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The Journal of Immunology, 2006, 177: 6634-6641.
Copyright © 2006 by The American Association of Immunologists, Inc.

Antibody Responses to Mycobacterial and Self Heat Shock Protein 65 in Autoimmune Arthritis: Epitope Specificity and Implication in Pathogenesis1

Hong Ro Kim*, Eugene Y. Kim*, Jan Cerny2,* and Kamal D. Moudgil2,3,*,{dagger}

* Department of Microbiology and Immunology and {dagger} Division of Rheumatology, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1l) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1l) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The effector mechanisms involved in the induction and/or propagation of the immunopathology in various autoimmune diseases are generally categorized as predominantly T cell or Ab mediated. For example, multiple sclerosis (MS),4 insulin-dependent diabetes mellitus, and rheumatoid arthritis (RA) are considered to be prototypes of primarily T cell-driven diseases, whereas systemic lupus erythematosus (SLE) and myasthenia gravis are typical Ab-mediated disorders (1, 2, 3, 4). It is conceivable that in the latter category of diseases, T cells provide the appropriate help to B cells in the generation of pathogenic Abs. Interestingly, there is increasing information pointing toward the role of Abs in the disease process in the former group of diseases. Studies in patients with MS (5, 6, 7, 8) and in the experimental autoimmune encephalomyelitis (EAE) model of this disease (9, 10, 11, 12) have revealed that Abs might be important in the pathogenesis of this disorder previously considered to be a predominantly T cell-mediated disease. Similarly, studies in a spontaneous model of autoimmune arthritis in the K/BXN mice have revealed that T cells initiate the disease, but its progression and maintenance is dependent on Abs (13, 14, 15). Likewise, there is suggestive evidence that Abs might be involved in the disease process in type 1 diabetes mellitus (16, 17).

In this study, we have examined the kinetics, the Ag/epitope specificity, and the functional attribute of Abs directed against the disease-related Ags in the rat adjuvant-induced arthritis (AA) model of human RA (18, 19). AA is inducible in Lewis rats (RT.1l) by s.c. immunization with heat-killed Mycobacterium tuberculosis H37Ra (Mtb) (18), and T cells directed against mycobacterial heat shock protein (hsp) 65 (Bhsp65) are believed to play a pivotal role in disease induction (20). Other studies (20, 21, 22, 23) and our own (24, 25) have defined various T cell epitopes within Bhsp65 that are involved in the induction or regulation of this disease. There is limited information in the AA model regarding the Ab response to Bhsp65 in arthritic Lewis rats. Naparstek and colleagues (26, 27) have reported the kinetics, peptide reactivity, and disease-modulating activity of anti-Bhsp65 Abs in naive vs arthritic Lewis rats, and also compared the Ab response of Lewis (RT.1l) rats with that of disease-resistant Brown Norway (BN) (RT.1n) rats. However, the quantitative and/or qualitative differences, if any, in the anti-Bhsp65 and anti-self (rat) hsp65 (Rhsp65) Abs produced in AA-susceptible vs AA-resistant rat strains possessing identical MHC haplotype, and the implication of such differences in disease susceptibility need further examination.

We have undertaken a comprehensive testing of Ab response of arthritic Lewis rats to Bhsp65 as well as to its self homolog, the rat hsp65 (Rhsp65). Also tested were Ab responses to the control hsp65 from Escherichia coli (GroEL) as well as to another arthritis-related Ag, type II collagen (CII). Furthermore, the Ab responses of Lewis rats were compared with those of AA-resistant WKY rats, which have the same MHC class II haplotype (RT.1l) as the Lewis rat (24). Finally, the physiologic significance of Abs induced during AA was tested by serum adoptive transfer. Our results highlight the role of Abs to hsp65 in the pathogenesis of AA, which is believed to be a typical T cell-mediated disease. These findings are of significant relevance to further understanding of the disease process in human RA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Inbred, 5–6 wk old (150–200 g), male Lewis (RT-1l) and Wistar Kyoto (WKY/NHsd) (=WKY) (RT-1l) rats were purchased from Harlan Sprague Dawley. Animals were maintained under a conventional housing environment in the Central Animal Facility of the University of Maryland School of Medicine (Baltimore, MD), in accordance with the guidelines of the Institutional Animal Care and Use Committee.

