody, YI-2, (Dudani & Gupta 1989) binds with all mycobacterial species and several other bacterial genera, which is characteristic of antibodies directed to the amino terminal end of the molecule (Thole et al. 1988, Ivanyi et al. 1988). Finally, a monoclonal antibody to the "arthritis related" peptide 180-188 was shown to stain human tissues in a similar way, though less consistently than ML30 and without demonstrable binding to the intact mycobacterial hsp60 molecule (de Graeff-Meeder et al. 1990). When tested on human, mouse and rat cells using Western blot or immunoprecipitation procedures, the ML30 mAb recognizes at least one molecule (for details see further below) with a molecular weight of approximately 60 kD (Fig. 2. and

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Evans et al. 1990, Kleinau et al. 1991), making it highly likely that this mAb recognizes a molecule corresponding to the human hsp60 homologue. When ML30 was used in immunohistochemical stainings of frozen sections from biopsy specimens of patients suffering from RA, we found a strong reactivity in the cartilage-pannus junction of the joints, as well as in reumatic noduli (KarlssonParra et al. 1990). This staining was particularly intense at the interface between cartilage and synovial tissue, where synovial mononuclear cells appear to erode the cartilage. The staining was seen both within synovial mononuclear cells and in the extracellular space. This would therefore indicate the presence of hsp60 at the site where the inflammatory reaction is most active, the extracellular hsp60 possibly being ascribable to dead or dying ceils leaking this protein into the extracellular space. There was also a correlation between high expression of hsp60 and HLA-DR expression, a molecule previously described to be over-expressed in inflamed joints (Klareskog et al. 1982), possibly due to the influence of local cytokine production. We have also tested whether high hsp60 expression was induced in experimental situations in rats with inflamed joints due to AA or collagen-induced arthritis (CIA) (Kleinau et al. 1991). Findings obtained here were very similar to those in human RA, with more intense staining in joints of arthritis rats suffering from AA or CIA than in normal rat joints. The staining with ML30 was seen within the cartilage-pannus junction, the sites of bone erosion and in chondrocytes in the inflamed joints, whereas staining in normal joints was located in the synovial Hning cells and in occasional chondrocytes. In a recent study of synovial tissues from patients with RA or osteoarthritis and AA rats, ML30 staining was found to be the strongest for synovial lining cells (de Graeff-Meeder et al 1990), in line with our findings.

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1

2

Figure 2. Enhanced levels of hsp60 delerminants in the THP-1 cell line itiduced by cytokine treatment. Western biot analysis was performed with ML30 on SDS-PAGE-separated proteins from THP-1. Cells were either untreated (I) or cultured for 48 h in 100 U/ml each of IFN-gamma/TNF-alpha (2).

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In view of the conclusion that hsp60 expression is markedly enhanced during the inflammatory process in the joint, we have asked whether hsp60 is a general marker for any inflammatory tissue. Arguing against this, however, human biopsies from kidneys undergoing acute graft rejection expressed only weak levels of hsp60 (Karlsson-Parra et al. 1990). Also, stainings of skin biopsies from sites of a delayed-type hypersensitivity reaction in the rat were totally unreactive with ML30 (Kleinau et al. 1991), arguing against this monoclonal being a general marker for inflammation. On the other hand, in tissues other than the joint, hsp60 expression does not appear to depend on the presence of any reactive process. Thus, Evans et al. (1990) observed a clear staining of certain types of cells in human liver, skin, small intestines and reproductive organs. Moreover, they also found an inflammation, dependent hsp60 expression in the synovia and alveolar lining. Taken together, it appears that inflammation is associated wilh high hsp60 expression only in certain tissues, such as the joint and the lung. This conclusion has obvious implications for the possible role of hsp60 in autoimmunity. What is the identity of the cell expressing high level of hsp60 in the arthritic joint? The histochemical studies demonstrated that a large part of the cells expressing hsp60 expressed the macrophage marker LeuM3, and most ofthe cells were also HLA-DR-positive (Karlsson-Parra et al. 1990). Therefore, it seems very likely that the cell type with high local hsp60 expression in the inflamed joint is a synovial monocyte/macrophage type of cell. Whilst the majority of peripheral blood mononuclear cells and the synovial cells in normal non-inflamed joints showed only low-intensity ML30 staining (Karlsson-Parra et al. 1990, and unpublished observation), the factors which drive the induction of hsp60 at sites of contact between the inflamed synovium and cartilage are not yet understood. It is interesting to note that the interface between the pannus and the bone/cartilage represents a site with an extraordinary concentration of denatured proteins, among them collagen type II, that are released from the vascular tissue under destruction. Such a situation has been reported to induce production of certain hsp (Anathan et al. 1986). An alternative possibiUty is that high local concentrations of cytokines, which have been found in synovial fluid or synovial tissue (Husby et al. 1985, Saxne et al. 1988), may induce hsp expression in the monocyte/ macrophage-like synovial cells. To study the latter, we have developed a model where hsp60 induction on human monocytic cell hnes could be analyzed, as will be outlined in the next section.

