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. 2001 Aug;125(2):323–331. doi: 10.1046/j.1365-2249.2001.01482.x

Amphipathic variable region heavy chain peptides derived from monoclonal human Wegener's anti-PR3 antibodies stimulate lymphocytes from patients with Wegener's granulomatosis and microscopic polyangiitis

E Peen *, C Malone *, C Myers *, R C Williams Jr *, A B Peck †, E Csernok ‡, W L Gross ‡, R Staud *
PMCID: PMC1906128  PMID: 11529926

Abstract

Amphipathic variable-region heavy chain 11-mer peptides from monoclonal human IgM antiproteinase-3 antibodies were studied for peripheral blood lymphocyte stimulation in 21 patients with Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA), connective tissue disease controls and normal control subjects. Positive T-cell activation was observed in most experiments with WG patients' lymphocytes using amphipathic VH-region peptides from four different human monoclonal anti-PR3 antibodies. Control peptides of the same length but without amphipathic characteristics along with other amphipathic peptides not derived from monoclonal anti-PR3 sequence were employed as controls. No significant lymphocyte stimulation was observed with normal controls, but positive stimulation with amphipathic VH peptides was also recorded in other connective tissue disease controls mainly patients with rheumatoid arthritis. Amphipathic peptides not derived from anti-PR3 sequence did not stimulate WG lymphocytes. Our findings indicate that lymphocyte reactivity as an element of cell-mediated immunity may be activated by amphipathic VH-region amino acid sequences of autoantibodies which are themselves associated with diseases such as WG.

Keywords: amphipathic, T-cell epitopes, Wegener's granulomatosis

Introduction

Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) with antibodies to neutrophil cytoplasmic antigens (PR3-ANCA) represent systemic diseases of unknown aetiology which often produce extensive inflammatory lesions within the sinuses and bronchopulmonary system as well as destructive renal lesions [1–3]. Much of the attention regarding pathogenesis has been directed at possible mechanisms of tissue damage related to the autoantibodies reacting with neutrophil granule components [4–7] such as proteinase-3 (PR-3). The present report now provides experimental results which indicate that autoantibody variable region peptides derived from monoclonal anti-PR-3 antibodies from WG may actually serve as T-cell epitopes capable of augmenting humoral immune responsiveness through T-cell help, or otherwise influencing immune mechanisms regulating the course of disease.

Materials and methods

Wegener's granulomatosis and microscopic polyangiitis patients

Twenty-one patients (ages 27–79) with well-documented C-ANCA-positive, WG or diffuse MPA were the main patient population studied. Nineteen patients had WG confirmed by bronchopulmonary, sinus or renal biopsy and two showed a clinical picture of MPA with arthralgias, peripheral mononeuritis multiplex and systemic illness. Most patients studied were taking moderate doses of corticosteroids (5–10 mg of prednisone/day) and varying amounts (25–200 mg) of cyclophosphamide/day. At the same time each WG or MPA patient was studied, a similar blood sample was collected from a healthy normal donor ages 22–70 and lymphocytes isolated in parallel by Ficoll-Paque ® centrifugation. Blood sample collection and lymphocyte isolation were performed at the same intervals for patients and normal controls studied together.

Stimulation of PBMC from other autoimmune disease patients

Twenty-three additional patients with a variety of other rheumatic diseases were also studied for lymphocyte stimulation using the same sets of VH-region peptides employed with the MPA or WG patients. Patient disease controls included 15 with active rheumatoid arthritis (RA) (ages 36–75), and one each with clinically active reactive arthritis (age 49), scleroderma–CREST syndrome (age 74) and osteoarthritis with gout (age 66), and three with active systemic lupus erythemotosus (SLE), one with Bechets disease, one with temporal arteritis, and one patient with Churg–Strauss syndrome with eosinophilic vasculitis but negative PR3-ANCA test. All RA control patients studied were receiving methotrexate and low-dose prednisone. All disease control patients studied showed negative PR3-ANCA assays. Control patients with reactive arthritis, SLE, Bechets and temporal arthritis were receiving 5–20 mg of prednisone per day. The patient with gout and osteoarthritis received no therapy and the patient with Churg-Strauss was untreated.

