In vitro cytokine production and proliferation of T cells from patients with anti-proteinase 3- and antimyeloperoxidase-associated vasculitis in response to proteinase 3 and myeloperoxidase.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 46, No. 7, July 2002, pp 1894–1904 DOI 10.1002/art.10384 © 2002, American College of Rheumatology In Vitro Cytokine Production and Proliferation of T Cells From Patients With Anti–Proteinase 3– and Antimyeloperoxidase-Associated Vasculitis, in Response to Proteinase 3 and Myeloperoxidase E. R. Popa, C. F. M. Franssen, P. C. Limburg, M. G. Huitema, C. G. M. Kallenberg, and J. W. Cohen Tervaert IL-10 and low production of IFN␥ in patients and controls. Conclusion. PR3 and MPO promote proliferation of CD4ⴙ T cells from patients with ANCA-associated vasculitides, but also cross-stimulate T cells from healthy individuals. Strong IL-10 production elicited by PR3 in vitro may act as an inhibitory signal for T cell proliferation and may have an important immunoregulatory function in vivo. Objective. To investigate in vitro proliferative responses of CD4ⴙ T cells and generation of specific cytokines induced by stimulation of peripheral blood mononuclear cells (PBMCs) from patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis with the autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO). Methods. PBMCs from vasculitis patients with PR3 ANCA or MPO ANCA and from healthy controls were stimulated for 7 days with PR3, MPO, or control stimuli. Proliferation of CD4ⴙ T cells was assessed by flow cytometry, using the proliferation marker Ki-67. Levels of the pro-proliferative cytokines interleukin-2 (IL-2) and IL-6 and of the Th1 and Th2 cytokines interferon-␥ (IFN␥) and IL-10 in culture supernatants were determined. Results. PR3 and MPO induced proliferative responses in CD4ⴙ T cells from individual patients with ANCA-associated vasculitides and healthy controls in vitro. Neither PR3 nor MPO elicited significant IL-2 production. Levels of IL-6 were highest after stimulation with PR3 but low after stimulation with MPO, independent of study group. Stimulation with PR3, and to a lesser extent with MPO, induced a Th2 cytokine milieu, characterized by high production of IL-6 and dation. Wegener’s granulomatosis (WG) and microscopic polyangiitis (MPA) are forms of pauci-immune necrotizing small-vessel vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs) (1–5). Whereas in WG the ANCAs are predominantly directed against proteinase 3 (PR3) (1,2,5), in MPA myeloperoxidase (MPO) is the main target for ANCAs (3–5). The clinical, i.e., diagnostic and prognostic, value of ANCAs and their potential pathophysiologic significance in these vasculitides have been investigated extensively (6–11), and results suggest that humoral autoimmunity plays a major role in these diseases (12). Moreover, the input of cellular immunity has been inferred from various findings, including the presence of T cells in granulomatous lesions of the upper and lower airways (13) and in the renal interstitium (14,15), increased T cell activation as reflected by cellular and soluble T cell activation markers (16–20), and responsiveness of disease activity to treatment with suppressors of cellular immunity, such as cyclosporin A and T cell–directed monoclonal antibodies (mAb) (21–23). Evidence of a role for the autoantigens PR3 and MPO has also begun to emerge. In vitro studies have suggested that both molecules are able to induce proliferation of peripheral blood mononuclear cells (PBMCs) Supported by grant C-97-1627 from the Dutch Kidney Foun- E. R. Popa, MSc, C. F. M. Franssen, MD, PhD, P. C. Limburg, PhD, M. G. Huitema, C. G. M. Kallenberg, MD, PhD, J. W. Cohen Tervaert, MD, PhD: University Hospital Groningen, Groningen, The Netherlands. Address correspondence and reprint requests to E. R. Popa, MSc, Department of Clinical Immunology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail: E.Popa@med.rug.nl. Submitted for publication July 2, 2001; accepted in revised form March 18, 2002. 