Human monocytic cell lines as a model for hsp60 induction To facilitate the study of humoral as well as cellular immune responses against human hsp60, we have established a model where this protein could be induced in human monocytic cell lines (Ferm et al. 1991). To develop this model, we have

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screened a panel of human tumor cell lines by Western blot, using the ML30 mAb. The majority of the 21 human cell lines tested showed a distinct band of the expected 60 kD MW, thus showing that transformed cells in general have a high expression of this protein. Particularly high hsp60 expression was found among certain Burkitts lymphomas and Epstein-Barr virus (EBV)-transformed cells. The expression of high hsp60 levels in transformed cell lines is in line with previous findings that other hsp, including hsp90 and hsp70, have been found to accumulate to abnormally high levels in transformed cells (Bensaude & Morange 1983, Macnab et al. 1985). Also, a murine tumor-specific transplantation antigen was identified as a member ofthe hsp90 family (Ullrich et al. 1986), and members of the hsp70 family were shown to interact with nuclear oncogenes such as p53, and the stability of this interaction was proposed as influencing transformation (Finlay et al. 1988). We found, however, that high hsp60 expression was not a general feature of all transformed cells, since some cell lines (the myelomonocytic leukemias) expressed little or no detectable hsp60 protein. Consequently, we could ask if treatment of these cells with cytokines or differentiation inducers would enhance the levels of hsp60 mRNA or protein. The pattern summarized in Table II and in Fig. 2 emerged (shown for the THP-1 monocytic leukemia line). While the cell line grown only in medium showed very weak hsp60 expression, a dramatic increase in protein as well as mRNA production was seen with a combined treatment with IFN-gamma/TNF-alpha (Fig. 2 shows a Western blot performed with ML30 on THP-1 cells). IFN-gamma alone also induced relatively high levels of hsp60, while the differentiation inducer retinoic acid and heat-shock at 42°C gave only a moderately increased expression of this protein or its mRNA. These results therefore agree with previous observations that cytokines are potent inducers of hsp (Ferris et al. 1988. Granelli-Piperno et al. 1986), and offer an attractive model for how changes in hsp60 expression could affect humoral and cellular effector mechanisms. The enhanced level of HLA-DR expression in tissues

TABLE TI

Inducible hsp60 expression in the human cell line THP-1 Agents used for induction' 42''C heat-shock Retinoic acid IFN-y IFN-y, TNF-g

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' A 48 h treatment was used, with 100 U/mi of the respective cytokine and 48 h of 2 10"^" M of retinoic acid. ' As measured with Northern blot, as described in Jindal et al. (1989). ' As measured in Western blot with the ML-30 mAb.

KIESSLING ET AL. of inflamed joints expressing high levels of hsp60 (Karlsson-Parra et al. 1990) was paralleled in this tumor model by a high level of HLA-DR expression in IFN-gamma/TNF-alpha-treated monocytic cell lines.