Isolation of peripheral blood mononuclear leucocytes and proliferation assays

For the isolation of PBMC, phosphate buffered saline pH 7·4 (GIBCO BRL, Life Technology, N.Y.), RPMI-1640 Medium (GIBCO BRL), and Ficoll Paque® (Pharmacia Biotech, Uppsala, Sweden) were used. For the cell cultures RPMI with 10% fetal calf serum added (FCS) was used. Pokeweed mitogen (PWM) and Ionomycin (Iono) (Sigma Chemical Co., St. Louis, MO) were both used as 0·5 µm solutions, each of them by adding 25 µl to each microwell containing isolated lymphocytes. Eight × 104 cells were cultured in 0·2 ml RPMI/FCS with or without stimulants (3·75 nmole peptides, or 25μl 0·5μm PWM plus 25μl 0·5μm lono) in flat bottom 96-well microtitre plates (Costar, Cambridge, MA) for four days at 37°C in a humidified incubator containing 6% CO2. All tests were performed in triplicate or duplicate if not enough cells were available for triplicate study. Sixteen hours before harvesting the cells, 1 mCi of Thymidine (3H) (New England Nuclear, Life Science Products, Inc., Boston, MA) was added to each microwell. Cells were harvested onto glass fibre filters (Inotech Biosystems International, Lansing, MI) and counted in a liquid scintillation counter.

Final results (stimulation ratios) were derived from the mean counts of the two closest values (cpm) of the triplicates stimulated with the same peptide, divided by the mean cpm without peptide stimulation. The stimulation index (SI) was defined as the stimulation ratio for the patient divided by the stimulation ratio of the parallel healthy control for each peptide. This procedure compared the relative stimulation of the patients' PBMC to stimulation of the healthy control by the peptides. In all instances when the conventional stimulation index was calculated comparing thymidine incorporation of cells alone with that of cells cultured with peptide, normal controls showed no evidence for stimulation. Using this comparison to a normal subject with lymphocyte stimulation and each peptide studied, a stimulation index of two was set as positive and a value of three or over, considered strongly positive. In virtually all instances the counts/min recorded for thymidine incorporation using cultured cells alone from patients or controls were low varying between 200 and 1000 cpm (mean value of 223 ± 80). By contrast, cells from patients or controls stimulated with mitogen (PWM + Iono) showed extremely high stimulation responses. Thirty-one normal subjects stimulated with mitogens (Iono plus PWM) showed a mean stimulation of 61 718 cpm ± 23 300. Twenty-one WG/MPA patients showed a mean mitogen stimulation of 47 791 cpm ± 18 650.

Identification of lymphocyte subpopulations proliferating after peptide stimulation

An attempt was made to determine what T-cell population was proliferating after exposure to test peptides by studying T-cell subsets (CD3 +, CD4 + and CD8 + cell) proportions before and after positive lymphocyte proliferation and positive thymidine (3H) incorporation after four days of incubation with test VH-region peptides. Baseline whole blood lymphocytes isolated by Ficoll-Paque® gradient from a WG patient in parallel with cells from a normal control subject were examined by flow cytometry for CD3, CD4 and CD8 positivity as well as activated blast cells generally segregating as larger cells. Five days following in-vitro WGH-1 VH region peptide stimulation, lymphocytes derived from wells showing high stimulation indices were examined by flow cytometry for CD3, CD4 and CD8 in parallel with unproliferated cells from the normal control subject. Cells examined from stimulated lymphocyte wells were harvested to include adherent cells as well. This experiment was repeated once with the same WG patient and control 10 days later with virtually identical results.

