Heat Shock Proteins in the NOD Mouse

1. Immunostimulation Reduces the Incidence of IDDM in the NOD Mouse

Several studies have established that the incidence of IDDM in the NOD mouse can be reduced by exposure to mycobacterial preparations including complete Freund's adjuvant (killed Mycobacterium tuberculosis in oil emulsion) (41-43) and the bacille Calmette-Guerin vaccine (BCG, live attenuated Mycobacterium bovis) (44). The mode of action is unclear, but hypotheses have included nonspecific immune stimulation, activation of suppressor lymphocytes or macrophages (45), alteration of cytokine profiles from TH1 to TH2 (46), or possibly by the common presence of mycobacterial hsp 65 in the administered antigen.

2. M. bovis Hsp 65 Cross-Reactive Antigen Identified as Target of Cellular Immune Response

Support for this last hypothesis came in a report from Elias and coworkers which suggested that the pathogenesis of IDDM in the NOD mouse was associated with a T-cell and antibody reactivity to an antigen which was cross reactive with mycobacterial hsp 65 (47). These investigators reported that the primary event appeared to be the release into the serum of the hsp 65 cross-reactive antigen, which preceded the onset of diabetes and the other autoimmune phenomena. It was implied that the hsp 65 cross-reactive antigen was a p-cell protein. The onset of insulitis was associated with the spontaneous development of T-cell responses to hsp 65. This group reported a reduction in the incidence of IDDM in mice treated at the age of 30 days with a preparation of mycobacterial hsp 65 in saline.

At this age, insulitis is frequently observed, but clinical diabetes does not usually occur before 120 days. T-cell clones were generated against mycobacterial hsp 65, and transfer of a single hsp-65-reactive CD4+ T-cell clone to young prediabetic NOD mice accelerated the onset of IDDM suggesting that the clone may have elicited an autoimmune response against a host protein. Although the implication was clearly that a heat shock protein was the target of the T-cell clones, it was postulated that the specific P-cell destruction observed in IDDM could result from the expression of a p-cell stress protein or that an unrelated protein shared sequence homology with the mycobacterial hsp 65. Immunization with mycobacterial hsp 65 in incomplete Freund's adjuvant (ICFA) lead to transient insulitis, but the incidence of IDDM was also reduced compared with the control population. This unexpected result will be discussed later.

3. CD4+ T Cells Responsive to an Immunodominant Epitope of Human Hsp 60 Transfer IDDM

In a later paper, Elias et al. repeated a number of elements of the experiments using human hsp 60 in place of the mycobacterial hsp 60 (48). The major difference was that immunization with hsp 60 did not reduce the incidence of IDDM but instead rapidly induced hyperglycaemia. An epitope of human hsp 60 was also identified which stimulated a T-cell response in NOD mouse. This peptide, p277, comprised amino acids 437-460 and differs by only one amino acid from mouse hsp 60, but it contains more limited sequence homology with mycobacterial hsp 65. Using the T-cell clones generated against mycobacterial hsp 65, it was found that in several cases the responses to human hsp 60 were stronger, implying that this antigen was more closely related to the P-cell target protein than mycobacterial hsp 65. Three T-cell clones responded to the peptide p277, and only these clones produced insulitis and hyperglycemia in transfer experiments. The other T-cell clones, which responded more strongly to mycobacterial hsp 65 than human hsp 60 were not able to transfer insulitis. This important peptide could be used in a nonimmunogenic form to downregulate the immune response to hsp 60 in the NOD mouse, and T-cell clones recognizing this peptide could induce insulitis and hyperglycemia. Further, the same clones, when attenuated by gamma irradiation, could be used as therapeutic vaccines. This paper is of interest as it clearly shows that autoreactive T-cell clones which recognize a peptide derived from the ubiquitous hsp 60 are able to exert a p-cell-specific effect. Although the protein is expressed in most or all the cells in the body, it is only the p cells that are destroyed by the p277 reactive T-cell clones.

4. Difference Between Mycobacterial Hsp 65 and Human Hsp 60 Immunization

Immunization with human hsp 60 in ICFA led to hyperglycemia, whereas mycobacterial hsp 65 under the same conditions reduced the later incidence of IDDM.