Evidence for cell surf ace location ofhsp60 Studies of a human lung carcinoma cell line, using a polyclonal antiserum specific for the hsp 58 of Tetrahymena thermophila, showed a mitochondrial pattern of staining (McMuUin & Hallberg 1988). This was confirmed by subcelluiar fraetionation and electron microscopy in tetrahymena and yeast cells. Mitochondrial distribution of hsp60 was also suggested by the granular pattern of staining of most tissues with the ML30 mAb, and by the observation that this was most intense in cells which are known to contain many mitochondria (Evans et al. 1990). Recently, cell surface location of hsp60 has been described with a polyclonal rabbit anti-hsp60 antiserum by immunoprecipitation of surface-iodinated Daudi cells (Fisch et al. 1990). The hsp60 homologue expressed on Daudi cells induced something that resembled superantigen responses in the Vgamma9/Vdelta2 subset of peripheral T cells. In our studies, however, Daudi cells were not cell surfacepositive when stained with ML30 or with an hsp60-specific, anti-peptide rabbit serum. Also, none of 21 other human cell lines investigated was positive for cell surface expression of ML30 determinants even when pretreated with IFN-gamma/ TNF-alpha, although they all had high levels of intracellular hsp60 expression as measured by Western blot analysis (Ferm et al. 1991). Inititai studies using ascitesderived ML30 mAb demonstrated that three of the tested myelomonocytic cell lines when induced by IFN-gamma/TNF-alpha treatment contained a large proportion of cell surface-positive cells. However, subsequent Facscan analysis of these cell lines with ML30 purified from the supernatant of in vitro cultured ML30 hybridoma, as well as with the above-mentioned hsp60-specinc rabbit serum, were consistently negative. Also Kaufman et al. (personal communication, see this volume) have similar findings of surface expression of ML30 determinants on murine mononuclear phagocytes as measured with the ascites-derived monoclonal, but not with the in vitro cultured hybridoma supernatant. The reason for the positive cell surface staining with the ascites-derived ML30 remains yet to be determined. In view of our negative results with the tissue culture-derived ML30 and with our rabbit anti-hsp60 peptide antibody, however, we are of the opinion that the existence of a cell surface-located hsp60 molecule on human cells still remains to be conclusively demonstrated. Cell-mediated effector mechanisms, of course, do not need the expression of the full size hsp60 molecule on the cell surface, as hsp60-derived peptides associated with HLA antigens may be recognized by specific T cells, as will be discussed in the next section.

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CELLULAR IMMUNITY TO BACTERIAL AND HUMAN hsp60 HOMOLOGUES Several distinct types of cell-mediated immune mechanisms have been described as being activated by and/or speciflc for hsp60, some of which may be relevant to discuss in the context of autoimmunity. It has generally been assumed that intracellular microbial pathogens are stressed within the hostile environment of macrophages and that the consequently enhanced synthesis or altered cellular distribution of stress proteins could influence the intensity or nature of the ensuing host immune response. However, there is so far no compelhng evidence that this dogma is applicable to anti-hsp60 T-cell immunity. It should be noted that the oflen quoted high frequency of proliferating anti-hsp60 T cells (20% of all mycobacteria-reacting T cells) in "M. tuberculosis-'xmmwne' mice (Kaufman et al. 1987) was obtained using draining lymph nodes, following immunization with 1 mg killed tubercle bacilli in incomplete Freund's adjuvant rather than from infected animals. Therefore, the resulting magnitude of the immune response is hardly relevant to the putative stress response of the infeecting organism. In fact, the proliferative response of spleen cells from M. tuberculosis-Mecitd mice to SDS-PAGE-separated antigen was distributed evenly between multiple fractions (Brett & Ivanyi 1990). There is also no published evidence of significant differences in the magnitude or epitope specificity of the T-cell response to hsp60 in either tuberculosis or leprosy and several T-cell clones have been successfully generated from lymphocytes of both patients and healthy subjects (Lamb et al. 1989, Van Schooten et al. 1989). In view of the highly conserved and crossreactive nature of T-cell stimulatory epitopes of hsp60 and their expression in many bacterial genera (Thole et al. 1988, Ivanyi et al. 1988) it would be plausible to assume that natural priming of individuals by protracted exposure to the various commensal organisms, all having the hsp60 constituent, could be mandatory for the activated as well as for the memory anti-hsp60 immune repertoire. Similar conclusions apply also to antibody responsiveness (see below).