1894 CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS from patients with PR3 ANCA– or MPO ANCA– associated vasculitis, but also from healthy controls (24–28). These findings were taken as evidence for the existence of autospecific T cells in the peripheral blood of vasculitis patients and, possibly, of healthy subjects. In vivo, increased serum levels of PR3 have been found in patients with active WG or polyarteritis nodosa (29), implying that this molecule is accessible for interaction with various cell types. One of these interactions is with endothelial cells, which have been shown to produce interleukin-8 (IL-8) (30) and monocyte chemoattractant protein 1 (31) upon in vitro incubation with PR3. These findings suggest that PR3, and possibly MPO, may exert functions other than antigenic stimulation of T cells. One of the functions that has not yet been investigated is the potential capacity of PR3 and MPO to modulate the cytokine milieu elicited by immunocompetent cells. We hypothesized that, besides acting as antigens for CD4⫹ T cells from patients with ANCA-associated vasculitis, the autoantigens PR3 and MPO can influence cytokine production by PBMCs in vitro. At the end of 7 days of in vitro stimulation of PBMCs with PR3, MPO, or control stimuli, expression of the nuclear proliferation marker Ki-67 in CD4⫹ T cells was assessed by flow cytometry, as an indicator of autoantigen-induced proliferation of CD4⫹ T cells. In addition, levels of the pro-proliferative cytokines IL-2 and IL-6 and of the Th1 and Th2 cytokines interferon-␥ (IFN␥) and IL-10 were determined. Implications of our findings with regard to the pathophysiology of these two ANCA-associated vasculitides are discussed. PATIENTS AND METHODS Patients and controls. Thirteen consecutive patients with WG (6 men, 7 women; median age 60 years, range 28–77 years), 7 consecutive patients with MPA (3 men, 4 women; median age 37 years, range 32–72 years), and 3 patients with idiopathic necrotizing crescentic glomerulonephritis (idiopathic NCGN) (1 man, 2 women; median age 70 years, range 41–78 years) were included in the study. Patients with WG fulfilled the American College of Rheumatology criteria for diagnosis of the disease (32). Patients with WG and MPA also fulfilled the definitions for those diseases as formulated by the Chapel Hill Consensus Conference (5). The diagnosis of idiopathic NCGN was based on renal biopsy showing focal or diffuse segmental NCGN and absence or paucity of immune deposits by immunofluorescence (2). In order to exclude T cell anergy due to immunosuppressive treatment or disease activity, only untreated patients whose disease was in complete remission were included. At the time of initial diagnosis and at the time of the in vitro tests, all patients had ANCA, as determined by indirect 1895 immunofluorescence and by enzyme-linked immunosorbent assay (ELISA) (2,33). Twelve WG patients had PR3 ANCA and 1 had MPO ANCA. All MPA patients, as well as all patients with idiopathic NCGN, had MPO ANCA. The median duration of disease was 6 years (range 1–22 years) in the PR3 ANCA patient group and 4 years (range 1–14 years) in the MPO ANCA patient group. The median time since the last period of active disease was 3.5 years for both groups (range 2.5–5 years in the PR3 ANCA group and 2.5–6 years in the MPO ANCA group). Extrarenal organ involvement was categorized as described previously (33). Patients had had 1–5 episodes of active disease prior to enrollment. Demographic and clinical characteristics of the patients are shown in Table 1. The control group consisted of 9 healthy volunteers (6 men, 3 women; median age 49 years, range 37–62 years). The study was approved by the Medical Ethical Committee Board at University Hospital Groningen. Antigens. PR3. PR3 was purified from human neutrophilic granulocytes, as previously described (7). The only modification consisted of disruption of polymorphonuclear cells (PMNs) by nitrogen cavitation at 4°C for 20 minutes at 350 psi in a nitrogen bomb (Parr Instruments, Moline, IL), in order to avoid lysis of PMNs by detergents. Purified PR3 was analyzed as described elsewhere (7). MPO. MPO was isolated and characterized according to a previously described protocol (7). To avoid a possible mitogenic and toxic influence of enzymatically active PR3 and MPO, both antigens were inactivated by heating