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SUMMARY
The development of arthritis induced in mice by intraperitoneal injection of the non-antigenic mineral oil pristane (2,6,10,14-tetramethylpentadecane) was shown to depend on the presence of CD4+ T cells. Initial experiments assessed the influx of lymphoid cells into the peritoneal cavity of CBA/Igb mice after pristane injection. Both CD4+ and CD8+ cell numbers were maximal around 50 days. Other experiments confirmed our original observation that irradiated pristanetreated mice failed to develop arthritis unless they were reconstituted with spleen cells from normal donors. This finding has been extended by showing that the population of transferred splenic lymphoid cells must contain CD4+ T cells, while CD8+ T cells and B cells were not required for reconstitution. Conventionally housed and hsp 65-immunized animals are known to harbour T cells reactive with hsp 65. In addition, hsp 65-immunized mice are resistant to the development of pristane-induced arthritis (PIA). Thus, additional experiments assessed the population of splenic T cells activated and proliferating against mycobacterial 65 000 MW heatshock protein (hsp 65). In cultures of purified splenic T cells derived from both conventional and hsp 65-immunized mice, removal of CD4+ T cells significantly reduced the proliferative response to hsp 65, while removal of CD8+ T cells often enhanced the response. These proliferative restricted. responses were also shown to be major histocompatability complex (MHC) class The present findings demonstrate that PIA is CD4+ T-cell mediated, and immunodominant environmental antigens such as hsp 65 activate this population of lymphocytes. The CD4+ hsp 65-reactive population may be pathogenic or protective in PIA, depending upon the route of sensitization.
INTRODUCTION Mice injected in the peritoneum with the paraffin oil pristane (2,6,10,14-tetramethylpentadecane) develop a chronic arthritis resembling rheumatoid arthritis (RA) in a number of respects. For example, pristane-induced arthritis (PIA) is characterized histologically by polymorphonuclear cell infiltration and the formation of pannus in affected joints.' Both PIA and RA are characterized by circulating rheumatoid factor,2 elevated levels of agalactosyl immunoglobulin G (IgG)"4 and interleukin (IL)-6.5-7 The PIA model is unique among the animal models of RA in that the inducing agent, pristane, is non-antigenic. Further, the onset time is very long, with disease appearing between 60 and 200 days later, depending on the strain of mice."12'8 RA is thought to be an immunologically mediated disease.
Received 7 May 1996; revised 8 September 1996; accepted 8 September 1996.
Abbreviations: hsp, heat-shock protein; PIA, pristane-induced arthritis. Correspondence: Dr S. J. Thompson, Pathology and Microbiology, The Medical School, University Walk, University of Bristol, Bristol BS8 1TD, UK.
Genetic analyses have shown an association between immune response genes and disease in both RA and PIA.2'9"0 That the immune system is involved in PIA has been corroborated experimentally, by showing that an intact immune system is necessary for development of disease. Thus irradiating animals prior to pristane injection prevents disease development. Furthermore, transfer of naive spleen cells to irradiated animals reconstitutes their ability to develop PIA."" Twin studies have suggested that the genetic contribution to RA disease susceptibility may be at most 50%.12 Therefore, environmental factors must make a substantial contribution, suggesting that RA is triggered by exogenous antigens which are immunologically cross reactive with self proteins."' Studies using the PIA model indirectly support this hypothesis, showing susceptibility to PIA is influenced by the environment."' In searching for a possible cross-reacting environmental antigen, proteins from the heat-shock protein (HSP)60 gene family have received much attention. These proteins are known to be very immunogenic and act as the dominant antigens in infectious diseases.'5 The HSP60 gene family includes both mammalian hsp 58 and mycobacterial hsp 65, which share 50% sequence identity. Consequently, epitopes on hsp 65 may be shared by self hsp 58, and thus hsp 65 would constitute a cross-reactive environmental antigen. Evidence demonstrating
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Where stated, groups of mice were irradiated sublethally with 500 rad from a caesium source (Gravatom Industries, Gosport, UK).
