Minke G. Huitema

Detection of autoantibodies against myeloid lysosomal enzymes: A useful adjunct to classification of patients with biopsy-proven necrotizing arteritis☆

PURPOSE Assessment of the value of determination of antineutrophil cytoplasmic antibodies (ANCA) and its specificities for classification of patients with biopsy-proven necrotizing arteritis. PATIENTS AND METHODS The serum samples of 28 consecutive patients with biopsy-proven vasculitis involving medium- and/or small-sized arteries were tested for ANCA by an indirect immunofluorescence technique, by neutrophil extract enzyme-linked immunosorbent assay (ELISA), and by catching ELISA. RESULTS Eight patients had Churg-Strauss syndrome; six had myeloperoxidase (MPO) antibodies, and in the other two patients, ANCA were not detected. Six patients had polyarteritis nodosa (PAN) limited to the skin and the musculoskeletal system; ANCA were not detected in these patients. Two patients had systemic PAN and both had MPO antibodies. The remaining 12 patients had overlapping clinical features of the different forms of vasculitis. Five patients had polyarteritis in combination with chronic nasal inflammation and glomerulonephritis compatible with Wegeners granulomatosis (WG) but without granulomas in the respiratory tract. All five patients had 29-kd serine protease antibodies. Two patients had polyarteritis in combination with nasal polyposis and asthma compatible with Churg-Strauss syndrome, but eosinophilia was not detected. Both patients had MPO antibodies. Three patients with unclassified granulomatous arteritis had either elastase antibodies or ANCA of unknown specificity. One patient with unclassified systemic vasculitis had 29-kd serine protease antibodies, and one patient with necrotizing arteritis of the bowel in combination with Schönlein-Henoch purpura was negative for ANCA. CONCLUSION Determination of ANCA and its specificities is a useful adjunct to the classification of patients with biopsy-proven necrotizing arteritis. Within the spectrum of idiopathic vasculitides, 29-kd serine protease antibodies are associated with WG, MPO antibodies are associated with Churg-Strauss syndrome and systemic PAN, and PAN limited to the skin and the musculoskeletal system is not associated with ANCA.

Serum markers of T cell activation in relapses of Wegener's granulomatosis

Levels of soluble IL‐2 receptor (sIL‐2R). soluble CD4 (sCD4) and CDS (sCD8) were measured by sandwich ELISA as markers for T cell activation in serial serum samples from 16 patients showing 18 histologically proven relapses of Wegeners granulomatosis (WG). Levels of sIL‐2R increased from 1065 U/ml (median, range 373–2345 U/ml) 6 months before the relapse to 1684 U/ml (median, range 486–3404 U/ml) at the moment of relapse for the whole group (P= 0.10). The eight major relapses showed a profound rise in sIL‐2R levels, from 1008 U/ml (median, range 686–1553 U/ml) 6 months before the relapse, to 1994 U/ml (median, range 1469–3404 U/ml) at the moment of relapse (P < 0.01). The levels of sIL‐2R at the moment of relapse were significantly higher at the eight major relapses than at the time of the 10 minor relapses (P<0.05). Minor relapses were not accompanied by a significant rise in sIL‐2R levels. Titres of antineutrophil cytoplasmic antibodies (ANCA) rose by two or more titresteps or from negative to positive in 15/18 patients during the 6 months period before the relapse. In all seven cases with both a rise of the ANCA titre and an at least 25% increase in sIL‐2R levels, the rise in ANCA preceded the rise in sIL‐2R by at least I month. The level of slL‐2R at the moment of relapse correlated with the level of C‐rcactive protein (P= 0.488, P<0.05) and with the disease activity score (r= 0.824, P<0.002). There were no significant changes in levels of sCD4 or sCD8. although the levels of sCD4 tended to be higher at the time of major relapses. We conclude that major relapses of Wegeners granulomatosis arc accompanied by systemic T cell activation. T cell activation, however, does not appear to precede the rise in ANCA titre.