Bacterial hsp60 is an immunodominant target for cytotoxic CD4^ T cells In studies by Berhane Kaleab et al. from the AHRI institute (Addis Ababa, Ethiopia), also reported elsewhere in this volume by Ottenhof et al., we have found that the recombinant hsp60 protein of BCG/M. tuberculosis is an important target molecule for CD4+ CD8' cytotoxic cells, and can activate CTL precursors against this molecule (Ottenhof et al. 1988, Kaleab et al. 1990b). The cytotoxic activity of the CD4+ T cell specific for bacterial hsp60 was shown to be HLA-DR-restricted. This was found with T cells both from immune healthy donors and from leprosy patients, stimulated in vitro with hsp60 or PPD, using antigen-pulsed macrophages as target cells. In the same macro-

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phage cytotoxicity assay, experiments using truncated hsp fusion proteins suggested that the N-terminal portion of this molecule was critical for the expression of the cytotoxic target epitope(s), when tested with T cells from 2 individuals (Kaleab et al. 1990b). The role of CD4'^ cytotoxic T cells against bacterial hsp60 in autoimmune phenomena caused by exogenous infectious agents, such as leprosy, tuberculosis and possibly even reactive arthritis triggered by exogenous microbial agents, still needs to be established. In these conditions, epitopes of bacterial hsp may be expressed on infected macrophages, and/or, alternatively, soluble hsp produced locally, or even systemically, could be endocytosed by synovial cells, making them susceptible to lysis by hsp-specific CD4"^ killer cells. Therefore, even T cells that are not specific for hsp60 epitopes shared between the bacterial and human protein may be active in the autoimmune process. This interpretation is further emphasized by the fact that such 004"*^ T cells in response to bacterial hsp60 can produce interferon and TNF-alpha (Kaleab et al. 1990b), both of which are produced also in the inflamed joints of RA patients (Husby et al. 1985, Saxne et al. 1988). Evidence for hspSO-specific CD8* T cells Although CD4'^ T cells have long been considered to play the main role in protection against intracellular bacteria, more recent evidence from murine experimental systems suggest that also CD8'^ T cells have a role in these infections (Libero & Kaufman 1986). This has recently been supported also by results from the above-mentioned human macrophage cytotoxicity assay, where mycobacteria were shown to induce cytotoxic T cells of both the CD8^ and CD4* phenotypes which specifically could kill infected macrophages (Kaleab et al. 1990a). Also supporting this notion, Rees et al. (1987) isolated a CD8^ T-cell line from the pleural effusion of tuberculosis patients which crossreacted with hsp70 of M. tuberculosis, E. coli and humans. Therefore, for intracytoplasmatic infections, bacterial hsp may also be accessible for the class I pathway. More relevant to consider in the context of autoimmunity, however, is the role of 008"^ cells as a cytotoxic mechnaism against host cells expressing endogenous hsp60. Koga et al. (1989) have provided evidence that stressed host cells can serve as targets for murine CD8"*^ T cells raised against mycobacterial hsp60 peptides. These T cells were shown to lyse not only macrophages primed with hsp60 peptides, but also lysed stressed macrophages in the absence of exogenous peptides. We have found that T cells stimulated against whole intact bacterial hsp60 or trypsin-cleaved peptides of this molecule (kindly provided by van Embden and van der Zee, Bilthoven) lysed human in vi7ro-cultured macrophages (Kaleab et al. 1990 and unpublished observation), but not more so if stressed by heatshock of IFN-gamma treatment (unpublished observation). It is possible, however, that