Antigens The recombinant mycobacterial hsp 65 for immunization and cultures was kindly provided by the World Health Organization Gene Bank. Irrelevant control antigens, bovine serum albumin (BSA) and human IgG were also used along with the mitogen concanavalin A (Con A) as controls in vitro.
a role for HSP60 proteins in experimental arthritides includes the finding that splenic T cells from mice, whether arthritic, non-arthritic or naive, can mount in vitro proliferative responses to hsp 65,11 whereas only T cells derived from arthritic mice respond to mammalian hsp 58.16 Hence, arthritic mice harbour autoreactive T cells, targetting self hsp 58, an antigen which is found to be up-regulated in affected joints.'7 In addition, preimmunization with mycobacterial hsp 65 in incomplete Freund's adjuvant (IFA) protects otherwise susceptible animals against PIA,1""8"19 adjuvant arthritis20 and streptococcal cell wall arthritis.21 The mechanism of protection by hsp 65 immunization is not yet fully understood, but is highly antigen specific," and can be transferred using splenocytes to naive susceptible recipients.22 Previous work, in relation to pristane-induced plasmacytomas, analysed the cells infiltrating into the peritoneum of mice injected with pristane, and showed that both CD4' and CD8 + cells enter. In mice maintained in specific pathogen-free (SPF) conditions, there was a significantly reduced CD4+ cell influx after pristane injection.23 As SPF mice are resistant to PIA, this suggested that a peritoneal influx of CD4+ cells could be involved in disease induction. However, in vivo depletion studies showed there to be no significant difference in the incidence of PIA in mice treated with depleting antibodies against CD4 or CD8 antigens,24 leaving the role of these T-cell subsets in PIA unclear. The present work phenotypes the T cells involved in PIA disease induction and resistance. Normal (susceptible) mice were compared with pristane-injected mice, or mice made resistant to PIA, either through irradiation or preimmunization with hsp 65. Three approaches were taken. Firstly, the pristane-induced cellular influx into the injection site (peritoneal cavity) was established in susceptible mice, hsp 65 protected animals and resistant irradiated mice. Secondly, studies in vivo compared the ability of different lymphocyte populations to reconstitute disease susceptibility in irradiated recipients. Thirdly, in vitro assays were used to phenotype the cells activated and proliferating against hsp 65, again comparing protected, susceptible and arthritic mice.
Preparation of cells Peritoneal cells were collected by injecting 7 ml of Hanks balanced salt solution with heparin (10 units/ml), gentle massaging of the abdomen, followed by aspiration. Peritoneal cells were analysed by dual colour flow cytometry (Becton Dickinson FACScan, Oxford, UK) using Pharmingen antibodies against CD4, CD8, xB-T-cell receptor (TCR) and yb-TCR. Spleens of individual mice were aseptically removed and single cell suspensions made in RPMI-1640 medium supplemented with 20 mm HEPES (pH 7 2, Flow Labs, Irvine, UK). For transfer experiments, spleen cell suspensions were depleted of B cells by immunomagnetic cell separation using Dynabeads M-450 (sheep anti-mouse IgG) and a Dynal magnetic particle concentrator (Dynal Co., Wirral, UK) according to the manufacturer's instructions. T-cell depletions were carried out utilizing anti-Thyl 2 monoclonal antibody (Seralab, Crawley Down, Sussex, UK). CD4+ T-cell depletion was carried out using 2 il per 107 cells of anti-murine CD4 monoclonal antibody (YTS-191 1), depletion of CD8 + T cells was carried out using supernatant from clone YTS-166-4, an antibody specific for CD8a (kind gifts from both Professor H. Waldmann and Dr S. Cobbold, University of Cambridge, UK). After 45 min incubation at 40, complement mediated lysis was carried out using 0-1 ml of rabbit complement (Low Tox-M, Cedarlane Laboratories Ltd, Ontario, Canada) per 107 cells for 45 min at 37. Depletion efficiency was determined by flow cytometry using fluorescein isothiocyanate (FITC/phycoerythrin (PE)-conjugated CD4/CD8 antibodies (Pharmingen, San Diego, CA). After depletion, CD4+ and
CD8+ T cells made up less than 0-6% and 0-7% of the total cell number, respectively. For cultures, T cells were enriched according to the panning method of Engleman et al.25 A purity of > 85% was achieved as assessed by anti-Thy 1-2 staining using flow cytometry (FACScan, Becton Dickinson Ltd). CD4 and CD8 depletions were carried out as above. Normal mouse spleen cells were used as antigen presenting cells (APC). The APC were irradiated with 1000 rads from a caesium source (Gravatom Industries).
