C. G. M. Kallenberg

Prediction of relapses in Wegener's granulomatosis by measurement of antineutrophil cytoplasmic antibody levels: a prospective study.

OBJECTIVE Prediction of relapses in Wegeners granulomatosis (WG) by measuring levels of antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) or myeloperoxidase (MPO) remains a controversial issue. To assess the value of serial quantification of ANCA by indirect immunofluorescence (IIF) and antigen-specific enzyme-linked immunosorbent assay (ELISA) for monitoring disease activity in patients with WG, a prospective observational study was conducted in patients with WG attending an outpatient clinic in the Netherlands. METHODS One hundred patients with WG (85 with PR3-ANCA, 15 with MPO-ANCA) were studied prospectively from 1996 to 1998. Serum samples were obtained and analyzed every 2 months for ANCA levels. Disease activity was prospectively assessed without knowledge of the ANCA levels. RESULTS Relapses occurred in 37 of 100 patients (37%). Thirty-four (92%) of the 37 patients showed a rise in the level of ANCA preceding their relapse, as detected by ELISA or IIF. The predictive value of an increase in ANCA titers for relapse was 57% (17 of 30) for cytoplasmic/classic ANCA (cANCA; by IIF), 71% (27 of 38) for PR3-ANCA (by ELISA), and 100% (3 of 3) for MPO-ANCA (by ELISA). The predictive value of a rise in ANCA as measured by ELISA or IIF did not substantially improve following concomitant measurement of the IgG3 subclass of PR3-ANCA. Forty-three percent of patients who showed a rise in cANCA (by IIF) and 29% with a rise in PR3-ANCA (by ELISA) did not subsequently experience a relapse. CONCLUSION Serial measurement of ANCA levels is valuable for the early prediction of relapses in patients with WG.

Prevention of relapses in Wegener's granulomatosis by treatment based on antineutrophil cytoplasmic antibody titre

58 patients with biopsy-proven Wegeners granulomatosis (WG) were prospectively screened for clinical evidence of the disease 3-monthly, with antineutrophil cytoplasmic antibody (ANCA) measurements every month. Over 24 months, ANCA rose in 20 patients, 9 of whom were randomly assigned to receive combined 9 and 3 month courses of cyclophosphamide and prednisolone, respectively, at the time of ANCA rise; and 11 patients who were untreated except if there was a clinical relapse. 6 of 11 untreated patients relapsed within 3 months of ANCA rise. 3 of the remaining 5 patients relapsed after 3 months. There were no early or late relapses in patients randomised to treatment. Patients receiving no treatment at the time of ANCA rise took more cyclophosphamide and prednisolone than patients who were treated. Side-effects did not significantly differ between the two groups.

Activation of granulocytes by anti‐neutrophil cytoplasmic antibodies (ANCA): a FcγRII‐dependent process

ANCA have been demonstrated to induce the respiratory burst in primed neutrophils. In this study we have extended the investigations on neutrophil activation by ANCA directed against proteinase 3 (PR3). Myeloperoxidase (MPO) and lactoferrin (LF), and we have analysed the underlying mechanisms. All three ANCA antigens were expressed on the cell surface of primed neutrophils. Superoxide production assayed by both cytochrome c reduction and oxidation of dihydrorhodamine 123, was induced by heterologous polyclonal anti‐MPO and anti‐LF antibodies, and ANCA‐positive plasma samples. Induction of superoxide production was dose‐dependent. F(ab)2 fragments did not induce the respiratory burst. Blockade of Fc receptors by specific oAbs showed that anti‐FcγRII antibodies were able to turn off the ANCA‐induced respiratory burst, whereas anti‐FcγRII antibodies did not. Plasma samples that induced the respiratory bursl did not differ from samples that did not induce superoxide production with respect to ANCA titre, but had higher levels of the lgG3 subclass of ANCA. Levels of the other subclasses of ANCA were comparable between those samples. We conclude that ANCA‐induced activation of primed neutrophils is FcγRII‐dependent, and appears to be facilitated by antibodies of the IgG3 subclass.