Strategy for identification of T-cell epitopes

T-cell stimulating epitopes were selected from amphipathic portions of the VH-region sequence of WGH-1 a monoclonal human IgM anti-PR3 antibody produced by a B-cell line derived from peripheral blood lymphocytes of an active WG patient [8–10], as well as three VH sequences recently reported by Sibilia et al. [11]. The VH sequences were arbitrarily chosen as the most likely source of T-cell stimulating epitopes and are shown in Fig. 1a,b. These VH sequences were next examined using several computer programs designed to help identify likely T-cell epitopes based on the occurrence of typical amphipathic motifs characterized by presence of strongly hydrophobic regions followed by appropriately spaced hydrophilic residues in characteristic alignment so as to satisfy the alpha-helical arrangement emphasized by Berzofsky et al. [12–14].

Fig. 1.

Fig. 1

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(a) The heavy chain variable region for monoclonal human IgM anti-PR3 antibody WGH-1 [10] is shown as framework and CDR regions. Portions of VH sequence containing highly amphipathic scores are underlined and the residues of greatest amphipathicity shown in bold type. Control 11-mer peptides tested in parallel are shown below the VH WGH-1 sequence. (b) Primary VH region sequences for three additional monoclonal human IgM anti-PR3 antibodies reported by Sibilia et al. [11] are shown as WG1, WG2, and WG3. In similar fashion to WGH-1 above, again regions containing sequence motifs of high amphipathic score were selected for overlapping peptide synthesis and are underlined. The residues showing highest amphipathic scores are represented in bold type. Control peptides are shown below.

The putative T-cell epitopes within the VH region of mAb WGH-1, as well as those from the VH region sequences reported by Sibilia et al. [11] were then synthesized as 11-mer peptides using the pin synthesis method previously described by Maeji et al. [15]. This method has previously been employed to identify T-cell determinants within VH-regions of anti-DNA antibodies in murine lupus [16,17]. The peptides synthesized representing overlapping peptides of 11 amino acids containing and surrounding the putative amphipathic T-cell epitopes are shown in Fig. 1a,b. Additional control peptides were synthesized which included amino acid sequences completely different from the hydrophobic/hydrophilic motifs employed as potential T-cell stimulators. None of these control peptides showed any amphipathic score.

We also selected a set of two amphipathic peptides and four nonamphipathic peptides from a panel of 24 peptides ranging between seven and 24 residues which we were available from previous studies. These additional control peptides are shown in Table 1 and were also studied for stimulation of WG, RA, SLE, MPA patients and normal control lymphocytes.

Table 1.

Additional control peptides employed in lymphocyte stimulation studies

Peptide designation Souce Actual amino acid sequence Amphipathic score
PHS-2 analogue Shigella CRQTDRHSESY 4·4*
WLMS 12 V region of mouse Kappa chain [18] YCQQYSKLPRTF 7·4–9·2
WLMS 16 Herpes virus Fc receptor protein gE ASTWTSRLA 0
WLMS 17B Human IgG Cγ3 residues 439–445 KSLSLSP 0
WLMS 18 Human B2 microglobulin LSQPKIVKWR 0
WLMS 20A Human IgG (Cγ3) EGLHNHY 0
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*

Amphipathic score derived from algorithm and computer procedure described in [13].

Statistical analysis

Average stimulation for the two sets of peptides studied (WGH-1 and WG-1, −2 and −3) as well as the two sets of control peptides were analysed using the Wilcoxon signed rank test. Repeated measures analysis of variance was also used on the logarithm of the stimulation indices of the various groups studied.

Results

Results of peripheral blood lymphocyte stimulation experiments in 17 WG or MPA patients showed positive PBMC stimulation with 14 of 17 patients studied in 19 different experiments. Two patients were serially studied with the WGH-1 peptides during two periods of their illness. Several other patients were studied with the WGH-1 peptides and later with the second set of VH peptides [11]. Composite experimental results with the WGH-1 peptides are shown in Fig. 2. Only two patients with untreated active disease were included in this initial study. Nevertheless, a majority of the patients studied showed various degrees of positive lymphocyte activation often with multiple individual peptides derived from the parent VH WGH-1 sequence. Of particular interest were results obtained with a previously untreated WG patient who presented with weight loss, unilateral conjunctivitis, sensory neuropathy, proteinuria and rapidly progressive renal failure accompanied by strongly positive serum PR3-ANCA assay. Renal biopsy performed a few days before the lymphocyte stimulation assay, showed extensive crescentic glomerulonephritis and granulomatous changes typical of florid WG. Surprisingly, it can be seen that no lymphocyte stimulation was recorded with multiple VH-region peptides, despite his critical clinical condition. However, when this patient was studied five months later after immunosuppressive treatment had induced some clinical improvement, marked stimulation with seven amphipathic peptides was recorded.