Culture and assay ofproliferation This was carried out as described previously. The medium employed was alpha modification of Eagle's medium (alpha MEM) (Flow) supplemented with 4 mM 1-glutamine (Flow), 100 U/ml benzyl penicillin (Glaxo Ltd, Greenford, UK), 100 Mg/ml streptomycin sulphate (Evans Medical Ltd, Greenford, UK), 5 x 10-5 M 2-mercaptoethanol (Sigma, Poole, Dorset, UK), 20 mm HEPES and 0-5% fresh normal mouse
"
Animals Male CBA/Igb mice aged between 4 and 8 weeks were used. CBA/Igb mice were originally a gift from Professor H.S. Micklem, Department of Zoology, Edinburgh, UK. They were obtained by back-crossing (101 strain x CBA) Fl hybrids to CBA mice and selecting those mice with Igb allotype in their serum.
Induction andprevention of arthritis Arthritis was induced by two intraperitoneal injections of 0 5 ml of pristane 50 days apart (Aldrich Chemical Co., Milwaukee, WI). The animals were examined for the incidence of arthritis in the ankle joints at various time points, with the final incidence being assessed 200 days post pristane injection. 11.18,19 Groups of mice were immunized (i.p.) 10 days before pristane challenge with 50 pg of mycobacterial hsp 65 administered as an emulsion in IFA. This regime has previously been reported to specifically block the development of PIA."1"18"19
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of ml
16xI"
Mg/ml).
After the periods of incubation indicated, 100 u1 samples were pulsed with 2 mCi of [3H]thymidine (specific activity 70-85 Ci/mmol; Amersham International Ltd, Amersham, UK) for 6 hr. The [3H]thymidine incorporated was measured using conventional liquid scintillation procedures with a LKB rackbeta counter (KB-Wallac Ltd, Pharmacia, Uppsala, Sweden).
Analysis of MHC class II antigen requirements for proliferation Proliferation assays were carried out as described above, with the addition of anti-class II H-2' antibodies ( 1-5, 3 or 6 yg/ml ), or isotype matched control anti-H-2qv (3 or 6 jug/ml) (all from Pharmingen, Cambridge Biosciences, Cambridge, UK). Results were compared using Student's t-test.
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Figure 1. Effect of pristane on cellular influx in the peritoneal cavity of (a) total cells (b) CD4+ cells and (c) CD8+ cells. Six to eightweek-old male CBA/Igb mice were given a single i.p. injection of 0 5 ml pristane and, at regular intervals, animals were killed for the collection of peritoneal exudate cells. All cells were stained directly with FITC-anti-CD4 and PE-anti-CD8 antibodies and immediately analysed on a Becton Dickinson FACScan. All data points represent the mean of up to 13 mice+SEM.
Table 1. Cell recovery from peritoneal cavities (n = 3 per group), assessed by differential cell counts, 20 days after pristane injection. Percentages are calculated relative to the 100% found in normal mice
PIA cannot be induced in irradiated mice but can in irradiated recipients of normal spleen cells."" Cell transfer studies were used to determine the phenotype of the reconstituting population. Groups of irradiated animals received no cells, spleen cells, or spleen cells depleted of different lymphocyte populations, prior to pristane injection. These groups were compared to that of a control group of non-irradiated pristaneinjected animals. Figure 2 confirms the previous finding that irradiation blocks development of PIA, while reconstitution with naive spleen cells restores PIA susceptibility. In two experiments, spleen cells depleted of B cells were able to
1997 Blackwell Science Ltd, Immunology, 90, 81-86
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cultures were a mixture of the two depleted cell preparations, to give the original ratio of CD4+ to CD8 + cells. Figure 4 illustrates representative proliferation data showing that the depletion of CD4+ cells prior to culture greatly decreases a population's ability to proliferate in response to hsp 65. In contrast, depletion of CD8+ T cells enhanced antigen specific proliferation in four out of six trials (Fig. 4).