for 15 minutes at 100°C. This procedure inhibited the enzyme activity of PR3 and MPO completely, as determined by using MeO-Suc-Ala-Ala-Pro-Val-pNa (Sigma, St. Louis, MO) as a substrate for PR3 and the guaiacol assay (5) for the measurement of MPO activity. Since T cells recognize linear epitopes and do not rely on antigen conformation, heat inactivation should not reduce the antigenicity of the proteins for antigen-specific T cell proliferation. All tests were performed with the same batch of purified PR3 and MPO. The PR3 and MPO preparations were tested for endotoxin by the Limulus assay (Bio-Whittaker, Vervier, Belgium). Endotoxin concentrations were ⬍0.5 units/ ml. Heat-inactivated PR3 and MPO at a concentration of 10 g/ml did not reduce the anti-CD3 mAb response in PBMCs from healthy controls and from a PR3 ANCA–positive patient (results not shown). This excludes the possibility of toxicity of the PR3 and MPO preparations at the concentrations used in our experiments. In vitro T cell stimulation. Blood was drawn in heparinized vacutainers (Becton Dickinson, Eerembodegem-Aalst, Belgium), and PBMCs were isolated by Lymphoprep density centrifugation (Nycomed, Oslo, Norway). Proliferation assays were performed in duplicate, in sterile, 5-ml, loosely capped tubes (Falcon; Becton Dickinson). PBMCs (1 ⫻ 106) were stimulated in a volume of 1 ml containing a final concentration of 15% normal human pooled serum, 10% CD28 (mAb) (supernatant of clone 20–4996; Central Laboratory of the Blood Transfusion Service [CLB], Amsterdam, The Netherlands) as a costimulatory factor, and 60 mg/liter gentamycin (Gibco Life Technologies, Paisley, UK) in RPMI (Gibco Life Technologies). Stimuli were added to this basic medium. Stimuli were either PR3 (final concentration 10 g/ml), MPO (final concentration 5.0 g/ml), a cocktail of recall antigens 1896 POPA ET AL Table 1. Demographic data of the vasculitis patients* Patient Age/sex Disease type† 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 70/M 28/F 50/M 60/M 73/M 66/F 32/F 60/F 66/F 52/M 63/F 45/M 32/F 70/M 41/F 65/M 32/F 72/M 70/F 77/F 37/F 78/M 36/F WG WG WG WG WG WG WG WG WG WG WG WG MPA MPA INCGN MPA MPA MPA INCGN WG MPA INCGN MPA Organ involvement† K, K, K K, K, K, K, K, K, K, K, K, K, K, K K, K, K, K K, K, K K, ENT, L ENT, S ENT, L, E ENT, E, CNS L E, L ENT, E L, CNS ENT, L E, S, CNS ENT ENT CNS L L L ENT ENT L Years of disease ANCA No. of relapses‡ 6 9 7 2 2 1 4 3 6 22 11 13 1 4 2 3 4 5 4 8 5 7 14 PR3 PR3 PR3 PR3 PR3 PR3 PR3 PR3 PR3 PR3 PR3 PR3 MPO MPO MPO MPO MPO MPO MPO MPO MPO MPO MPO 3 4 1 1 2 1 2 1 1 3 2 5 1 1 2 1 1 1 1 2 1 1 2 * ANCA ⫽ antineutrophil cytoplasmic antibody; WG ⫽ Wegener’s granulomatosis; K ⫽ kidney; ENT ⫽ ears, nose, throat; L ⫽ lung; PR3 ⫽ proteinase 3; S ⫽ skin; E ⫽ eyes; CNS ⫽ central nervous system; MPA ⫽ microscopic polyangiitis; MPO ⫽ myeloperoxidase; INCGN ⫽ idiopathic necrotizing crescentic glomerulonephritis. † At first presentation. ‡ Number of relapses (including first presentation) before enrollment in the study. (tetanus toxoid and diphtheria toxoid at final concentrations of 15 limes flocculates/ml; RVMI, Bilthoven, The Netherlands), or 10% CD3 mAb (supernatant from clone WT-32). Stimulation with PR3 at 1 g and MPO at 0.5 g was also performed. However, since at these concentrations T cell proliferation and cytokine production were low-to-undetectable, 10 g/ml PR3 and 5 g/ml MPO were used to obtain the present data. PBMCs were cultured for 7 days in an incubator containing 5% CO2. At the end of the culture period, cells were spun down (5 minutes at 300g), and supernatants were collected and stored at ⫺20°C. The culture supernatants were tested for endotoxin by the Limulus assay. Endotoxin concentrations were ⬍0.5 units/ml. Flow cytometric assessment of T cell proliferation. Cells were collected from cultures by centrifugation (see above). The cells were washed and incubated in fluorescenceactivated cell sorter permeabilizing solution for 10 minutes at room temperature, according to the instructions of the manufacturer (Becton Dickinson). After washing, cells were incubated with directly conjugated mAb against the T cell markers CD3 (allophycocyanin; Becton Dickinson), CD4 (cyanine 5.18–rhomadine–phycoerythrin; Immuno Quality Products, Groningen, The Netherlands), and the proliferation marker Ki-67 (fluorescein isothiocyanate; Coulter Immunotech, Mi- ami, FL) in the presence of 5% normal human serum for 30 minutes in the dark. After washing, the cells were fixed for 10 minutes in 0.5% paraformaldehyde, washed, and resuspended in phosphate buffered saline (PBS) for analysis on a Coulter Immunotech Elite flow cytometer. Flow cytometric data were analyzed with WinList software (Verity; Software House, Topsham, ME). Results were expressed as the percentage Ki-67⫹ T cells in the CD4⫹ T cell population, with background values subtracted. Cytokine assays. All cytokine assays were performed on culture supernatants obtained 7 days after treatment with various stimuli. This time point was chosen because maximal T cell proliferation was observed, allowing correlation between proliferation and cytokine production data. IL-2. IL-2 concentrations in culture supernatants were assessed by means of a cytotoxic T lymphocyte (CTL) proliferation assay. Briefly, 50 l of culture supernatant was added to 5 ⫻ 104 CTLs in a Costar 3790 tissue culture plate (Costar, Badhoeverdorp, The Netherlands) and incubated for 2 days at 37°C. Subsequently, 0.5 Ci/well 3H-thymidine was added, and the cells allowed to incubate for 16 hours at 37°C. The cultures were subsequently harvested on glass-fiber paper, and incorporation of 3H-thymidine was determined by beta-scintillation counting. Results are expressed as disintegrations per second with background values subtracted or as stimulation indexes, CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS 1897 Figure 1. Proliferative response of CD4⫹ T cells from vasculitis patients with proteinase 3 antineutrophil cytoplasmic antibody (PR3 ANCA), vasculitis patients with myeloperoxidase (MPO) ANCA, and healthy controls (ctrls.), after stimulation with anti-CD3/anti-CD28 (a), recall antigens tetanus toxoid (t.t.) and diphtheria toxoid (d.t.) (b), PR3 (c), or MPO (d). Proliferative responses were measured after a culture period of 7 days. For each patient, the value obtained with unstimulated culture was subtracted from the value obtained after addition of one of the stimuli. Horizontal lines indicate median values; circled symbols indicate patients with significantly increased T cell proliferative responses. calculated by the ratio between the test values and background values. IL-6, IL-10, and IFN␥. IL-6, IL-10, and IFN␥ were measured by ELISA. Microtiter plates (Costar 9018), coating buffer (0.1M carbonate buffer, pH 9.6), blocking buffer (0.01M PBS, 2% bovine serum albumin, 0.05% Tween 20), incubation buffer (0.01M PBS, 0.05% Tween 20, 0.2% gelatin), washing buffer (0.025M Tris HCl, 0.15M NaCl, 0.05% Tween 20), detection substrate (TMB; Braunschwig Chemie, Amsterdam, The Netherlands), substrate reaction buffer (0.1M acetate buffer, pH 6.0), and stopping solution (2N H2SO4) were used for all ELISAs. Appropriate calibration dilutions were performed. The microtiter plates were coated overnight at room temperature. Cytokine-specific mAb used for coating were CLB.MIL6/16 (IL-6; CLB) and rat anti-human IL-10 (Becton Dickinson). IFN␥ was measured using the PeliKine Compact human IFN␥ ELISA kit, according to the instructions of the supplier (CLB). After incubation of the plates with appropriate dilu- tions of culture supernatants containing the cytokines of interest, detection antibodies in the appropriate dilutions were applied. These detection antibodies were sheep anti-human IL-6–biotin polyclonal antibody (CLB.SIL6-D; CLB), rat antihuman IL-10 mAb–biotin 18562 (IL-10; PharMingen), and the detection antibody provided in the IFN␥ detection kit. After appropriate incubation and washing steps, bound conjugates were detected with appropriately diluted streptavidin-labeled horseradish peroxidase (CLB). Optical densities were measured at 450–575 nm, using SOFTMAX software. For statistical analyses, values from unstimulated cultures for each patient were subtracted from values obtained from culture with various stimuli. Statistical analysis. Data are presented as the medians. Differences in Ki-67 expression, disintegrations per second, stimulation indices, and supernatant cytokine concentrations between PR3 ANCA– and MPO ANCA–positive patients and controls were tested by Mann-Whitney U test. Two-tailed P values less than 0.05 were considered significant. 