Antigens

Native hsp65 proteins. Mycobacterial hsp65 (Bhsp65) (28, 29) was purified from pET23b-GroEL2 vector (Colorado State University) transformed into E. coli strain BL21 (DE3) pLysS (Novagen). Rat hsp65 (Rhsp65) (30) and E. coli GroEL (28, 29) were purified from Rat60TA-PtrcHisA-transformed E. coli Top-10 cells and Ecgroel-PtrcHis-transformed E. coli Top-10 cells, respectively (provided by Dr. R. S. Gupta, McMaster University, Hamilton, Ontario, Canada). After overexpressing each of the recombinant proteins with 1 mM isopropylthio-beta-D-galactoside for 4 h at 37°C, it was applied to a TALON metal affinity column (BD Clontech) followed by recovery with an elution buffer (300 mM NaCl and 50 mM sodium acetate (pH 5.0)). The pooled eluate samples were concentrated and desalted using a high-performance concentrator, 30-kDa APOLLO 20 ml (Orbital Biosciences). Any endotoxin contaminating the purified recombinant proteins was removed by using an ActiClean Etox column (Sterogene Bioseparations), and its depletion was confirmed by Limulus Amebocyte detection kit (BioWhittaker). The recombinant proteins were further characterized by SDS-PAGE and Western blot analysis (data not shown).

Peptides. The peptides spanning the amino acid sequences of Bhsp65 or Rhsp65 were obtained from Macromolecular Resources and Global Peptide Services.

Other Ags. Keyhole limpet hemocyanin (KLH) and CII were purchased from Sigma-Aldrich. Denatured CII was obtained by heating native CII at 56°C for 30 min.

Induction and evaluation of AA

Lewis/WKY rats were s.c. at the base of the tail with 200 µl (1 mg/rat) of heat-killed Mtb (Difco) suspended in mineral oil (Sigma-Aldrich) (24, 31). Beginning on day 6 after immunization, rats were observed regularly for clinical signs of arthritis in their paws. The severity of arthritis was evaluated on the basis of erythema, swelling, and induration as follows: each of the four regions (wrist/ankle joints, metacarpals/metatarsals, carpal/tarsal joints, and interphalangeal joints) of fore/hind paws was graded on a scale of 0–1. The highest score for each paw was 4, and the total maximum score for each rat was 16. The course of AA in the Lewis rat consists of the following phases: incubation (Inc; up to day 9 after Mtb injection); onset of disease (Ons; days 9–11); peak (Pk; days 15–18); and recovery (Rec; days 21–29) phase. For ease of comparison of immune parameters with Lewis rats, the corresponding time points post-Mtb injection in AA-resistant WKY rats were also presented as phases of AA. In each case, day 0 or another day before Mtb challenge was labeled as the "naive" state. Lewis rats with moderate to severe AA frequently develop ankylosis and deformities of the paw. In this regard, the regression of inflammation in the latter part of the disease course is not necessarily the same as complete recovery from the disease process. Unlike gradually increasing inflammation in the early (acute) stage of AA, there is a gradual regression of inflammation in the late stage of the disease, which we have labeled as the Rec phase.

Preparation of sera for ELISA and adoptive transfer

Blood was collected from a cohort of naive and arthritic Lewis rats at defined time points after Mtb challenge, and from WKY rats at the corresponding time points as described above. The collected blood was allowed to clot, and was then centrifuged at 2700 x g for 15 min at 4°C. The separated sera were stored in a plastic tube at –20°C or below until used. For production of a control hyperimmune serum, LEW rats were first immunized s.c. with KLH (0.1 mg/rat) emulsified in IFA and 14 days later, injected i.p. with soluble KLH (0.1 mg/rat) in PBS. After 9 days postboost, blood was collected from these rats, tested for the presence of anti-KLH Abs, and then processed further for adoptive transfer as described above.

For adoptive Ab transfer, pooled sera were concentrated using Centriplus YM-100 with 100,000 Da cutoff to remove low molecular mass material, and sterilized by filtration through 0.45-µM Millipore membranes. Naive, 5- to 6-wk-old male Lewis rats were injected i.p. each with 10-ml equivalent of concentrated serum that was adjusted to 5 ml with sterile PBS. After 24 h of serum transfer, the recipient Lewis rats were immunized s.c. at the base of the tail with 200 µl (1 mg/rat) of Mtb (Difco) suspended in oil (Sigma-Aldrich), and the rats were observed regularly for clinical signs of arthritis in their paws as described above.