Disturbed Th1, Th2, Th17 and T-reg balance in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease accompanied by disturbed T-cell homeostasis. Dysbalance of T-helper-cell (Th) subsets (Th1/Th2/Th17) and regulatory T-cells (T(regs)) is suggested to contribute to the pathogenesis of SLE. Recent reports suggest functional deviation of T(regs) in terms of producing IL-17A, a process that may be aberrant in SLE. Therefore, we analyzed these T-cell subsets in SLE to test the hypothesis that aberrant T-cell subset skewing is present in SLE-patients. We investigated simultaneously the intracellular cytokines IFN-γ, IL-4 and IL-17A in CD4(+)T-cells as well as in T(regs). Skewing of T-cell subsets towards Th17 cells was observed in SLE-patients. Although the proportion of T(regs) was similar between SLE-patients and healthy controls, the ability of T(regs) to express IFN-γ and IL17A was impaired in SLE-patients. Even in quiescent SLE-patients T-cell homeostasis is aberrant in terms of skewing towards IL-17 producing T-cells.

Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes

Aim: In this study, we employed chimeric human/mouse Proteinase 3 (PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice. Method: Rats and mice were immunised with recombinant human PR3 (HPR3), recombinant murine PR3 (mPR3), single chimeric human/mouse PR3 (HHm, HmH, mHH, mmH, mHm, Hmm) or pools of chimeric proteins. Antibodies to mPR3 and HPR3 were measured by ELISA. Antibodies to rat PR3 were determined by indirect immunofluorescence (IIF) on rat white blood cells. Urinalysis was performed by dipstick analysis. Kidney and lung tissue was obtained for pathological examination. Results: In mice, immunisation with the chimeric human/mouse PR3 Hmm led to an autoantibody response to mPR3. Rats immunised with the chimeric human/mouse PR3 Hmm, HmH and mmH, or a pool of the chimeric human/mouse PR3 proteins, produced antibodies selectively binding to rat granulocytes as detected by IIF. No gross pathological abnormalities could be detected in kidney or lungs of mice or rats immunised with chimeric human/mouse PR3. Conclusion: Immunisation with chimeric human/mouse proteins induces autoantibodies to PR3 in rats and mice. Chimeric proteins can be instrumental in developing experimental models for autoimmune diseases.

Coexpression of CD177 and membrane proteinase 3 on neutrophils in antineutrophil cytoplasmic autoantibody–associated systemic vasculitis: Anti–proteinase 3–mediated neutrophil activation is independent of the role of CD177-expressing neutrophils

OBJECTIVE Wegeners granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation. METHODS Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n=53), those with SLE (n=30), those with RA (n=26), and healthy controls (n=31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorhodamine assay. RESULTS Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor alpha, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177- neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils. CONCLUSION Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.

Serum levels of BAFF, but not APRIL, are increased after rituximab treatment in patients with primary Sjögren's syndrome: data from a placebo-controlled clinical trial

B cell depletion therapy with rituximab (RTX; 2 weekly infusions of 1000 mg, premedication: 100 mg prednisolone) in primary Sjogrens syndrome (pSS) patients is effective in reducing subjective and objective symptoms.1 As B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are important cytokines involved in B cell survival and activation, we assessed in pSS patients included in a double-blind, randomised, placebo-controlled trial1 the effects of RTX on serum BAFF and APRIL levels up to 48 weeks after RTX. Serum concentrations of BAFF and APRIL were measured by ELISA using kits from RD median 1277 pg/ml (range 907–3802 pg/ml)) compared with healthy controls (n=10; median 983 pg/ml (range 600–1564 pg/ml)); p<0.01; figure 1A). Also, baseline serum APRIL levels were significantly higher in pSS patients (median 15 098 pg/ml (range 1891–228 591 pg/ml)) than in healthy controls (median 1965 pg/ml (range 889–4567 pg/ml); p<0.05; …

Disturbed B cell homeostasis in newly diagnosed giant cell arteritis and polymyalgia rheumatica

Several lines of evidence indicate that B cells may be involved in the immunopathology of giant cell arteritis (GCA) and polymyalgia rheumatica (PMR). This study was undertaken to examine the distribution of defined B cell subsets, including effector B (Beff) cells and regulatory B (Breg) cells, in patients with GCA and patients with PMR before and after corticosteroid treatment.