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Figure 2. Effect of the reconstitution with normal spleen cells, B-depleted (A and B), or T-depleted (b) spleen cells on the incidence of arthritis in irradiated pristane-challenged mice. Control animals were challenged with pristane or irradiated and challenged with pristane. Test mice were irradiated (day -2), reconstituted (day-i) and challenged with pristane (day 0).
reconstitute the ability of mice to develop PIA (Fig. 2a and b). In contrast, T-depleted spleen cells were unable to reconstitute susceptibility to recipients (Fig. 2b). In order to determine which T cells are responsible for reconstitution, irradiated animals were given naive spleen cells or spleen cells depleted of CD4+ or CD8+ cells. Recipients of CD4+-depleted spleen cells had a significantly reduced incidence of PIA, relative to the recipients of CD8+-depleted spleen cells, with the same number of cells transferred (Fig. 3).
Phenotype of cells proliferating against hsp 65 In hsp 65-activated splenic cell cultures from normal mice, arthritic mice and hsp 65-protected mice, both CD4+ and CD8 + T cells show signs of cellular activation, upregulating CD71 (transferrin receptor) and CD25 (IL-2R) antigens. The CD4+ population has a much larger percentage of activated cells, with up to four times more CD4+ cells than CD8 + cells becoming activated (data not shown). In order to determine which of these activated cells are the proliferating population, splenic T cells from PIA-susceptible (normal) mice and PIAresistant (hsp 65-immunized) mice were depleted of either CD4+ or CD8 + T cells prior to culture. Positive control
Antigen presentation in the context of MHC class IT molecules is required for a CD4+ T-cell mediated response. Thus, in order to corroborate the evidence for CD4 + T cell proliferation to hsp 65, the effect of blocking MHC class II-mediated antigen presentation was assessed by the addition of MHC class TI-blocking antibodies to splenic cultures. T cells from CBA/Igb mice (H-2k) were cultured with hsp 65 and APC in the presence of blocking antibody against H-2k, or isotype matched control blocking antibody against H-2 sv. While the control antibody had no significant effect, a clear dosedependent decrease in proliferation was seen with increasing doses of anti-H-2k antibodies, in cultures of cells from normal mice, hsp 65-protected animals and arthritic mice (Fig. 5). 400001I
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Figure 4. T-cell proliferative responses to hsp 65 in CD4 or CD8 depleted cultures of cells from (a) a normal mouse and (b) an hsp 65-immunized mouse. The results are presented as c.p.m. where the background proliferation with no antigen has been subtracted. [3H]thymidine uptake by (A) CD8-depleted cultures (@) CD4-depleted cultures and (U) positive control cultures of remixed depleted populations giving the original cell ratios. Data presented as c.p.m. of 100 pl aliquots.
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Figure 3. Effect of reconstitution with normal spleen cells, CD4-depleted, or CD8-depleted spleen cells on the incidence of arthritis in irradiated pristane-challenged mice. Control animals were challenged with pristane only. Test mice were irradiated (day-2), reconstituted (day-1) and challenged with pristane (day 0). (*P < 0.025, X2-test).
Figure 5. T-cell proliferative responses to hsp 65 in the presence of MHC class II-blocking antibodies. Splenic T cells from normal, hsp 65-protected and pristane-injected mice were cultured in the presence of hsp 65 and irradiated APC. From 1 5 jg/ml to 6 yg/ml of antiH-2k or 6 jg/ml anti-H-2q ,, (except cultures of cells from arthritic mice, which received 3 pg/ml) was added to the test cultures during the entire culture period. Bars represent means of duplicate aliquots where the sample error is less than 10%. *P<0.05; **P<0.01; ***P<0 005, relative to cultures where no antibody was added.