1898 POPA ET AL Figure 2. Cytokine production in relation to autoantigen concentration. Peripheral blood mononuclear cells from vasculitis patients with PR3 ANCA or MPO ANCA were stimulated with PR3 at 10 g/ml or 1 g/ml or with MPO at 5 g/ml or 0.5 g/ml, and production of interleukin-6 (IL-6) (a and b), IL-10 (c and d), and interferon-␥ (IFN␥) (e and f) was measured. IL-2 production was not detectable after stimulation with low-dose PR3 (1 g/ml) or MPO (0.5 g/ml). See Figure 1 for other definitions. Correlations between different parameters were tested with the Spearman’s rank test. Test outcomes for individual patients were considered significantly increased when they were more than 2 SD above the mean in the control group. RESULTS T cell proliferative response. We first studied the capacity of the autoantigens PR3 and MPO to induce specific proliferation of CD4⫹ T cells from patients with vasculitis. Anti-CD3 and the recall antigens tetanus toxoid and diphtheria toxoid were used to test the proliferative capacity of T cells from patients and control subjects. In the 2 patient groups as well as in controls, both stimuli elicited expression of Ki-67 in CD4⫹ T cells, demonstrating unimpaired proliferative potential of T cells in all 3 groups. Mitogenic stimulation resulted in comparable percentages of Ki-67⫹,CD4⫹ cells in vasculitis patients and controls (Figure 1a). Upon stimulation with recall antigens, the percentage of Ki67⫹,CD4⫹ T cells was highest in patients with MPO ANCA (P ⫽ 0.027 versus patients with PR3 ANCA [Figure 1b]), but did not differ significantly between CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS 1899 Figure 3. Profiles of interleukin-2 (IL-2) (a) and IL-6 (b) production after in vitro stimulation of peripheral blood mononuclear cells from vasculitis patients and healthy controls for 7 days in the presence of various stimuli, measured by cytotoxic T lymphocyte proliferation assay (IL-2) and by enzyme-linked immunosorbent assay (IL-6). For IL-6, values obtained with unstimulated cultures were subtracted from values obtained after addition of the stimuli. Horizontal lines indicate median values. SI ⫽ stimulation index (see Figure 1 for other definitions). vasculitis patients and controls. There was no correlation between T cell proliferative response to recall antigens and age of the patients. Upon stimulation with PR3, a significant increase in the median percentage of Ki-67⫹,CD4⫹ cells, as compared with unstimulated cultures, was seen in patients with PR3 ANCA (P ⫽ 0.042) and healthy controls (P ⫽ 0.031), but not in patients with MPO ANCA. There were no significant differences in the percentage of Ki-67⫹,CD4⫹ cells between the various groups (Figure 1c). Two patients with PR3 ANCA and 1 patient with MPO ANCA showed significantly increased proliferative responses to PR3, defined as values more than 2 SD above the median in the control group (Figure 1c). Upon stimulation with MPO, proliferative responses of T cells from vasculitis patients and controls were not significantly higher than in the unstimulated cultures. No significant difference in the percentage of Ki-67⫹,CD4⫹ cells was found between the 3 groups (Figure 1d). One patient with MPO ANCA had a significantly higher response as compared with the con- trol group (Figure 1d). Notably, this patient also had an increased proliferative response upon stimulation with PR3. Effect of autoantigen concentration on cytokine production. To assess whether autoantigen concentration affected levels of cytokine production, PR3 was used in concentrations of either 10 g/ml or 1 g/ml, and MPO in concentrations of either 5 g/ml or 0.5 g/ml. Lower concentrations of PR3 and MPO yielded significantly lower–to-undetectable levels of IL-6, IL-10, IFN␥ (Figure 2), and IL-2 (data not shown). The Th1/Th2 balance (IL-10 versus IFN␥) was not altered when lower concentrations of the autoantigens were used. IL-2 and IL-6 production. One of the early