Fig. 2.

Fig. 2

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A separate colour was used for each patient studied in the Wegener's granulomatosis (WG) and the disease control (Control) group. The peptide sequences used for the stimulation experiments are shown and the control peptides indicated by *. The values of the stimulation indices are shown as the height of the vertical bars. The WG group showed more stimulation in relation to normal controls compared to the disease control group as demonstrated in the figure by the high bars in the WG group, although there was no statistical significance between the disease control and WG groups. Stimulation by neighbouring peptides was observed in several patients suggesting some element of epitope spreading.

T cell stimulation also did not always appear to correlate with estimates of a patient's degree of clinical disease activity. Thus two patients considered still to have slight disease activity showed stimulation with only one or two peptides. On the contrary, five patients clinically judged to have no disease activity showed positive lymphocyte responses to 4–17 different VH-region peptides. Two patients also judged to have mild residual clinical disease activity showed considerable lymphocyte stimulation with a broad range of 5–9 amphipathic peptides.

Two patients were studied serially using the WGH-1 peptides during the evolution of their disease. One was studied initially when her disease was still clinically active with mononeuritis multiplex, arthritis and positive PR3-ANCA. At this juncture, she showed strong lymphocyte stimulation with five amphipathic VH-region peptides. However, several months later with better apparent clinical control of her disease following initiation of cyclophosphamide treatment, her repeat lymphocyte stimulation studies were even more positive than before showing stimulation with 19 of 24 VH-region peptides. By contrast the other WG patient who was studied before any therapeutic immunosuppression showed one of the highest levels of absolute lymphocyte stimulation seen throughout the course of the study with stimulation indices 20–43 fold greater than the normal controls studied at the same time.

If a stimulation index of 2·0 was considered positive when results with each WGH-1 peptide were compared to parallel results obtained with a normal donor run at the same time, then 16 of 19 experiments with WG patients showed positive results. If a stimulation index of at least 3·0 was considered positive, then 14 of 19 experiments with WG patients were positive. Of particular interest was the broad number of peptides from the WGH-1 sequence which induced lymphocyte stimulation. Eight patients showed significant stimulation with many WGH-1 amphipathic regions, whereas four other patients showed very little in the way of significant broad stretches of peptide sequence stimulation. Results using the same WGH-1 peptides with the disease control group were also of interest. A substantial proportion of these 23 individuals showed significant lymphocyte stimulation with test WGH-1 peptides. Most of the positive lymphocyte stimulation was recorded in the patients with rheumatoid arthritis. Peptide-stimulation of at least half of the disease control population of 23 was recorded with 5 of the 24 test WGH-1 peptides. However, stimulation indices were 3–4 times higher in WG/MPA patients than in disease controls. Although some positive lymphocyte stimulation was noted with the 4 control peptides, generally this was recorded only in a minor proportion of experiments with the WG patients and disease controls. None of the 23 disease control patients studied showed positive stimulations with other amphipathic peptides not derived from anti-PR3 VH region sequences (Table 1) or the 4 nonamphipathic control peptides studied.