1997 Blackwell Science Ltd, Immunology, 90, 81-86
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This report details studies on peritoneal influxes of normal mice after pristane injection, showing B cells, CD4' and CD8+ cells able to enter. To distinguish the role of these lymphocytes in PIA, different populations of cells were purified and used in reconstitution experiments. Disease resistant, irradiated mice, which were shown here to have no peritoneal lymphoid influx, were used as recipients. Only CD4' T cells reconstituted disease susceptibility in these mice (see below). Taken together these results suggest that the CD4+ component of the peritoneal infiltrate is necessary for the disease. Nevertheless, the presence of an intact CD4+ cell peritoneal infiltrate alone does not lead to disease, as shown by its presence in the hsp 65-protected animals. We demonstrate here that in both pre-immunized and susceptible mice, the T cells proliferating against hsp 65 are MHC class II-restricted CD4+ T cells. We propose therefore that it is this hsp 65-specific population of CD4' T cells that mediate resistance in immunized mice, and the non-immunized CD4' hsp 65-specific population which mediates disease in susceptible mice. Under this scheme, as neither SPF23 nor irradiated mice have an influx of CD4' cells, the hsp 65-specific CD4' population is absent, and the mice are resistant. There is a critical phase which requires the presence of lymphocytes, shortly after pristane injection, in order for the mice to be susceptible to the development of PIA. This window falls between the day of challenge and about 40 days later, as demonstrated using irradiation and reconstitution experiments.11 In looking at the peritoneal lymphoid populations present during this time frame, we found the influx to consist of equal numbers of CD4' and CD8+ T cells, together with some B cells.23 We carried out further reconstitution experiments in order to distinguish the role of each lymphoid population in disease. As previously, naive spleen cells were used to restore disease susceptibility in resistant (irradiated) recipients. Initially, T- and B-cell depleted populations were transferred directly into the peritoneum of irradiated animals. B cells were not required, as pre-depletion of B cells did not alter the capacity of the cells to reconstitute susceptibility. In contrast, the removal of T cells ablated the competency of splenic cells to restore susceptibility. This data accords with the observation of Wooley et al. that mice carrying the nu/nu mutation are not susceptible to PIA.2 The role of different T-cell subsets in disease susceptibility is clear from Fig. 2, which shows that CD4+ T cells were required for PIA development, whereas CD8+ cells were not essential. How could the inflammation-inducing irritant and adjuvant effects of pristane26 and the presence of CD4+ T cells in the peritoneum be linked in mediating disease? Several theories have been proposed. Firstly, irritation caused by pristane could result in altered gut permeability, allowing bacterial antigens to enter the peritoneum from the intestines."1'4 Release of gut antigens has previously been implicated in leading to joint inflammation in patients with Crohn's disease,27 and those having undergone jejuno-ileal bypass surgery.28 This type of contamination from the intestines would include immunodominant bacterial antigens such as HSP60 proteins. These antigens could activate CD4+ T cells,29'30 which could then cross react with the upregulated joint hsp 58,'7 causing joint inflammation. Secondly, inflammation upregulates self
C 1997 Blackwell Science Ltd, Immunology, 90, 81-86