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if in vitro culture in itself activates hsp60 expression, as seen with certain longterm in vi/ro-cultured myelomonocytic cell lines (Ferm et al. 1991), this additional stress stimulus may not further enhance hsp expression. Nonspecific NK/LAK cells are also activated by hsp60 Apart from the specific HLA-restricted cytotoxic cells, also nonspecific NK/LAK types of effector cells are stimulated by bacterial hsp60 (Kaleab et al. 1990b). Thus, both hsp60- and PPD-stimuIated effector cells were found to mediate strong MHC-unrestricted cytotoxic activity against various allogeneic tumor target cells (Kaleab et al. 1990b). Induction of this nonspecific cytotoxicity is strictly antigendependent, since virtually no lysis of these targets is seen with nonstimulated cells cultured in vitro without antigen. Nonspecific lysis of tissue macrophages and other target cells by activated NK cells or MHC-nonrestricted T cells, such as Schwann cells or synovial cell, may be part ofthe mechanism during autoimmune phenomena in infectious mycobacterioses or chronic arthritis. How abundant are T cells reacting with the conserved epitopes of hsp60? T-cell clones reacting with conserved epitopes of hsp60 shared between the human and bacterial protein have been isolated from healthy subjects (Lamb et al. 1989), but the question as to whether these occure at a higher frequency during autoimmune conditions is not known. As reviewed extensively elsewhere in this volume, there is evidence for a role of hsp60 in chronic infiammatory arthritis. Studies in RA patients demonstrated an association between T-cell responses to the mycobacterial hsp60 and the early stages of joint infiammation (Res et al. 1988). Also, our group has demonstrated that T cells from synovial fiuid proliferate in response to mycobacterial hsp60 more than those from the peripheral blood of patients with reactive arthritis and RA (Soderstrom et al. 1990). The enhanced reacti.vity of synovial T cells was also seen for BCG but not for tetanus toxoid, P H A or IL-2, thus implying some antigen selectivity in this phenomenon. However, others have demonstrated that the higher responsiveness of mononuclear cells in arthritic inflammation is not confined to mycobacterial antigens but can be seen also with other bacterial antigenic preparations (Gaston et al. 1989, Life et al. 1990, Res et al. 1990). It is possible that local clonal expansion of T cells as a result of exposure to antigenic tissue determinants crossreactive with bacterial antigens such as hsp, as well as enhanced capacity of synovial cells to present bacterial antigens, may explain this (Life et al. 1990). The epitopes of hsp60 recognized by the synovial T cells have been analyzed using synthetic peptides. T-cell clones of a patient with acute arthritis were shown to recognize epitopes in the amino terminal portion of this molecule that is not conserved between bacteria and eukaryotes (Gaston et al. 1989). We have recently

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analyzed the proliferative capacity of synovial T cells from patients suffering from RA as well as reactive arthritis to a semi-purified preparation of recombinant human hsp60 (obtained in collaboration with S. Jindal and R. Young, Cambridge, USA). Similar to what was seen with bacterial hsp60, in a large proportion of the patients, synovial T cells responding to human hsp60 were seen, wliile the PBMC from the same patients gave no or much lower responses. As we cannot exclude that some of the T-cel! reactivity to the human hsp60 could be ascribed to minor E. coli contaminants or to bacterial hsp60, synovial T-cell lines from these patients are presently being screened also against synthetic peptides to exclude a possible role of minor E. coli contaminants or bacterial hsp60.

Recognition of hsp60 by yj6 cells and the evidence for a different subtype distribution ofyjS cells in inflammatory joint diseases Many groups have recently shown that yjd T cells can be stimulated by mycobacterial antigens, including hsp60 (for review see Young & Elliot 1989). As hsp60 is expressed locally in the joints of RA patients (Karlsson-Parra et al. 1990), and as y!S T-cell clones recognizing bacterial hsp60 were isolated by Holoshitz et al. (1988) from the synovial fluid of a patient with RA, this would merit further investigations into the role of yjd cells in inflammatory joint diseases. There is, however, no evidence supporting a higher frequency of hsp60-reactive yjd cells in RA patients or locally in the synovial tissue. Also, the question of whether hsp60 is a dominant antigen for yjd cells in general is not resolved. Kabelitz et al. (1990), using limiting dilution assays, concluded that the majority of Vy9/V(52 T-cell clones from the peripheral blood of normal individuals recognize determinants other than hsp60 on mycobacteria. Fisch et al. (1990) recently suggested that the stimulatory epitopes on the surface of Daudi cells for human \y9j\62 cells depend on the conformation or suitable presentation of hsp60 molecules or their peptide fragments. Paradoxically, however, the purified recombinant mycobacterial hsp60 antigen did not stimulate Wy^jWdl cells. Also the question whether yjd cells are more abundant locally during inflammatory joint diseases is controversial. T yjd cells in the synovial fluid have been reported either increased (Brennan et al. 1988), the same, or slightly elevated (Soderstrom et al. 1991, Smith et al. 1990, Kjeldsen-Krag et al. 1990) in the synovial fiuid of RA patients as compared to peripheral blood. Very high yjd cell values were, however, found in the synovial compartment of some but not all children with juvenile rheumatoid arthritis (Kjeldsen-Kragh et al. 1990). Studies using mAb specific for defined subsets of yjd cells have recently yielded more consistent results, and the V(51 + subset, positive for the monoelonals t5TCS1 (T-cell Sciences, Cambridge, USA) and A13 (Ferrini et al. 1989), has attracted particular interest in this regard. Thus, a different distribution of subsets of yjd cells was found in synovial fluid as compared to peripheral blood of the same