The mechanism of action was unclear at the time of publication of the papers describing hsp 60 as an autoantigen in NOD mouse IDDM (47,48). It was well established that the administration of the native peptide in a nonimmunogenic form (either supraoptimal or oral route) is able to anergize the responding T-cell population, but immunization in adjuvant would have been expected to stimulate a response. However, it was recently shown that presentation of an altered peptide ligand could induce anergy in T cells even when administered in a system in which a response would normally be stimulated (49). The mycobacterial homologue of the mouse/human p277 peptide may therefore constitute an altered ligand which is able to tolerize the p277-responsive CD4+ T-cell population even when administered in IFA.

5. Disease Transfer to NON.H-2NOD Mice

The p277-responsive T-cell clones were also transferred to NON.H-2nod mice (48). This strain of mice does not develop spontaneous IDDM but shares a common MHC class II genotype. These mice, however, developed IDDM after administration of this T-cell clone. Importantly, although these mice do not spontaneously develop IDDM, immunization with human hsp 60 resulted in hyperglycemia and insulitis. Thus, activation of a population of T-cell clones which react with a host heat shock protein is the key event in the autoimmune process. In the NOD mice, this event occurs spontaneously, but in the related NON mice, the T-cell clones must be activated experimentally by immunization. At the time this paper was published, the concept of TH1 and TH2 subsets of CD4+ T cells was not well established. It would be of interest to examine whether the T-cell clones were of a TH1 or TH2 phenotype.

6. Treatment of Established IDDM in NOD Mice with p277

More recently, the peptide p277 has been used to treat NOD mice with established insulitis in 84-, 105-, and 119-day-old mice (50). Of 30 mice treated with the control peptide, bovine serum albumin (BSA), 28 had died by 280 days. Conversely, none of 21 mice treated before 105 days with a 50-^g dose of peptide p277 died, although many exhibited hyperglycemia. Similar results were also obtained in a larger series of 157 NOD mice: Total mortality in the pep-tide-treated group was 3% compared with 84% in the control BSA group.

Together these reports clearly identify hsp 60 as a key autoantigen in the etiology of IDDM in the NOD mouse.

B. Hsp 60 in Human IDDM

1. Cellular Stress Induces Increased Hsp 60 Expression in Rat

Insulinoma Cells and Purified Human Islets Our initial experiments with a rat insulinoma (RIN) cell line showed that hsp 60 was expressed at a low level in unstressed cell. Cellular stress induced by hyperthermic incubation or by treatment with the cytokines interleukin-ip (IL-ip), IFN-y, or TNF-a led to increased synthesis of hsp 60 following a prolonged recovery phase (Fig. 1) (51).

More recently, we have used purified human islet tissue in the same system. Hsp 60 expression was considerably higher in the unstressed islets than the insulinoma cell line, probably as a consequence of the procedure used to remove the islets from the pancreas, but again, a qualitative increase in expression of hsp 60 was observed. The expression of GAD 65 appeared to be reduced under the same conditions.

2. Antibody Reactivity to 64-kDa Antigen is Reduced by Pretreatment with Anti-Hsp 60 Antibody The earliest evidence for involvement of hsp 60 in human IDDM came from our work using stressed RIN cells as a target (51). These experiments demonstrated that expression of a -64-kDa protein was induced and that antibodies present in the serum of patients with insulin-dependent diabetes bound to this region in Western blots. Further, the binding of anti-64-kDa antibodies in the serum of newly diagnosed patients with IDDM was inhibited by the preincubation of the cell suspensions with antibodies against mycobacterial hsp 65. These experiments suggested that the 64-kDa antigen, a common target of hum-

Figure 1 Expression of hsp 60 in heat shocked RIN cells. (From Ref. 51.).

oral immunity in IDDM, may be a heat shock protein or was an unrelated antigen with sequence homology to a heat shock protein.

This study was performed before the identity of the 64-kDa antigen was known. The 64-kDa antigen was subsequently demonstrated to be the 64-kDa islet isoform of GAD 65. However, several studies have established that 64-kDa antibody reactivity may not be composed only of GAD 65-reactive immunoglobulin: We have proposed that hsp 60 autoreactivity may constitute a significant fraction of 64-kDa reactivity in some subjects (52).