Studies with additional amphipathic peptides from WG-1, WG-2 and WG-3 monoclonal human anti-PR3 antibodies

Results with the second set of amphipathic peptides derived from the sequences reported by Sibilia et al. [11] also showed positive stimulation. In many instances the WG or MPA patients represented the same group as had been previously studied with the first set of amphipathic WGH-1 peptides and in four instances patients studied with the second set of amphipathic VH-region peptides were subjects representing entirely different new patients presenting to clinic or admitted for definitive therapy. Lymphocyte stimulation studies performed with this second set of peptides are shown in Fig. 3. Of great interest was the overall strongly positive stimulation results seen with WG/MPA when compared always in each separate experiment with a normal control. Results showed that if a stimulation index of 2 is taken as a positive result, then 17 of 19 experiments of lymphocyte stimulation were positive in the Wegener/MPA group. If a stimulation index of 3 fold was considered a positive result, then 15 of 19 experiments with the WG group were positive. Again, several patients considered to be either slightly active or in long-term remission showed positive peptide lymphocyte stimulation. A number of the WG patients were studied first for stimulation with the WGH-1 peptides and later in the patients' course with the second set of WG-1, WG-2, or WG-3 VH peptides. When results were compared in 14 patients who were tested individually first with WGH-1 peptides and subsequently with the second set of WG-1, −2, or −3 peptides, similar positive stimulation results were recorded in 12 of 14 patients, whereas 2 WG/MPA patients who initially showed positive stimulation with the WGH-1 peptides were negative using the second WG-1, WG-2 or WG-3 panel. Several patients who showed broad patterns of lymphocyte stimulation with the first WGH-1 amphipathic peptides also showed the same broadly spread reaction pattern with the second set of peptides. Four entirely new patients were studied with the second WG-1, −2 and −3 peptides. Patient R showed no lymphocyte stimulation and was clinically inactive, taking no immunosuppressive drugs. The remaining 3 patients all showed clinical evidence of active disease and varying degrees of positive peptide lymphocyte stimulation. Control nonamphipathic peptides showed positive stimulation in 4 of 19 experiments with WG patients.

Fig. 3.

Fig. 3

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A separate colour was used for each patient studied in the Wegener's granulomatosis (WG) and the disease control (Control) group. The peptide sequences used for the stimulation experiments are shown and the control peptides indicated by *. The values of the stimulation indices are shown as the height of the vertical bars. The WG group showed more stimulation in relation to normal controls compared to the disease control group as demonstrated in the figure by the high bars in the WG group, with a statistical significance between the two groups (P = 0·0104). Stimulation by neighbouring peptides was observed in several patients suggesting some element of epitope spreading.

When 9 additional connective tissue disease controls were also studied with the additional WG-1, −2, and −3 amphipathic peptides, 5 of 9 showed positive stimulation with 1–8 different peptides if a stimulation index of 2 fold was considered a positive result. Four of the 5 disease control patients showing positive stimulation had rheumatoid arthritis. However, if 3·0 was considered positive, only 2 of 9 disease control subjects showed positive stimulation. Of particular interest were the negative lymphocyte stimulation results recorded using the second set of WG VH peptides with disease control patient no. 9 with Churg–Strauss syndrome, who had extensive vasculitis but a negative PR3-ANCA test result. The relative increment in stimulation index recorded for WG/MPA patients compared to disease controls is clearly illustrated in Figs 2 and 3.

A parallel series of control experiments were also performed using lymphocyte stimulations in 5 additional WG patients in parallel with 6 normal controls and 8 disease controls – 6 with rheumatoid arthritis and 2 with SLE. In these separate expts 2 other strongly amphipathic peptides. CRQTDRHSESY and YCQQYSKLPRTF (Table 1) derived from non-Wegener related sequences as well as 4 other control nonamphipathic peptides were also tested with the 5 Wegener, 6 RA patients, 2 SLE and 6 additional normal control subjects. In no instance was positive lymphocyte stimulation recorded – either with non-Wegener related strongly amphipathic peptides or with the additional 4 control nonamphipathic peptides.