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RA patients, with a high proportion of V(51 + cells (positive for the mAb 1 or A13) in synovial T cells or in corresponding short-term T-cell hnes (Smith et al. 1990, Kjeldsen-Kragh et al. 1990, Soderstrom et al. 1990). We have found that short-term T-cell lines from the synovia! fluid of patients with inflammatory arthritic diseases (mainly RA patients), activated by mycobacterial hsp60 or IL2 alone, have a high proportion of yjd T cells. A significant predominance of V(51 + cells (positive for the (5TCS-I mAb) was found in these T-cell lines, exceeding the yjd cell subset most common in peripheral blood ofthe same patients (positive for the mAb BB3 and TiyA). The finding that the WS\ + cells from synovial fluid expand rapidly when grown in IL-2, or when stimulated with hsp60, may be indicating that this subset is preactivated in vivo. We have therefore analyzed the expression of activation markers expressed on V(51 T cells of paired samples of synovial and blood T cells from RA patients. As can be seen from the result of a representative Facscan analysis shown in Fig. 3, a much higher proportion of the synovial fluid-derived Wdl subset expresses HLA-DR and CD45RO as compared to the blood-derived \5\ cells fromt he same patient. This argues that synovial VSl cells are activated in vivo, and may explain why this yjS cell subset rapidly expands upon culture in IL-2. In line with these findings, the expression of activation markers (CD69, HLA-DR, and CD25) on yjd cells was significantly higher in the synovial compartment compared with the peripheral blood in children with juvenile rheumatoid arthritis (Kjeldsen-Kragh et al. 1990), although the expression of these activation markers on V^l cells was not analyzed. Interestingly, however, the V^Sl"^ subset was also reported to predominate in the human

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Figure 3. The percentage of \-d-l* yS T cells from SFMC (open bars) and PBMC (filled bars) also positive for HLA-DR, CD45RA and CD45RO. Fresh cells from an RA patient were double-stained with FITC-Idbelled ^TCS-I (anti-V-5-I) and phycoeryihrin-labelled antibody as indicated in the figure, and analyzed by Facscan.

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intestinal mucosa, particulariy in celiac disease (Spencer et al. 1989, Halsten et al. 1989), possibly reflecting the greater migratory tendency of this subset to sites of inflammation (Grossi et al. 1989). HUMORAL RESPONSE TO hsp60 The antigenic structure of mycobacterial hsp60 has been investigated extensively with the aid of murine mAb. Using the competition assay between pairs of mAb, 14 distinct epitopes have been identified (Buchanan et al. 1987) and the sequences of some of the corresponding epitopes have been established using recombinant DNA clones (Mehra et al. 1986) and synthetic peptides (Anderson et al. 1988). The great majority of these epitopes are expressed in all members of the mycobacterial genus; only one (111E9) is of strictly M. leprae- and one (TB78) of partially M. tubercUlosis-restncted specificity. It would be of interest to determine the mAb-defined epitopic specificity of the abundant anti-hsp60 antibodies in humans. Serologicai studies based on the competition of test sera with the antigen binding of labelled mAb showed that the TB78 specificity is elevated above healthy controls in about 20% of tuberculosis patients (Jackett et al. 1988), whilst the M. /eprae-restricted inE9 specificity could not be detected in patients with lepromatous leprosy who produced high antibody levels to other M. leprae antigens (Mwatha et al. 1988). Surprisingly, human sera with high whole hsp60-binding antibody levels also failed to compete with the binding of several anti-hsp60 mAb (Cl.l, IIH9, ML30, 1108, II1C8) directed against the cross-reactive hsp60 epitopes (Ivanyi, unpubhshed results). Hence, it appears that the majority of anti-hsp60 antibodies in the sera from normal or diseased humans are directed to difTerent, possibly conformational epitopes, which do not overiap with those identified by mAb. The overwhelming bias of mAb towards sequential epitopes could be attributed to their selection from mice which had been hyperimmunized with crude mycobacterial extracts containing largely proteolytically degraded (typically "multibanded") hsp60 and therefore not reflecting the epitope repertoire of antibodies produced in response to natural exposure to commensal or pathogenic microorganisms. Analysis of antibody levels against the whole recombinant mycobacterial hsp60 antigen demonstrated large individual variation in serum titers (1/5-1/500) between apparently healthy subjects from the U.K. (Fig. 4, Jackett et al. 1988). In view of these variations, no significant increase of titers was found in patients with tuberculosis. In contrast, antibody levels to the mycobacterial 38 kD antigen or its two epitopes (TB71 and TB72) showed a pronounced increase from low background in healthy subjects to high titers in tuberculosis patients (Fig. 4). Furthermore, there was no correlation between antibody levels to mycobacterial hsp60 and those to the immunodominant species-specific 38 kD and 19 kD antigens in tuberculosis patients (Jackett et al. 1988). These results suggest that