consequences of antigen- and mitogen-induced stimulation of T cells is the production of IL-2 (34). Subsequently, IL-2 supports rapid proliferation of T cells originally activated by antigen (34). Therefore, we measured IL-2 concentration in the supernatants of PBMC cultures after stimulation for 7 days in vitro with the autoantigens PR3 and MPO or control stimuli, in order 1900 POPA ET AL Figure 4. Profiles of interleukin-10 (IL-10) (a) and interferon-␥ (IFN␥) (b) production after in vitro stimulation of peripheral blood mononuclear cells from vasculitis patients and healthy controls for 7 days in the presence of various stimuli, measured by enzyme-linked immunosorbent assay. Values obtained with unstimulated cultures were subtracted from values obtained after addition of the stimuli. Horizontal lines indicate median values. See Figure 1 for other definitions. to investigate 1) the effect of PR3 and MPO stimulation on IL-2 production and 2) differences in IL-2 induction by PR3 and MPO in vasculitis patients compared with controls. PR3 and MPO did not elicit significantly higher concentrations of IL-2 than were found after administration of other stimuli. Only anti-CD3 stimulation of PBMCs from patients with MPO ANCA yielded increased levels of IL-2 when compared with PR3 stimulation (Figure 3a). Comparison of the 3 study groups in terms of IL-2 production upon treatment with different stimuli showed that only stimulation with anti-CD3 yielded concentrations of IL-2 that were significantly higher in vasculitis patients than in healthy controls (P ⫽ 0.02, patients with PR3 ANCA versus controls; P ⫽ 0.01, patients with MPO ANCA versus controls). There was no correlation between the percentage of Ki-67⫹, CD4⫹ cells and IL-2 concentration in the same cultures, irrespective of the stimulus. IL-6 is involved in T cell activation and prolifer- ation (35) and acts in synergy with IL-2 by providing a second signal for the production of IL-2 (36) and by inducing expression of the IL-2 receptor (37,38). PBMCs from patients in all 3 study groups produced high amounts of IL-6, with all of the stimuli tested. In all 3 groups, stimulation with PR3 evoked the highest levels of IL-6. Concentrations of IL-6 were lower upon MPO stimulation (Figure 3b). When comparing vasculitis patients with healthy controls, we found no differences in IL-6 concentrations, irrespective of the stimulus. With none of the stimuli was there a correlation between the percentage of CD4⫹,Ki-67⫹ cells and IL-6 concentration in the same cultures. IL-10 and IFN␥ production. To investigate whether PR3 or MPO stimulation of PBMCs from vasculitis patients or controls results in a skewed cytokine pattern (Th1 versus Th2), we assessed the production of 2 prototypical Th1 and Th2 cytokines, IFN␥ and IL-10, respectively, in supernatants from PBMCs that CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS 1901 Figure 5. Th1/Th2 skewing in vitro. Peripheral blood mononuclear cells from vasculitis patients with PR3 ANCA (a–d), vasculitis patients with MPO ANCA (e–h), and healthy controls (i–l) were cultured for 7 days in the presence of various stimuli. Interleukin-10 (IL-10) and interferon-␥ (IFN␥) were measured by enzyme-linked immunosorbent assay. Values obtained with unstimulated cultures were subtracted from values obtained after addition of the stimuli. Horizontal lines indicate median values. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.005; ⴱⴱⴱ ⫽ P ⬍ 0.0005; ⴱⴱⴱⴱ ⫽ P ⬍ 0.0001. rel. units ⫽ relative units (see Figure 1 for other definitions). were cultured for 7 days in the presence of PR3, MPO, or control stimuli. Both in vasculitis patients and in healthy controls, stimulation with PR3 elicited the highest IL-10 production, whereas MPO induced low IL-10 production (Figure 4). Upon stimulation with the mitogen anti-CD3 or the recall antigens tetanus toxoid and diphtheria toxoid, IL-10 concentrations were lower than those induced by PR3 but higher than those induced by MPO (Figure 4). When comparing IL-10 production between vasculitis patients and healthy controls after addition of various stimuli, we found significantly higher production in the group of PR3 ANCA patients after treatment