These investigations (51) were not supported by studies by Atkinson and coworkers (53). Using a similar model, they found that hyperthermic incubation of islets stimulated the expression of heat shock proteins with Mrs of 72, 75, and 90-kDa. The 60-kDa heat shock protein was constitutively expressed, and it was not upregulated by hyperthermic stress in their system. In addition, Western blot analysis with 64-kDa antibody-positive individuals did not identify hsp 60 as a target. However, our initial observations were dependent on a prolonge recovery time of the heat shocked RIN cells, and the data obtained by Atkinson and coworkers did not include an recovery phase, suggesting that the time scale of production of an islet-specific hsp 60 homologue is not immediate and may follow a cellular response and recovery.

3. Hsp 60 Antibody Reactivity Is Significantly Higher in IDDM Subjects More recently, we have studied the humoral autoreactivity to hsp 60 in IDDM using recombinant human hsp 60. A total of 167 sera from 26 cases newly diagnosed IDDM, 45 long-standing cases of IDDM, 46 non-insulin-dependent diabetic patients (NIDDM), and 50 control subjects were assayed in an ELISA for antibodies to human and mycobacterial hsp 60. Significantly higher reactivity was found both in newly diagnosed patients and in those with long-standing IDDM than in those with NIDDM and control subjects (Table 1). The demonstration of increased autoantibody reactivity to human hsp 60 in IDDM subjects has established hsp 60 as a potential autoantigen in IDDM. However, the earlier discussion of effector mechanisms clearly shows that CD4+ T-cell reactivity against an antigen is more etiologically important.

4. Proliferative T-Cell Responses to Hsp 60 in IDDM

We have therefore measured CD4+ T-cell responses to human hsp 60 in IDDM subjects in an in vitro T-cell proliferation assay (Fig. 2). In this assay, autoreactive CD4+ T cells are stimulated by antigen, and the proliferative response is measured on the basis of thymidine incorporation into the dividing antigen-responsive clones. A positive proliferative response is defined by a stimulation index greater than 3, (where the stimulation index = proliferation with antigen/ proliferation with medium alone). This cut-off is widely used, and it represents

>25 -i

as

Proliferative T

cell

O

response to Human

HSP60

20 -

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18 -

-a

16 -

c

oo

o

c

14 -

o

°

12 -

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D

10 -

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IDDM Control

Patient GrouDS

New Non diabetic

IDDM Control

Patient GrouDS

Figure 2 T-cell proliferative responses to hsp 60 in IDDM.

the mean plus 3 SD of the wells containing medium alone. In a series of 22 patients with IDDM, 20 showed positive proliferative responses to recombinant human hsp 60. Conversely, only 4 of 17 nondiabetic subjects showed proliferative responses. The difference in both frequency of positive response and magnitude of response were significant between the two groups.

Table 1 Serum Antibody reactivity to human and mycobacterial hsp 60 in newly diagnosed anc long-standing IDDM, NIDDM, and Nondiabetic control subjects

Subject groups

Newly diagnosed

Long

Nondiabetic

Hsp type

IDDM

duration

NIDDM

control

Human hsp 60

median

0.108

0.178

0.092

0.057

range

0.026-0.306

0.037-0.368

0.010-0.373

0.010-0.137

significance (p)

p < 0.02

p < 0.001

ns

-

Mycobacterial hsp 65

median

0.106

0.367

0.150

0.066

range

0.023-0.720

0.029-0.610

0.032-0.687

0.10-0.268

significance

p < 0.04

p < 0.001

ns

ns, not significant.

In the most recent study of p277 in the NOD mouse, Cohen and Elias outlined preliminary data which showed that in human IDDM, the human homo-logue of the p277 peptide appeared to be a common T-cell epitope. In their experimental system, 20 of 23 newly diagnosed patients with IDDM showed proliferation in response to this peptide (50).

Hsp 60 has not been studied as extensively as GAD 65 in human IDDM. Nonetheless, the emerging pattern is that hsp 60 appears to be a common target of the cellular immune response in IDDM, and that the dominant T-cell epitope may be conserved with the NOD mouse model. It is to be hoped that the therapeutic effects of p277 tolerization in this animal model will be extended to the treatment of human disease. The recent paper by Shehadeh et al. may be of particular interest in this respect (54). In a small series of newly diagnosed IDDM subjects, BCG immunization appeared to influence the 'honeymoon period' frequently observed following clinical diagnosis of IDDM, with a significantly increased frequency of subjects remaining insulin independent 3-6 months after treatment. However, this study showed a lower than expected frequency of the subjects treated with placebo in remission at these time points; more extensive studies are clearly required.

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