hsp 581731 and consequently activates CD4' Tcells specific for self hsp 58.29 As with the cross-reactive T cells above, these hsp 58-reactive T cells could lead to joint inflammation. Thirdly, the pro-inflammatory cytokines induced by pristane, such as interferon-y (IFN-y),32 may alter the antigen processing and presentation of either endogenous or exogenous antigens, leading to cryptic epitopes being revealed, and activating otherwise quiescent autoreactive T cells (for further discussion, see Elson et al. 16). It is clear from our results that CD4+ T cells in the spleens of susceptible, arthritic or protected mice (hsp 65 preimmunized) are responsive to hsp 65.1117 We propose that it is these hsp 65-specific CD4' T cells, as T-helper 1 (Thl) cells in susceptible mice, which are capable of inducing an inflammatory response in the joints leading to arthritis. In support of this proposition, T cells from arthritic mice, cultured with hsp 65, produce IFN-y and IL-2, in the absence of IL-4 and IL-5 production (Beech et al. manuscript submitted). On the other hand, we propose that the cells recognizing hsp 65 which arise in pre-immunized (protected) animals, are Th2 CD4' T cells which would protect by virtue of secretion of antiinflammatory cytokines. A Th2 phenotype for these cells is supported by the finding that they produce IL-4 and IL-5, when cultured with hsp 65 (Beech et al. manuscript submitted). We have not excluded the potential role of hsp 65-activated CD8 + T cells in PIA as they could clearly contribute to a proinflammatory disease mechanism. However, it was clear from the reconstitution studies that CD8 + T cells cannot transfer disease susceptibility, and thus a purely cytotoxic response does not initiate PIA. In conclusion, the present study demonstrates a critical role of CD4+ T cells, and a potential role of hsp 65-reactive CD4+ T cells in both the development of and protection from PIA. The protective mechanisms of irradiation and hsp 65 pre-immunization probably do not act by the same means, as the hsp 65 protection seems to rely on T-cell responses to the antigen in an inhibitory fashion, whereas irradiation ablates the peritoneal T-cell infiltration necessary for disease initiation. Further understanding of disease processes will aid in developing new strategies for immuno-intervention in the treatment of inflammatory joint diseases.
ACKNOWLEGMENTS
S.J. Thompson is an ARC Research Fellow and L.M. Stasiuk was also supported by a grant from the Arthritis and Rheumatism Council, UK. M. Ghoraishian was supported by the Iranian Government. This investigation also received financial support from the UNDP/World Bank/WHO Special Programme for research and training in tropical diseases (TDR).
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4. ROOK G., THOMPSON S., BUCKLEY M. et al. (1991) The role of oil and agalactosyl IgG in the induction of arthritis in rodent models. Eur J Immunol 21, 1027. 5. HOUSSAIu F.A., DEVOGELAER J.P., VAN DAMME J., DE DEUXCHAISNES C.N. & VAN SNICK J. (1988) Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 31, 784. 6. HIRANO T., MATSUDA T., TURNER M. et al. (1988) Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. Eur J Immunol 18, 1797. 7. HITSUMOTO Y., THOMPSON S.J., ZHANG Y.W., ROOK G.A.W. & ELSON C.J. (1992) Relationship between interleukin 6, agalactosyl IgG and pristane-induced arthritis. Autoimmunity 11, 247. 8. POTTER M. & WAx J.S. (1981) Genetics of susceptibility to pristane induced plasmacytomas in BALB/cAn: reduced susceptibility in BALB/cJ with a brief description of pristane induced arthritis. J Immunol 127, 1591. 9. WATANABE Y., TOKUNGA K., TAKEUCHI F. et al. (1989) Putative amino acid sequence of HLA-DR/3 chain contributing to rheumatoid arthritis susceptibility. J Exp Med 169, 2263. 10. STASNEY P. (1978) Association of the B cell alloantigen DRw4 with rheumatoid arthritis. New Engl J Med 298, 869.
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drome. Arthritis Rheum 24, 684. 29. ANDERTON S.M., VAN DER ZEE R. & GOODACRE J.A. (1993) Inflammation activates self hsp 60-specific T cells. Eur J Immunol 23, 33. 30. ZUGEL U., SCHOEL B., YAMAMOTO S., HENGEL H., MOREIN B. & KAUFMANN S.H.E. (1995) Crossrecognition by CD8 T cell receptor aB cytotoxic T lymphocytes of peptides in the self and the mycobacterial hsp 60 which share intermediate sequence homology. Eur J Immunol 25, 451. 31. POLLA B.S. (1988) A role for heat shock proteins in inflammation? Immunol Today 9, 134. 32. McDONALD A.H. & DEGRASsI A. (1993) Pristane induces an indomethacin inhibitable inflammatory influx of CD4+ T cells and IFN-y production in plasmacytoma-susceptible BALB/cAnPt mice. Cell Immunol 146, 157.