Levels of soluble VCAM-1, soluble ICAM-1, and soluble E-selectin during disease exacerbations in patients with systemic lupus erythematosus (SLE); a long term prospective study

Active SLE is characterized by immune deposits and subsequent vascular inflammation in many organs. Expression and up‐regulation of adhesion molecules is basic to migration of inflammatory cells into the tissues. Recently, soluble isoforms of these molecules have been described which might be an expression of their up‐regulation in the tissues and, as such, of disease activity. The purpose of this study was to evaluate whether changes in levels of soluble adhesion molecules reflect disease activity. We analysed serial sera in a 6‐month period preceding 22 consecutive exacerbations of SLE for levels of soluble vascular cell adhesion molecule‐l (sVCAM‐1). soluble intercellular adhesion molecule‐l (slCAM‐1), and sE‐selectin. Levels were related to clinical disease activity (SLEDAI). and levels of anti‐dsDNA and complement. At the time of maximal disease activity, levels of sVCAM‐1 in patients with SLE were higher than those in controls (P < 0.0001), levels in patients with renal involvement being higher than in those without (P < 0.02). Levels of sVCAM‐1 correlated with SLEDAI scores (P < 005) and, inversely, with levels of C3 (P = 0.01). In addition, in the presence of anti‐dsDNA, levels of sVCAM‐1 tended to correlate with levels of these autoantibodies (P < 0.1). Levels of sICAM‐1 were normal and sE‐selectin levels even decreased compared with controls. Levels of sVCAM‐l were higher at the moment of relapse (P = 0.001) than at 6 months before this time point. This rise correlated with the rise in SLEDAI score (P < 0.02). Levels of sICAM‐l and sE‐selectin did not rise, and remained in the normal range in all exacerbations studied. In conclusion, in contrast to sICAM‐1 and sE‐selectin, levels of sVCAM‐l are increased, rise parallel to disease activity during exacerbations in SLE, and are associated with decreasing levels of complement factors. This favours the hypothesis of immune deposit formation, activation of the complement cascade and activation of endothelial cells. Concurrent up‐regulation of vascular adhesion molecules may thus result in transmigration of activated inflammatory cells inducing tissue damage.

Relevance of classic anti-neutrophil cytoplasmic autoantibody (C-ANCA)-mediated inhibition of proteinase 3-α1-antitrypsin complexation to disease activity in Wegener's granulomatosis

In the sera of patients with Wegeners granulomatosis (WG), C‐ANCA can be detected that are directed against proteinase 3 (PR3). We have previously observed that C‐ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor, α1antitrypsin (α1AT). In the present study we investigated whether this inhibitory effect of C‐ANCA on PR3‐α1 AT complexation correlates with clinical activity of WG. Serial serum samples of eight consecutive patients with histologically proven relapses of WG were tested. At the moment of relapse all sera revealed inhibitory activity towards PR3‐α‐1 AT complexation (median 22%, range 10–59%). Disease activity score (r=0.87, P<0.02) and C‐rcactive protein (CRP) levels (r= 0.66. P<0.1) correlated with C‐ANCA inhibition of PR3‐α1 AT complexation, while they did not correlate with the C‐ANCA titre detected by indirect immunofluorcsccnce (IIF) nor with IgG anti‐PR3 antibody level measured by ELISA. The inhibitory effect of C‐ANCA on PR3‐α1 AT complexation had risen significantly at the moment of relapse compared with values 3 months (P<0.05) and 6 months (P<0.01) before relapse. Eight patients with established WG and positive for C‐ANCA but without clinical evidence of relapse served as controls. In this group no inhibitory effect of C‐ANCA on PR3‐α1AT complexation was observed in 7/8 patients sera. Sera of one control patient contained moderate C‐ANCA inhibitory activity towards PR3‐α1AT complexalion, which remained at a constant level during the 6 months period of observation. Thus, disease activity in WG appears to be more closely related to C‐ANCA inhibitory activity towards PR3‐α1AT complexation.

Autoimmunity to lysosomal enzymes: new clues to vasculitis and glomerulonephritis?