Measurement of the level and isotype of serum Abs

A high-binding capacity 96-well ELISA plate (Greiner Bio-One) was coated with 100 ng/well of an Ag in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 7 H2O, and 1.4 mM KH2PO4 (pH 7.2)) overnight at 4°C, and then blocked with 5% BSA (Sigma-Aldrich) in PBST (PBS with 0.5% Tween 20) for 2 h and washed thoroughly. The test serum was added to the wells at various dilutions and then incubated for 1 h at room temperature. Following rinsing five times with PBST, the plate-bound total Ig, IgG, and IgM isotypes were detected by affinity-purified HRP-conjugated goat anti-rat Ig (BD Pharmingen) and HRP-conjugated goat anti-rat IgG and anti-IgM (Zymed Laboratories), respectively. The substrate, 3,3',5,5'-tetramethylbenzidine (Bio-Rad), was then used for development of the colorimetric reaction. After adding an equal amount of stop solution (0.18 M H2SO4), the color intensity was read at 450 nm using Vmax ELISA autoreader (Molecular Devices). {Delta} OD was calculated by subtracting background OD value obtained with secondary Ab from OD value for a test Ag.

Statistical analysis

The data were analyzed using the Student t test and the nonparametric Wilcoxon rank sum test as applicable. A p value of <0.05 was considered significant. In the pepscan analysis, a "peak" Ab response was represented by a peptide giving a significantly higher (p < 0.05) reactivity (mean OD ± SD) with serum Ig compared with that of at least two adjacent peptides giving a baseline reactivity with the same serum collected at a particular phase (e.g., the Rec phase) of the disease.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ab responses to native hsp65 proteins

The course of Mtb-induced arthritis in Lewis rats can be divided into approximately four stages (Fig. 1): an asymptomatic Inc phase (up to 9 days after Mtb injection), Ons (9–11 days), Pk (15–18 days), and a slow Rec phase (21–29 days). As shown in Fig. 1, the relatively resistant strain of WKY rats, which has the same class II haplotype as the Lewis rat, did not develop arthritis during the same period after the Mtb injection. Sera were collected from the naive Lewis and WKY rats and from the Mtb-injected rats of both these strains at the indicated time periods (Inc, Ons, Pk, and Rec) and tested by ELISA for specific Ab (either as total Ig or IgG) against the mycobacterial hsp65 (Bhsp65), rat hsp65 (Rhsp65), E. coli GroEL, and CII; wells coated with KLH were used for control of specificity. Both strains of rats developed specific Abs against the bacterial Ags as well as against the self hsp65 (Rhsp65), however, there were marked differences in the magnitude and the kinetics of these responses between the WKY and Lewis rats. Furthermore, Mtb-immunized Lewis and WKY rats did not raise any Ab response to another arthritis-related Ag, CII, as the level of response to CII was comparable to that to the irrelevant control Ag, KLH.


Figure 1
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  		FIGURE 1. The relative severity of AA in Lewis and WKY rats following challenge with Mtb. Lewis ({blacksquare}) and WKY ({square}) rats (5–6 wk old, male, n = 12 per group for each rat strain) were immunized s.c. with 1 mg/rat Mtb in oil. Thereafter, these rats were observed regularly, and the severity of arthritis was graded as described under Materials and Methods. The difference in the severity of disease during the entire course of AA in Lewis vs WKY rats was statistically significant (p < 0.05) as analyzed by the nonparametric Wilcoxon rank sum test. Different phases of AA in the Lewis rat are as follows: Inc (up to day 9 after Mtb injection); Ons (days 9–11); Pk (days 15–18); and Rec (days 21–29) phase.

 
The disease-resistant WKY rats developed higher titers of anti-Bhsp65 Ig faster than did the susceptible Lewis rats (Figs. 2–4). Indeed, even naive WKY rats demonstrated a strong anti-Bhsp65 binding at 1/100 dilution for either total Ig (Figs. 2 and 3) or for IgG fraction that contains bulk of the Ab (Fig. 4). Moreover, the WKY rats developed a robust anti-Rhsp65 Ab response early after the Mtb injection (during an Inc phase), whereas in the Lewis rats, such Abs remain undetectable until their recovery from arthritis (Rec phase) (Figs. 3 and 4). The strain differences in Ab responses were evident mostly among the IgG isotypes whereas the IgM contributed relatively little (Fig. 4). The sera from the naive WKY rats also showed a higher Ig binding to the self hsp65 as compared with the naive Lewis rats (Fig. 2). These data show that the resistance to Mtb-induced arthritis correlates with a rapid and robust IgG Ab response to both bacterial and self hsp65 proteins.