with anti-CD3, but not with other stimuli (P ⫽ 0.03). IFN␥ was virtually absent after 7 days in culture with PR3 or MPO (Figure 4). Mitogenic stimulation with anti-CD3 elicited the strongest IFN␥ production in all study groups (Figure 4). IFN␥ levels were low after stimulation with recall antigens (Figure 4). No differences in IFN␥ production between vasculitis patients 1902 POPA ET AL and healthy controls were observed, irrespective of the stimulus used. Subsequently, we investigated whether a preferential Th1 or Th2 response could be detected in cultures of PBMCs from vasculitis patients after administration of various stimuli. To this end, we compared IL-10 and IFN␥ production in supernatants of the same culture (Figure 5). Stimulation with both PR3 and MPO consistently elicited a Th2 cytokine milieu, characterized by high IL-10 and low IFN␥ production. Th2 skewing in response to stimulation with PR3 and MPO was detected in both groups of vasculitis patients, as well as in healthy controls. In response to anti-CD3, cytokine production in cultures from patients and controls was clearly skewed toward the Th1 type (Figure 5), marked by high IFN␥ production and low IL-10 production. No skewing of cytokine production was seen in any of the study groups in response to a combination of the classic antigens tetanus toxoid and diphtheria toxoid. DISCUSSION In the present study, we investigated the in vitro capacity of PR3 and MPO to induce proliferation of CD4⫹ T cells and to modulate cytokine patterns in PBMCs from patients with ANCA-associated vasculitis and healthy subjects. We studied ANCA-positive patients whose disease was in remission and was not being treated, in order to avoid skewing of our results by T cell anergy. We showed that CD4⫹ T helper cells from individual patients with ANCA-associated vasculitides proliferate in vitro upon stimulation with the vasculitisassociated autoantigens PR3 and MPO, as assessed by nuclear expression of the proliferation marker Ki-67 in CD4⫹ T cells. Stimulation with PR3 resulted in high production of IL-6 and IL-10 in vasculitis patients and controls. Furthermore, stimulation with PR3 and MPO, but not with the recall antigens tetanus toxoid and diphtheria toxoid, resulted in a Th2-skewed cytokine pattern, characterized by high IL-10 and low IFN␥ production, in all study groups. Mitogenic stimulation with anti-CD3 mAb induced a Th1-skewed cytokine response, marked by high IFN␥ and low IL-10 production. Since we were specifically interested in the proliferative responsiveness of CD4⫹ T helper cells, we used flow cytometric detection of the proliferation marker Ki-67 in the nuclei of CD3⫹,CD4⫹ T cells, as an indicator of proliferation. Expression of Ki-67 starts in the late G1 phase of the cell cycle and continues through the G2 and M phases (39,40), thus allowing detection not only of dividing cells, but also of cells that have been committed to, or completed, a division cycle. This is in contrast with the 3H-thymidine incorporation method, used previously to assess proliferation of PBMCs from vasculitis patients in response to autoantigens (25,28), which reflects cell division and postdivision events. In vasculitis patients as well as in controls, proliferative responses to PR3, but not to MPO, exceeded those found in unstimulated cultures. However, proliferative responses of cells from healthy individuals to these stimuli equaled those of cells from the patients with vasculitis. Since the proliferative capacity of T cells from vasculitis patients was assessed by stimulation with a mitogen (anti-CD3 mAb) and a combination of recall antigens (tetanus toxoid and diphtheria toxoid) and was found to be comparable with that of cells from healthy individuals, it is unlikely that our findings reflect hyporesponsiveness of T cells from vasculitis patients toward PR3 and MPO. More probably, both autoantigens can elicit a specific cross-stimulation of T cells. This notion is supported by the finding that individual healthy subjects showed strong T cell proliferative responses toward MPO. Specific responses of memory T cells toward PR3 and MPO are possibly reflected by the fact that T cells from 2 patients with PR3 