The demonstration of autoantibodies to neutrophil cytoplasmic antigens (ANCA) in (1) systemic vasculitides such as Wegeners granulomatosis, classic polyarteritis nodosa and the Churg Strauss syndrome and (2) idiopathic crescentic glomerulonephritis has placed these disorders within the spectrum of autoimmune diseases. The target antigens have been identified as myeloid lysosomal enzymes that are involved in tissue destruction by neutrophils. Recent data suggest a pathogenetic role for these autoimmune responses. The autoantibodies may activate neutrophils, resulting in extensive tissue necrosis. Alternatively, T-cell-mediated immunity to these positively-charged enzymes that localize to polyanionic basement membranes may, in concert with the presence of autoantibodies, result in glomerulonephritis and vasculitis.

Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener’s granulomatosis?

The majority of patients with Wegener’s granulomatosis (WG) are chronic nasal carriers of Staphylococcus aureus. Chronic nasal carriage of S. aureus is associated with an increased risk of developing a relapse of the disease. The mechanism by which this occurs is still unknown. We hypothesized that a cationic protein of S. aureus, staphylococcal acid phosphatase (SAcP), acts as a planted antigen and initiates glomerulonephritis and vasculitis in patients with WG. In order to test the hypothesis that SAcP can act as a planted antigen in WG, we studied the ability of SAcP to bind to human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells. We also studied whether this binding can be prevented by preincubation with an anionic protein, and whether binding of SAcP activates endothelial cells. We also evaluated whether antibodies in sera of patients with WG are able to bind to endothelial cell‐bound SAcP. The results show that SAcP can act as a planted antigen by binding to both types of endothelial cells in a concentration‐dependent manner. Binding of concentrations as low as 4 μg/ml can be detected on HUVEC within 5 min of incubation. Binding of SAcP to endothelial cells was charge‐dependent but did not activate endothelial cells. Finally, endothelial cell‐bound SAcP was recognized by sera of patients with WG. The data suggest a possible pathogenic role for SAcP by acting as a planted antigen thereby initiating glomerulonephritis and vasculitis in patients with WG.

European attempts to set guidelines for improving diagnostics of autoimmune rheumatic disorders

The rational way to set a diagnosis and estimate a prognosis in rheumatology is to start by setting a tentative diagnosis and then follow a fixed scheme for laboratory testing, eg, by using an agreed algorithm. The use of order algorithms can be extended to post-test algorithms that will assist clinicians in approaching the right diagnosis and prognosis. New methods used in autoimmune serology do not deliver results that can be directly compared to those of older methods, and thus the new methods need to be thoroughly tested with sera from differential diagnostically relevant disease controls to set a clinically meaningful cut-off for positivity. Borderline positive results need to be treated with special care to avoid misuse. Early diagnosis is of great importance, and serological results can be very useful if used the right way. European efforts to secure rational diagnostic work-up in autoimmune rheumatic disease have led to a better dialogue between clinicians and laboratory scientists in several countries.

Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis

Anti‐neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegeners granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3‐ANCA do not always correspond to clinical disease activity.

B cell activation in clinically quiescent systemic lupus erythematosus (SLE) is related to immunoglobulin levels, but not to levels of anti-dsDNA, nor to concurrent T cell activation

In clinically quiescent SLE hypergammaglobulinaemia, presence of autoantibodies, and increased soluble IL‐2 receptors (sIL‐2R) have been reported, suggesting persistent B as well as T cell activation. In contrast, the primary immune response lo test antigens is markedly decreased. To analyse these phenomena at a cellular level, we undertook a cross‐sectional study on 13 non‐active SLE patients and 15 controls. We determined the composition of lymphocyte subsets with special attention to activation markers (CD25, HLA‐DR, CD38) and the presence of naive T cells (CD45RO‐), and related those findings to serological parameters. In non‐active SLE patients the expression of activation markers on B cells and T cells was higher than in normal controls (P≤ 0·02), but was not interrelated. Percentages of activated B cells in SLB were related lo levels of total IgG (P < 0·02)and IgM (P < 0·02) but not to anti‐dsDNA, suggesting a disordered immune system also in clinically quiescent SLE. Numbers of CD4+ cells (P < 0·001) and CD4+CD45RO‐ cells (P < 0·05) were decreased. The latter finding might explain the anergy to primary test antigens in clinically quiescent SLE.