Figure 2
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  		FIGURE 2. Ab titers against hsp65 of sera of Lewis and WKY rats following challenge with Mtb. Lewis (left panel) and WKY (right panel) rats (5–6 wk old, male, n = 32 per group for each rat strain) were immunized s.c. with Mtb (1 mg/rat) for the induction of AA. Thereafter, sera were collected from arthritic Lewis rats at different time points after Mtb challenge (Inc, Ons, Pk, and Rec), and from WKY rats at the corresponding time points. Naive preimmune serum from the corresponding rat strain served as a control. These sera were tested at different dilutions in triplicates in ELISA using Bhsp65 (A), Rhsp65 (B), E. coli GroEL (C), and CII (E) as test Ags. KLH (D) was used as an irrelevant Ag (negative control). The total Ig reactivity was expressed as mean OD (450 nm).

 

Figure 3
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  		FIGURE 3. Ab responses to hsp65 of Lewis and WKY rats at different time points after Mtb injection. Sera collected from Mtb-immunized Lewis (left panel) and WKY (right panel) rats (5–6 wk old, male, n = 32/group for each rat strain) as described in the legend to Fig. 2 were tested (at 1/100 dilution each) in ELISA in triplicates using Bhsp65, Rhsp65, E. coli GroEL, and CII as test Ags. KLH was used as an irrelevant Ag (negative control). The results for total Ig binding were expressed as {Delta} OD 450 nm (mean ± SEM). *, p < 0.05 for Lewis/WKY test sera vs the corresponding naive sera; {dagger}, p < 0.05 for Lewis naive sera vs WKY naive sera; {ddagger}, p < 0.05 for Lewis test sera vs WKY test sera at the corresponding time point after Mtb challenge.

 

Figure 4
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  		FIGURE 4. The serum IgG and IgM reactivity to hsp65 in sera of Lewis and WKY rats following challenge with Mtb. Sera were collected from arthritic Lewis rats (left panel) at different phases of the AA, and from Mtb-immunized WKY rats (right panel) at the corresponding time points as described in Fig. 3. These sera (1/100) were tested in ELISA using Bhsp65, Rhsp65, E. coli GroEL, and CII as test Ags. The levels of bound IgG and IgM were expressed as OD 450 nm (mean ± SEM). *, p < 0.05 for Ab level in Lewis/WKY test sera compared with the corresponding naive sera; {dagger}, p < 0.05 for WKY naive sera vs Lewis naive sera.

 
In contrast to the high reactivity with the mycobacterial and rat hsp65, the serum Ab bound only weakly to the E. coli hsp65, GroEL (Fig. 2), despite the similar degree of homology between Bhsp65, Rhsp65, and GroEL (~50% amino acid identity) (28, 29, 30). The baseline binding to GroEL did not increase significantly in time after the Mtb immunization and there was no difference in the reactivity of Ab from the susceptible Lewis and the resistant WKY rats (Figs. 3 and 4). This reactivity pattern demonstrates the specificity of the Ab response to the hsp65 molecules (Bhsp65 and Rhsp65) that are linked to the AA pathogenesis.

Ab responses to hsp65 peptides

To obtain an insight into the repertoire of the anti-hsp65 response, we tested the above serum Ig for reactivity with linear peptides spanning the Bhsp65 and Rhsp65 amino acid sequences. The results (Fig. 5) showed a dramatic difference in the fine epitope specificity of Ab in WKY and Lewis strains, and suggested a process of Ab repertoire shift in time after the Mtb injection but in opposite directions.


Figure 5
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  		FIGURE 5. The serum Ig reactivity against peptides of Bhsp65 and Rhsp65 of Lewis and WKY rats following immunization with Mtb. Rats (5–6 wk old, male, n = 30/group for each rat strain) were immunized s.c. with Mtb (1 mg/rat). Sera were collected from arthritic Lewis rats (open symbols) at different phases of the disease, and from WKY rats (closed symbols) at the corresponding time points: Naive (A and F), Inc (B and G), Ons (C and H), Pk (D and I), and Rec (E and J). These sera were tested in ELISA (1/100 dilution) using peptides of Bhsp65 (A–E, left panel) and Rhsp65 (F–J, right panel). The total Ig reactivity was expressed as mean {Delta} OD at 450 nm. The peptides giving peak reactivity (details given under Statistical Analysis) with sera of both Lewis and WKY rats in the Rec phase of AA (E and J) are marked by an arrowhead.