Statistical analysis of results

We compared the average stimulation observed using the WGH-1 peptides and WG/MPA patients with the average stimulation for the control peptides using the Wilcoxon signed rank test and found that the WGH-1 peptides showed a significantly higher stimulation relative to the normal controls than was observed with the control peptides (P = 0·0039). The results with the disease control group also showed a significant degree of lymphocyte stimulation by the WGH-1 peptides (P = 0·032) compared with the control nonamphipathic peptides, but not as highly significant as with the group of WG/MPA patients. WG/MPA patients compared with normal controls studied for stimulation by WG-1, WG-2 and WG-3 peptides showed greater average stimulation than with the control peptides (P = 0·0062). However, there was no significant difference between results with the WG-1, WG-2, and WG-3 peptides and the control peptides in the disease control group (P = 0·91). When a repeated measures analysis of variance was applied to the logarithm of the stimulation indices of each patient studied, we found a significant difference between the WG/MPA group and the disease control group stimulated with the WGH-1 peptides. These latter findings correlated well with the results found using the Wilcoxon signed rank-test.

Lymphocyte profiles before and after peptide incubation

Cells collected from proliferation assays were analysed by FACS for cell surface markers. A comparison of cell profiles for cells from a WG patient vs. a healthy control is presented in Fig. 4. Results obtained in 2 separate assays were essentially identical. Whereas there were no differences observed at day 5 between cell populations of the control subject following stimulation with either a stimulating or nonstimulating VH-region peptide, there was a major shift in the peptide-stimulated population from the WG patient where a decrease in the proportion of small lymphocytes with a concomitant increase in activated larger CD3 + CD4 + lymphocytes was recorded.

Fig. 4.

Fig. 4

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Flow cytometry results comparing a normal control with a Wegener's granulomatosis (WG) patient whose lymphocytes were stimulated by one amphipathic peptide (P +) but not by a different peptide (P−). Parallel results are shown using these same peptides (P +) and (P−) with cells from a normal control. Two separate experiments gave virtually identical results. Results shown on the left indicate no difference at day five in CD3+/CD4 + cell populations in the normal control when peptide (P +) (▪) or peptide (P−) (Inline graphic) was added (□ Day 0). Also no change in large (activated) T cells was observed after either (P +) or (P-) peptide was present. Results shown on the right side with the WG patient indicate a major difference at day 5 in the small lymphocyte population, where CD3+/CD4 + cells were reduced after (P +) stimulation, compared to (P−) stimulation. However, in the large cell population from the WG patient sample at day 5, the reverse was seen. After stimulation with (P +) there was a relative increase in CD3+/CD4 + cells compared to (P−) stimulation. When this experiment was repeated using the same WG patient and control 10 days later, results were virtually the same.

Discussion

The present study shows that amphipathic regions present in VH segments of monoclonal anti-PR3 antibodies can behave as activators of peripheral blood lymphocytes from patients with PR3-ANCA positive WG or diffuse MPA. The primary B cell product represented by the antibody with specificity for granule constituents of PMN's such as anti-PR-3 may provide an amplification loop or adjuvant to the destructive autoimmune inflammatory process by encompassing amphipathic segments which activate T-cell receptors – presumably on helper T-cells. Data presented in this report indicate a surprising level of T-cell reactivity to VH-region amphipathic segments among a large group of patients with WG or MPA. Alternatively, T-cell activation could be involved in anti-idiotypic control mechanisms. Surprisingly, little stimulation was recorded among normal control subjects studied in each experimental protocol. Since the amphipathic V-region peptides showed marked stimulation with WG patients and a few disease control subjects, but not with normals, these amphipathic peptides cannot be considered as nonspecific mitogens. Other connective tissue disease controls consisting mainly of patients with rheumatoid arthritis did show positive lymphocyte stimulation with varying sets of the same amphipathic peptides. Nevertheless, when the WG/MPA group was compared with the disease controls, a greater proportion of WG/MPA individuals showed more pronounced peptide lymphocyte activation than disease controls. Positive lymphocyte reactivity to VH amphipathic residues found in patients with rheumatoid arthritis may be related to previous reports of positive serum antibody with C-ANCA specificity among such a population group [19–22].