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Figure 4. Antibody levels in patients with tuberculosis. Sera from patients with smearpositive pulmonary tuberculosis (S + ) and clinically healthy controls (H) were tested for antibody binding to whole antigens by ELISA (•) and for epitope-specific competition with the mycobacterial hsp60-directed TB78 (o) or the 38 kDa antigen-specific TB71 (O) and TB72 ( c ) monoclonal antibodies. (Modified from Jackett et al. 1988).

non-mycobacterial stimuli, possibly represented by protracted exposure to eertain commensal organisms, could play a major role in the stimulation of anti-hsp60 antibody levels. At least one of these stimuli could be represented by unrecorded superficial infections with Candida alhicans, most frequently manifested as oral or vulvovaginal candidiasis, in both of which elevated antibody levels to mycobaeterial hsp60 were reported (Ivanyi & Ivanyi 1990). In view of the common occurrence and mild clinical manifestations, it would seem desirable to monitor salivary Candida counts in future studies on the possible disease association of anti-hsp60 antibodies. As to the mechanism involved, it is of interest that the antibodies binding to mycobacterial hsp60 cannot readily be absorbed with an extract from C. albicans, hence indicating the possibility that Candida infections may act as a source of non-specific cellular stress, rather than as a cross-reactive antigenic stimulus. Significantly elevated antibody levels to mycobacterial hsp60 (but not to E. coli hsp60) had been reported also in about 25% of patients with rheumatoid

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arthritis but not in other patient groups including SLA and Crohns disease (Bahr et al. 1988, Tsouifa et al. 1989a, b). However, these results have been interpreted in terms of polyclonal activation in a subsequent study which found only the IgA antibody levels elevated, and even this difference was abolished by adjusting the serum antibodies to total immunoglobulin levels (McLean et al. 1990). There has been no published report as yet on the humoral reactivity to human hsp60 in relation to health and disease. Our preliminary study has demonstrated significant ELISA levels (with positive results to hsp60 confirmed in Western blots) of IgG antibodies to human hsp60 in some healthy controls as well as in patients with RA and leprosy (Gronberg et al., unpublished results). The origin of these antibodies is unknown but they most probably represent cross-reactive antibodies elicited by bacterial immunity. An alternative hypothesis is that autoantibodies to hsp60 may be formed during normal immune responses due to cytokine-induced synthesis and release of endogenous hsp60, as discussed above. Further studies are needed to establish whether there is a preponderance of antihuman hsp60 antibody levels in any particular disease. I