 
The AA-resistant WKY rats developed a rapid Ab response within the first week after the Mtb injection (Inc phase), which was directed against numerous peptides from the N to C termini of both bacterial and rat hsp65 molecules (Fig. 5, B and G). Interestingly, this heterogeneous Ig response then diminished and became gradually more restricted. At the later intervals, particularly after day 21 (Rec phase), the WKY Ig recognized only three peptides in the Bhsp65 (peptides 31–46, 211–226, and 349–364) (Fig. 5E) and only one peptide (61–80) in the Rhsp65 (Fig. 5J). Aside from these distinct peaks of Ab reactivity, the binding to other regions of either hsp65 did not increase above that in the preimmune sera (OD450 ~0.2).

In marked contrast, the susceptible Lewis rats failed to mount an Ig response to linear peptides of either the bacterial or rat hsp65 during the incubation (Inc) phase (Fig. 5, B and G); however, a weak binding to several regions of both hsp65 became detectable from the onset of the disease. The Ig repertoire of the Lewis rats at the recovery phase was focused on five peptides of Bhsp65, three of which–31–46, 211–226, and 349–364–overlapped with the peptides recognized by the WKY rat sera (Fig. 5E). Even more remarkably, the late Ab in Lewis rat was focused on only one epitope of the Rhsp65, represented by peptide 61–80, which is the only epitope also recognized by the WKY Ig (Fig. 5J). However, it should be noted that the Ig from recovery phase Lewis rats, in addition to the preferential reactivity with selected epitopes, also demonstrated a significantly higher binding to all linear hsp65 peptides (OD450 = 0.3–0.4, Fig. 5, E and J) as compared with the preimmune, naive Ig (OD450 = 0.1–0.2, Fig. 5, A and F) (p < 0.05), a pattern that was not seen with the WKY rats.

Thus, it appears that the AA-resistant WKY strain respond in a rapid, memory-like fashion to numerous hsp65 determinants, but this Ab response becomes gradually restricted to a few key epitopes. The repertoire of Abs from Lewis rats that have recovered from the disease is also focused on these epitopes, although it apparently spans other parts of the hsp65 molecule as well showing a trend toward diversification (epitope spreading).

Ab from rats in recovery phase protect against the Mtb-induced disease (AA)

We next examined whether an administration of the Rec phase sera that contained the distinctive patterns of Ab against hsp65 could modulate the Mtb-induced arthritis in naive animals. Groups of naive recipient Lewis rats were injected i.p. with pooled sera from either naive Lewis or WKY rats (control groups) or from Lewis or WKY rats that had recovered from arthritis (experimental groups). The recipients were challenged with Mtb 24 h later and observed regularly for signs of arthritis. The recipients of sera from the arthritis-recovered Lewis donors showed a significant reduction in the severity of Mtb-induced disease as compared with either of the controls that received serum from naive Lewis donors or no serum, respectively (p < 0.05) (Fig. 6, A and B). The experiment in Fig. 6A included also a group of recipients that received serum from arthritis-recovered Lewis donors without subsequent Mtb challenge. These rats did not show any signs of disease symptoms during five weeks of observation (data not shown), which rules out a direct arthritogenic effect of the anti-hsp65 Ab. These data suggest that the Abs that are produced in the course of AA in the susceptible Lewis rats may down-modulate the course of the disease. However, similar results were obtained with sera from the disease-resistant WKY rats. The naive recipients (Lewis) of sera from arthritis-recovered WKY donors showed significant reduction in disease severity as compared with the sera from naive WKY donors (p < 0.05) that did not appear to affect the disease (Fig. 6D). This was unexpected because the sera from naive WKY rats contain a robust Ig/IgG reactivity against the whole Bhsp65 (Figs. 2 and 4) although they did not show the peptide reactivity that is associated with Ab from the Rec phase (Fig. 5). Thus, we suggest that the resistance of WKY to AA is not due to the pre-existing "natural" anti-hsp65 Ab, but, rather owing to their ability to respond rapidly to the Mtb challenge (Fig. 5, B and G) and to develop protective Ab (see Discussion).