Constitutive membrane expression of proteinase 3 (PR3) and neutrophil activation by anti‐PR3 antibodies

Antineutrophil cytoplasm autoantibodies with specificity for proteinase 3 (PR3) are thought to play a major role in the pathogenesis of Wegener’s granulomatosis (WG), presumably by their potential to activate neutrophils. In patients with WG, high expression of PR3 on the surface of nonprimed neutrophils is associated with an increased incidence and rate of relapse. In this study, we analyzed the functional significance of constitutive PR3 expression for neutrophil activation as induced by anti‐PR3 antibody. Therefore, primed and nonprimed neutrophils were stimulated with the monoclonal anti‐PR3 antibody PR3G‐3. Activation was measured as actin polymerization by the phalloidin assay as an early, detectable activation event and oxidative burst by the dihydrorhodamine assay, as a late, detectable activation event. In contrast to the oxidative burst, we found that anti‐PR3 antibody‐induced actin polymerization could be triggered in neutrophils without priming with tumor necrosis factor α (TNF‐α). In addition, a correlation was found between the level of PR3 expression on the surface of these nonprimed neutrophils and the degree of actin polymerization. However, after priming with TNF‐α, no correlation was found between membrane expression of PR3 and the level of actin polymerization or respiratory burst as induced by anti‐PR3 antibody. These data suggest that the presence of PR3 on the surface of nonprimed neutrophils has consequences for their susceptibility to the initial activation step by anti‐PR3 antibodies. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.

In vitro T lymphocyte responses to proteinase 3 (PR3) and linear peptides of PR3 in patients with Wegener's granulomatosis (WG)

T cell‐mediated immunity is thought to play an important role in the pathogenesis of WG. In previous studies a minority of WG patients as well as some healthy controls showed in vitro proliferation of their peripheral blood mononuclear cells (PBMC) to PR3, the main autoantigen in WG. The relevant peptides responsible for this in vitro proliferation have not been identified. In order to define immunogenic peptides, PBMC of 13 WG patients in remission and 10 healthy controls were tested for proliferation to linear peptides of PR3 and to whole PR3. Fifty overlapping peptides spanning the whole PR3 sequence were synthesized. Peptides were tested in pools of five peptides and as single peptide. PBMC of two WG patients and one healthy control proliferated to whole PR3 and to peptide pools. In addition, 10 WG patients and eight healthy controls that did not proliferate to whole PR3 did proliferate to pools of PR3 peptides. Although more WG patients tended to react to particular peptide pools, no significant difference was seen between lymphocyte proliferation to PR3 peptides of WG patients and that of healthy controls. The pools of peptides recognized were mainly located at the N‐ and C‐terminus of PR3. No correlation was observed between HLA type and proliferation on particular peptide pools. No proliferation of PBMC was observed to single peptides. In conclusion, T cells of WG patients proliferate in vitro more frequently to PR3 peptides than to the whole PR3 protein. Peptides derived from the signal sequence, the propeptide or peptides located at the C‐terminus of PR3 induce highest levels of proliferation. No specific PR3 sequence could be identified that was preferentially recognized by PBMC of WG patients compared with controls.

Plasma levels of soluble IL-2R, soluble CD30, IL-10 and BAFF during follow-up in PR3-ANCA-associated vasculitis: associations with disease activity and relapse

Objectives: To evaluate whether T cell activation, as reflected by levels of soluble interleukin 2 receptor (sIL2R), soluble CD30 (sCD30), IL-10 and B cell activator of the tumour necrosis factor family (BAFF) at diagnosis and during initial follow-up, is predictive for persistent or renewed antineutrophil cytoplasmic antibody (ANCA) positivity and clinical relapse in patients with vasculitis associated with proteinase 3-antineutrophil cytoplasmic antibodies (PR3-ANCA). Methods: 87 Patients with PR3-ANCA-associated vasculitis and at least 2 years of follow-up were included in the study. At diagnosis, and at 3, 6, 12, 18 and 24 months after diagnosis, cytoplasmic ANCA titres were detected by indirect immunofluorescence (IIF), and PR3-ANCA, sIL2R, sCD30, IL-10 and BAFF levels were assessed by ELISA. 31 healthy volunteers provided plasma samples for comparison. Levels of immune markers were related to ANCA positivity and relapse during follow-up. Results: Plasma levels of sIL2R, sCD30 and BAFF were higher in patients than in controls at all time points. Plasma levels of sIL2R, sCD30 and IL-10 were higher at diagnosis and relapse than during remission. At 18 months, sCD30 (p<0.001) and sIL2R levels (p = 0.01) were significantly higher in PR3-ANCA-positive patients (detected by ELISA) than in PR3-ANCA-negative patients. ANCA-positive patients detected by ELISA or IIF at 24 months had significantly higher plasma sCD30 levels (p = 0.02 and p = 0.03, respectively) than ANCA-negative patients. Conclusion: Increased T cell activation in patients with ANCA-associated vasculitis in remission during and after immunosuppressive treatment is associated with persistent or renewed ANCA positivity.