SUMMARY AND CONCLUDING REMARKS In this review we have been particularly concerned with the question of where and when hsp60 is expressed, and about the subset - and activation - of T cells that may be involved in the recognition of hsp60. Participation of hsp60-reactive T cells in local tissue destruction can be inferred from the finding of an inflammation-related expression of hsp60 in certain tissues, mainly the arthritic joint of RA patients and of rats with experimentally induced arthritis. The demonstration of cytokine-inducible expression of hsp60 molecules, raises the question of whether additional non-mitochondrial members of an hsp60 family may be identified. One may also speculate whether a special pattern of hsp60 expression could explain how anti-hsp60 reactive T cells may have escaped thymic elimination. A multitude of potentially harmful immune mechanisms, such as cytotoxic CD4+ and CD8^ T cells, yjd T cells, NK cells and cytokines, has been shown to recognize or to be activated by hsp60. Until now, this has mainly been shown with bacterial hsp60, and the extent to which this also applies to the human hsp60 homologue is yet not resolved. The existence of activated yjd T cells, particulariy ofthe V^l+ subtype, at the local site of synovial inflammation should motivate further studies of their role in synovial destruction, possibly mediated via hsp60 recognition. Anti-hsp60 reactive T cells might be particularly important in cases hke arthritis, where endogenous hsp60 expression appears to be exceptionally high. A possible scenario could be that anti-hsp60 reactive T cells contribute to the secondary activation of resting, or even anergic T cells with other more restricted epitope specificities that might have an effector role in organ-specific autoimmune

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diseases. If so, this might constitute one part of the explanation of how antihsp60 reactivity may be involved in several different organ-specific autoimmune diseases. Such a situation would also be compatible with a role for anti-hsp60 reactivity both in the response to certain forms of cancer, and as a "first line" defence against certain microbes, particularly mycobacteria. In more general terms, the study of anti-hsp60 reactive T cells, as well as the study of endogenous hsp60 expression, might help us to understand how different parts of an anti-microbial defence system, involved during difierent phases of evolution, may act together; sometimes constituting an efficient combination of less specific/rapid and more specific/late responses towards microorganisms, at other times, as for example in arthritis, leading to an autoaggressive tissue damage, due to the synergy and overactivation of both types of system. I

I

ACKNOWLEDGMENTS

This work was supported by the Swedish Medical Research Council, the Swedish National Association against Rheumatism and the Swedish Cancer Society.

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larly sized ubiquitous bacterial common antigen. Microbial. Pathogenesis 41, 71. Thole, J. E. R., Keulen, W. J., Kolk, A. H. J., Groothuis, D. G., Berwald, L. G., Tiesjema, R. H. & van Embden, J. D. A. (1987) Characterization, sequence determination, and immunogenecity of a 64-Kilodalton protein of mycobacterial bovis BCG expressed in Escherichia coli K-12. Infect. Immun. 55, 1466. Thole, J. E. R. & van der Zee, R. (1990) The 65 kD antigen:molecular studies on a ubiquitous antigen. In: McFadden, J., ed. Molecular Biology of the Mycobacteria. Surrey University Press, London. Tsouifa, G.. Rook, G. A. W., Bahr, G. M., Sattar, M. A., Behbehani, K., Young, D. B., Mehlert, A., Van-Embden, J. D. A., Hay, F. C , Isenberg, D. A. & Lydyard, P. M. (1989a) Elevated IgG antibody levels to tbe mycobacterial 65-k.Da heat shock protein are characteristics of patients with rbeumatoid arthritis. Scand. J. Immunol. 30, 519. Tsouifa, G., Rook, G. A. W., Van-Embden, J. D. A., Young, D. B., Mehlert, A., Isenberg, D. A., Hay, F. C. & Lydyard, P. M. (1989b) Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis. Ann. Rheum. Dis. 48, 118. Ullrich, S. J- Robinson, E. A.. Law, L. W., Willingham, M. & Appella, E. (1986) A mouse tumor-specific transplantation antigen is a beat sbock-related protein. Proc. Nati Acad- Sci. USA 83, 3121. Welch, W. J. (1987) The mammalian heat sbock (or stress) response: A cellular defense mechanism. Adv. Exp. Med. Biol. 225, 287. Young, D., Lathigra, R., Hendrix, R., Sweetser, D. & Young, R. A. (1988) Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85, 4267. Young, R. A. & Elliot, T. J. (1989) Stress proteins, infection and immune surveillance. Cell 59, 5.

Role of hsp60 during autoimmune and bacterial inflammation.

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