Figure 6
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  		FIGURE 6. Modulation of AA in the recipient Lewis rat by adoptive transfer of sera before Mtb challenge. Recipient naive Lewis rats (n = 3/group) were injected i.p. with 5 ml of concentrated pooled sera (=10 ml of original serum) prepared from sera of either naive or Rec (recovery phase) Lewis rats (A). After 24 h of serum transfer, the recipient Lewis rats were challenged with Mtb. Another group of control Lewis rats received no serum at all, but was injected with Mtb. Thereafter, all rats were observed and scored regularly for signs of arthritis as described under Materials and Methods. The difference in the severity of AA in control Lewis rats vs each group of Lewis rats given Rec phase serum of Lewis rats was found to be significant (*, p < 0.05–p < 0.025) from days 7 through 15 as tested by the Student t test. Similar results were obtained in an independent repeat experiment (B). Using a similar protocol, two additional sets of serum transfer experiments were performed. In one (C), naive Lewis rats (n = 4/group) were injected with a control hyperimmune serum derived from Lewis rats immunized with KLH, whereas the control group received no serum before Mtb injection. The difference in the arthritic scores of the two rat groups was not significant. In another (D), naive Lewis rats (n = 4/group) received either sera of naive WKY rats (controls) or of WKY rats at Rec phase post-Mtb immunization (experimental group) followed by Mtb injection. Rats given Rec phase serum showed a significant reduction in the severity of AA (p < 0.05) from days 12 through 22 compared with those injected with naive serum.

 
To further examine the specificity of serum protection, groups of naive Lewis recipients received either sera from Lewis donors immunized repeatedly with KLH or PBS (control) and Mtb challenge 24 h later (Fig. 6C). There was no significant difference in the disease scores between the two groups, indicating the protection seen in Fig. 6 (A, B, and D) is not due to a nonspecific substance(s) in hyperimmune sera.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The mycobacterial hsp65 (Bhsp65) has been implicated in the pathogenesis of rat AA (20, 21, 22, 23, 24, 26, 32) as well as human RA (32, 33, 34, 35, 36, 37, 38). Several disease (AA)-regulating T cell determinants within Bhsp65 have been described by other studies and our own (21, 23, 24, 25, 26, 32). However, there is relatively meager information on Ag-specific Ab in AA (26, 39, 40). In the present study, we have shown that Mtb-immunized Lewis rats develop Abs not only against Bhsp65, as previously described (26, 39), but also to the rat’s own hsp65 (Rhsp65) that is a presumed target Ag in autoimmune arthritis. An intriguing feature of these anti-hsp65 Abs was their apparent heterogeneity. We did not observe a straightforward correlation between the serum Ig activities against the whole hsp65 molecules vs the linear peptides spanning these molecules, at specific times after the Mtb injection. For instance, the AA-susceptible Lewis rats showed binding to the whole Bhsp65 at the Inc phase (Fig. 3) with no detectable binding to the peptides (Fig. 5B). The anti-Bhsp65 Ig in the AA-resistant WKY rats reacted strongly to the whole molecule (Fig. 3) as well as to numerous peptides at early stage (Inc) (Fig. 5B), but 1 wk later (Ons) (Fig. 5C), the former reactivity remained strong, whereas the peptide binding was largely undetectable (compare Fig. 3 with Fig. 5, B and C). It is unlikely that such remarkable changes in Ab repertoire reflect merely a different sensitivity of the respective ELISAs. Rather, these data suggest that a variable portion of the anti-hsp65 Ab is directed against discontinuing (conformational) epitopes instead of the linear peptides, as would be expected with any large protein Ag. Thus, the changing patterns of the peptide-reactive Ab repertoire reveal only a portion of the response.