ANCA and 1 patient with MPO ANCA showed significantly higher proliferative responses after stimulation with these autoantigens than did cells from healthy controls. After a stimulation period of 7 days, we found low concentrations of IL-2 in the culture supernatants. Whether this phenomenon was due to consumption of IL-2 or to T cell anergy related to previous immunosuppressive treatment is not clear, since we have not assessed the kinetics of cytokine production in vitro. Since the proliferative responses in vasculitis patients were comparable with those in healthy controls, irrespective of stimulus, we expect that low IL-2 levels were not attributable to anergy. Surprisingly, IL-6 production in response to all stimuli was high in all 3 study groups, although the highest IL-6 levels were provoked by stimulation with PR3 (up to a median of 10,000 relative units/ml). IL-6 acts in synergy with IL-2, providing a second signal for the production of IL-2 (36) and inducing expression of the IL-2 receptor (37,38). Thus, we expected an inverse relationship between IL-6 and IL-2 levels, reflecting consumption of IL-6 leading to increased IL-2 production. In vasculitis patients, of all stimuli used, the mitogen anti-CD3, but not PR3 or MPO, elicited IL-2 levels that were significantly higher than in healthy controls. Interestingly, anti-CD3 also yielded the (com- CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS paratively) lowest IL-6 levels, suggesting that IL-6 may have been used for IL-2 production. The fact that the proliferative response of T cells from vasculitis patients at the same time point did not differ significantly from that of healthy controls suggests that the peak proliferative response to this stimulus may have occurred earlier. Our findings of a skewed Th1/Th2 cytokine pattern induced by stimulation with PR3 or MPO in vasculitis patients and controls may aid in the interpretation of the proliferative response and the IL-2 and IL-6 production pattern. Stimulation with anti-CD3 elicited a clear-cut Th1 cytokine response dominated by the production of IFN␥, a result that is consistent with findings by Ludviksson et al in a study of patients with WG (41). As mentioned above, anti-CD3 also elicited high levels of IL-2. Stimulation with PR3 resulted in a marked Th2 response, governed by high production of IL-10 and little or no production of IFN␥. It should be noted that detection of the prototypic Th2 cytokine IL-4 is desirable in investigating the Th1/Th2 balance, but this requires very sensitive assays that allow assessment of this cytokine after longer-term cultures. Although PR3 also stimulated the highest production of IL-6, no marked IL-2 production or T cell proliferation was seen. These findings can be interpreted in light of the immunosuppressive and antiinflammatory effect of IL-10 (42). Thus, we speculate that in our in vitro model, high levels of IL-10, elicited by stimulation with PR3 and to a lesser extent with MPO, suppress proliferation of autoantigen-responsive T cells, even in the presence of the proliferation-stimulating cytokine IL-6. The question of whether these cytokines were produced by responsive T cells present in our culture system cannot be answered by this study. In vivo, however, the source of these cytokines after stimulation with PR3 may actually be less relevant than is their impact on regulation of the autoimmune responses. Further in vivo investigation of the effect of the autoantigens PR3 and MPO on cytokine production in ANCA-associated vasculitides may reveal new aspects of immune regulation in these autoimmune disorders. One interesting question may relate to the extent to which the difference in the capacity of PR3 and MPO to induce IL-10 production may explain the clinical differences observed between PR3 ANCA– and MPO ANCA–associated vasculitis (43). Moreover, new treatment possibilities, aiming at modulation of cytokine profiles in vivo, may be envisaged. 1903 ACKNOWLEDGMENTS The authors are grateful to Y. M. van der Geld and I. K. Bouwman for their assistance. REFERENCES 1. Van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es LA, et al. 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