The lymphocyte reactivity to the amphipathic VH-region epitopes studied seemed to be relatively common among patients who were in relative disease remission and that lymphocyte reactivity did not always correlate with disease activity. This could point to the possibility that T-cell activation might be contributing to an anti-idiotypic control response. Lymphocyte stimulation to amphipathic peptides only became positive after potent immunosuppression had induced a remission. Analysis of our results in the WG/MPA patients indicates that positive lymphocyte reactivity is very common but did not correlate directly with concurrent assessments of disease activity. It must be emphasized that our clinical study was not ideal since only a few patients were studied before any immunosuppressive therapy and most of the patients studied here were receiving potent immunosuppressive regimens.

The precise requirements for amphipathicity necessary for T cell receptor engagement have previously been extensively analysed by Berzofsky et al. [12]. Amphipathicity relates to having hydrophilic residues and hydrophobic residues segregated in space on opposite sides of an alpha-helical structure. Previously it has been estimated that only about half of all crystallographically defined alpha-helices are amphipathic [23]. In this regard it has been suggested that the hydrophilic side of the amphipathic epitope may be more capable of encoding individual specificity through interaction with the highly specific T-cell receptor whereas the hydrophobic part of the amphipathic loop/helix might interact with the less specific binding region on Class II MHC molecules. However, in some instances the reverse situation might actually prevail [12]. A number of studies of this putative interaction appear to support the concept that immunodominant sites recognized by helper T cells often represent amphipathic structures [24–26]. Many of these studies have indicated that T-cell recognition required only short peptides and therefore was based only on primary or secondary structure and not tertiary folding of the native protein. The remarkably high frequency of positive lymphocyte reactivity to the amphipathic V-region peptides recorded during the present study cannot be directly related to HLA Class I or Class II specificity since extensive analyses of HLA correlations and WG or MPA in the past have shown no segregation.

A number of studies have shown that the actual tissue lesions in WG or MPA often show heavy infiltrates with T-cells [27–30], and therefore various T-cell subsets must be making a substantial functional contribution to local inflammatory reactions associated with these disorders. Several recent reports support the involvement of T-cells in WG. Ludviksson and coworkers [31] investigated the proliferative response as well as the cytokine profiles of T-cells from WG patients. Increased proliferation was recorded with either PWA/lonomycin or anti-CD2 and anti-CD28 compared with normal donors. Peripheral blood mononuclear cells (PBMC) from WG patients showed increased secretion of IFN-γ, but not IL4, IL5 or IL-10. Moreover, INF-Y production by WG PBMC was inhibited in a dose-related manner by exogenous IL-10. WG patients also produced increased amounts of IL-12. It was suggested that overproduction of IFN-and TNF-alpha might be due to disregulated IL-12 secretion, and that IL-10 administration might serve to down-modulate the disease process [31]. Recently, T cell responsiveness in MPA has been reported by one group [32] to PR3 and myclo-peroxidase as well as to PR3 peptides. Moreover, a surprising series of reports by Molldrem et al. [33,34] indicated that cytotoxic T cells directed against a peptide from PR3 (amino acids 169–177) were HLA restricted to HLA A2, 1 and could inhibit PR3-expressing colony forming units of chronic myeloid leukaemia.

The strategy of using T-cell activation or alternatively T-cell receptor down-modulation in autoimmune disorders has recently come into some degree of focus. It can be demonstrated that oligomerization of T-cell epitopes can actually induce superactivation of an immune response [35] whereas tolerance induction through nasal administration of synthetic acetylcholine receptor T epitope sequences has also been demonstrated to prevent experimental myasthenia gravis in C57B1/B mice [36]. Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein [37]. These observations suggest that a balance between two different T-cell repertoires specific for the same or very closely related epitopes could determine resistance or susceptibility to development of an autoimmune disease. It now seems important to focus on the precise elements of T-cell reactivity in diseases such as WG with the ultimate goal of being able to induce tolerance to putative disease-specific T-cell epitopes and thus perhaps down-modulate disease manifestations by subverting self-destructive natural immune cascades.

Acknowledgments

Supported in part by The Norwegian Council of Research project no. 111159/320 (E.P.), a grant from the Florida Chapter of the Arthritis Foundation and by the Marcia Whitney Schott endowment to the University of Florida for Research in Rheumatic Diseases

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