Nonetheless, it is intriguing that the specificity of the late Ab becomes restricted to a small set of hsp65 peptides. The significance of this apparent repertoire selection is underscored by three observations: 1) there was an overlap in peptide specificity of the Ab from the AA-resistant WKY rats with the Ab from the Lewis rats that become resistant during the recovery from the disease, both for the Bhsp65 (Fig. 5E) and the self hsp65 (Fig. 5J). Because the sera from the recovery phase Lewis rats can transfer a protection against AA to naive recipient Lewis rats (Fig. 6), we hypothesize that the disease resistance is mediated through a selection of B cell clones with reactivity against one to three key epitopes of hsp65. 2) The epitopes that were recognized by the late Ab from both WKY and Lewis rats in our study, represented by the Bhsp65 peptides 31–46, 211–226, and 349–364 and the Rhsp65 peptide 61–80, matched with those described earlier by Ulmansky et al. (26) as the peptides showing the highest reactivity with sera from postarthritic Lewis rats, indicating that the progressive focusing of Ab repertoire on these epitopes is well-conserved. One of these epitopes, human hsp65 31–50, has been also shown to be immunoregulatory in AA in another study (23) examining the T cell epitopes within human hsp60. 3) There appears to be an intriguing overlap between the narrow repertoire of Bhsp65-reactive B and T cells during the recovery from arthritis in Lewis rats. A comparison of present data from Fig. 5E with our earlier results on anti-Bhsp65 T cell responses (24) revealed that the Ab recognized the peptides that overlapped either partially or completely with those that stimulated the highest T cell reactivity (Table I). Such a commonality between T and B cell epitopes, which has been previously demonstrated for other protein Ags (41, 42), including the target autoantigens for multiple sclerosis (7, 8) and type 1 diabetes mellitus (43), suggests that a binding of Ab to key epitopes can directly modulate the effector T cell reactivity against the disease-related Bhsp65 possessing arthritogenic as well as regulatory epitopes. However, we could not perform a similar comparative study on the repertoire of B and T cells that react against the self hsp65 (Rhsp65) as the information regarding Rhsp65-specific T cell epitopes that might be responded to by Lewis rats during the course of AA is not yet available. If there is an overlap between the two, the self hsp65-specific Ab may impact the disease differently than the antibacterial Ab because the self-reactive T cells are involved in the remission/protection from AA (23, 31).


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  		Table I. Regions of Bhsp65 recognized by serum Ab and T cells from Lewis rats recovering from arthritis

 
It is reasonable to expect that a significant fraction of Abs react with the conformational hsp65 determinants. This notion is implicit in our observation that the immune sera from the incubation (Inc) phase of AA in Lewis rats reacted with the whole Bhsp65 (Figs. 2 and 3) but not with the linear peptides (Fig. 5B), and that the sera from the recovering Lewis rats demonstrated an increased baseline binding to peptides across the entire Bhsp65 and Rhsp65 (Fig. 5, E and J). Such Abs may indirectly boost or suppress the responses to distant T cell determinants on the same molecule (43, 44, 45).

In comparison to the previous reports by other investigators on the Ab response in AA (26, 39), our study has contributed the following novel aspects of Ag/epitope-specific Abs that have either not been addressed or not fully examined in those studies: 1) this is the first report describing Abs to truly self (rat) hsp65 (Rhsp65); in a previous study, human/mouse hsp65 were tested (26), but even 97.8% homology between human vs rat hsp65 (or mouse vs rat hsp65) (31) is not enough to render the former protein "self" for the Lewis rat. 2) We established comparative profiles of Ab reactivity to Bhsp65, Rhsp65, E. coli GroEL, and CII; only Bhsp65 and/or mammalian (human/mouse) hsp65 were tested in previous studies (26, 39). 3) We studied qualitative and quantitative differences in Ag-specific Ab response to the above-mentioned Ags in AA-susceptible Lewis vs AA-resistant WKY rats: the novelty of this pair of rats lies in the fact that these strains bear the same MHC class II haplotype (RT.1l); in an earlier study, rat strains of entirely different MHC haplotypes (RT.1l vs RT.1n) were compared (26). 4) We tested the Ag-specific Ab response in arthritic rats at defined time points that correspond to different stages of AA (Naive, Inc, Pk, and Rec); other investigators selected arbitrary time points. 5) We have independently validated the functional significance of Abs generated during the course of arthritis; these Abs are protective rather than being pathogenic.

We observed that the reactivity of Abs to different epitopes of Bhsp65 showed a trend toward diversification of response with the progression of AA in the Lewis rat (Fig. 5), particularly evident in the transition from Inc to Pk phase of AA. The phenomenon of epitope spreading involving Ag-specific Abs has previously been reported in other autoimmune diseases such as SLE, myasthenia gravis, MS/EAE, type 1 diabetes mellitus, etc. (3, 4, 9, 12, 16, 17, 46, 47, 48, 49, 50). The proposed mechanisms underlying spreading of Ab response include: 1) enhanced uptake of the Ag by B cells expressing the appropriate Ag receptor and subsequent processing and presentation of the Ag to specific T cells recognizing other epitopes/Ags (9, 48, 51, 52, 53); this property of B cells has also been invoked in explaining the beneficial effect of transient depletion of B cells in RA patients (53); 2) activated T cells of one epitope/Ag specificity provide help to B cells recognizing other epitopes/Ags (47, 52); and 3) modulation of the processing and presentation of T cell epitopes by the Ag-bound Ab (44, 45). Detailed mechanistic studies on the modulation of AA by Ab will require the characterization of conformational determinants and separation of the Bhsp65-specific and the self Rhsp65-specific Abs.

Unlike the demonstration of pathogenic Abs in the murine models of SLE and arthritis (3, 13, 14, 54), the results of serum Ab transfer in our study and that of Ulmansky et al. (26) showed that Abs produced during the course of AA confer "protection" against subsequently induced AA in Lewis rats. Such protective Abs have been also found in the sera of the naive AA-resistant Fisher and BN rats (26, 27) but not in the WKY rats in the present study (Fig. 6). This divergence probably reflects the genetic differences between the resistant strains, including the MHC haplotype (WKY, RT.1l vs BN, RT.1n) which influence the response to environmental Ags. Despite of these differences, however, these rat strains produced Abs with similar repertoire after immunization with hsp65. The influence of both MHC and non-MHC genes on the development of "natural" vs adaptive response to hsp65 and the mechanisms by which the respective Abs down-modulate the course of AA have yet to be determined. A recent study suggests that EAE may be also regulated by Ab (11).

In regard to RA, it has been reported that sera of these patients possess anti-Bhsp65 Abs (55, 56). However, the precise role of Abs in the pathogenesis of RA is not clear. It is conceivable that subsets of these Abs might be involved in the regulation of acute RA. Coupled with our previous study showing that the T cell response to Bhsp65 diversifies to the disease-regulating Bhsp65 C-terminal determinants during the late phase of AA (24), the above-mentioned results showing the generation of "protective" Ab with the progression of AA provide a novel aspect of T cell-Ab cooperation in recovery from acute AA. As reported in SLE, another aspect of T cell-Ab interplay that might be of significance in AA is the priming in vivo of the disease-regulating Ig-reactive CD4+CD25+ T cells by Ab-derived epitopes (57). However, contrary to these regulatory interactions, studies showing the beneficial effect of transient depletion of B cells in RA patients highlight the contribution of these cells to the propagation of the disease process presumably by production of pathogenic Abs, by secretion of cytokines, and by serving as APC (53, 58, 59). Taken together, the above results from the AA model and those from RA patients document that B cells and their products (Abs) contribute significantly to the pathogenesis of autoimmune arthritis. These findings in arthritis, combined with those from studies in MS and EAE mentioned above, would stimulate novel strategies for the management of these autoimmune diseases previously believed to be primarily T cell-driven in nature.


   Acknowledgments
 
We thank Malarvizhi Durai and Younus Mia for the preparation of Rhsp65 and GroEL, and Shailesh Satpute for help with experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (AI47790 and AI059623), the Arthritis Foundation (Atlanta, GA and the Maryland Chapter, Baltimore, MD), and the Maryland Arthritis Research Center (Baltimore, MD). Back

2 J.C. and K.D.M. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Kamal D. Moudgil, Department of Microbiology and Immunology, Howard Hall 323C, University of Maryland School of Medicine, 660 W. Redwood Street, Baltimore, MD 21201. E-mail address: kmoud001{at}umaryland.edu Back

4 Abbreviations used in this paper: MS, multiple sclerosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; EAE, experimental autoimmune encephalomyelitis; AA, adjuvant arthritis; Mtb, Mycobacterium tuberculosis; hsp, heat shock protein; Bhsp65, mycobacterial hsp 65; BN, Brown Norway; CII, type II collagen; Rhsp65, rat hsp 65; WKY, Wistar Kyoto (WKY/NHsd); KLH, keyhole limpet hemocyanin; Inc, incubation; Ons, onset; Pk, peak; Rec, recovery. Back

Received for publication August 30, 2005. Accepted for publication August 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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