Gerda Horst

Predominance of IgG1 and IgG4 subclasses of anti-neutrophil cytoplasmic autoantibodies (ANCA) in patients with Wegener's granulomatosis and clinically related disorders.

In view of the supposed hypersensitivity, the elevated levels of IgE, and the occurrence of eosinophilia reported in Wegeners granulomatosis and related conditions, we studied the IgG subclass distribution of ANCA directed against a 29‐kD serine protease and myeloperoxidase (MPO) in 41 untreated ANCA‐positive patients with several forms of active vasculitis and/or glomerulonephritis. We found that both 29‐kD ANCA and MPO ANCA were predominantly of the IgG1 and IgG4 subclass in all groups of patients. The additional presence of IgG3 subclass was associated with renal involvement. We compared the subclass distribution of ANCA with that of total IgG subclass levels, and with the IgG subclass distribution of antibodies to cytomegalovirus (CMV) as a persistent endogenous antigen and antibodies to tetanus toxoid (TT) as an exogenous recall antigen. Total levels of IgG4 were elevated in the majority of the patients together with elevated IgG1 levels. Antibodies to CMV and TT, however, had the same subclass distribution as found in normals and did not show enhanced IgG4 expression. ANCA belong predominantly to the IgG1 and IgG4 subclass, which may suggest that the production of ANCA is related to recurrent exposition to the antigen(s) involved, possibly as part of a hypersensitivity reaction.

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.

Expression of costimulatory molecules on peripheral blood lymphocytes of patients with systemic lupus erythematosus

OBJECTIVE In systemic lupus erythematosus (SLE) autoantibody production is T cell dependent. For a proper T and B cell interaction, signalling of costimulatory molecules on these cells is necessary. The expression of costimulatory molecules on peripheral blood lymphocytes in patients with SLE in conjunction with disease activity was measured to evaluate whether expression of costimulatory molecules in SLE is increased. METHODS Thirteen patients with SLE with active disease, 10 patients with inactive disease, and 14 controls entered the study. In addition, samples from 10 of the 13 patients with active disease could be studied at a moment of inactive disease as well. Isolated peripheral blood lymphocytes were stained for the lymphocyte subset markers CD4, CD8, CD19, their respective activation markers CD25, HLA-DR, CD38, and the costimulatory molecules CD40L, CD28, CD40, CD80, and CD86. Expression was measured by flow cytometry. RESULTS Peripheral blood lymphocytes of patients with SLE showed signs of increased activation at the moment of active disease. Almost all CD4+ T cells expressed CD28, both in patients and in controls. CD80 expression on CD19+ B cells was low in both groups and did not correlate with disease activity. In contrast, the percentage of CD19+ B cells expressing CD86 was increased in patients with SLE even in patients with inactive disease (p=0.04) and correlated with the SLEDAI score (p=0.0005) and levels of anti-dsDNA (p=0.006). No changes in CD40 or CD40L expression were found in the patients with SLE. CONCLUSION In patients with SLE the expression of CD86 on CD19+ B cells is increased and is associated with disease activity, B cell activation, and levels of anti-dsDNA. The increased CD86 expression will render (autoreactive) B cells more susceptible for T cells. This can facilitate autoantibody production and might be a target for immunosuppressive treatments.

Anti‐neutrophil cytoplasmic antibodies (ANCA) in inflammatory bowel disease: characterization and clinical correlates

ANCA were detected by indirect immunofluorescence in 34 out of 67 patients with ulcerative colitis (UC, 51%) and in 14 out of 35 patients with Crobns disease (CD, 40%). All but one ANCA‐positive sera produced a perinuclear pattern of fluorescence (P‐ANCA) on ethanol‐fixed neutrophils. On paraformaldehyde‐fixed neutrophils 76% of P‐ANCA‐positive sera in UC and 50% of P‐ANCA‐positive sera in CD produced cytoplasmic fluorescenec, indicating that, indeed, cytoplasmie antigens are recognized by a considerable number of these sera. By Western blot analysis using whole neutrophil extract as a substrate 46% of sera from patients with UC and 32%, of sera from patients with CD showed reactivity with either lactolerrin, polypeptides occurring as a doublet of 66/67 kD mol. wt, or polypeptides occurring as a doublet of 63/54 kD mol. wt, respectively. Identical patterns of reactivity have been observed among P‐ANCA‐positive sera from patients with autoimmune liver disease and rheumatoid arthritis. These data suggest that ANCA of restricted specificities are not specific for inflammatory bowel disease (IBD), but are present in diverse conditions characterized by chronic idiopathie inflammation.

Catalase and alpha-enolase: two novel granulocyte autoantigens in inflammatory bowel disease (IBD)

In IBD, the target antigens of anti‐neutrophil cytoplasmic autoantibodies (ANCA) have not been fully identified, which limits the analysis of the diagnostic significance as well as of the possible pathophysiological role of these antibodies. In this study, we identify the target antigens of ANCA in large groups of patients with ulcerative colitis (UC) and Crohns disease (CD). Apart from antibodies against lactoferrin and bactericidal/permeability‐increasing protein (BPI), which have been reported before, antibodies against two novel granulocyte antigens were identified: antibodies against a 57/56‐kD doublet were found in 38% of samples from UC patients and in 26% of samples from CD patients, whereas antibodies against a 47‐kD protein were found in 10% of samples from UC patients and in 18% of samples from CD patients. Partial purification and amino acid sequence analysis identified the 57‐kD protein as catalase and the 47‐kD protein as α‐enolase. This study is the first to report catalase and α‐enolase as granulocyte antigens for autoantibodies in IBD.

Anti-dsDNA production coincides with concurrent B and T cell activation during development of active disease in systemic lupus erythematosus (SLE)

The objective was to serially analyse T and B cell activation in relation to autoantibody production during the development of relapses in SLE. In a prospective study we serially analysed, by flow cytometry, T cell activation in relation to B cell activation and anti‐dsDNA production in quiescent SLE and during the development of a clinical relapse. In addition, we related changes in T and B cell activation to changes in levels of anti‐dsDNA and total IgG. During periods with clinically quiescent disease, the expression of activation markers on T cells (IL‐2R and HLA‐DR) and B cells (CD38) was persistently higher in SLE than in healthy controls (P < 0.001). Percentages of CD20+CD38+ B cells were related to levels of total IgG (P < 0.02), but not to levels of anti‐dsDNA. Development of disease activity was paralleled by an increase in the percentages of CD4+ T cells (P < 0.005) and CD20+CD38+ B cells (P < 0.001), which were interrelated. Increases in B cell activation were related to increases in levels of anti‐dsDNA (P < 0.005), but not to changes in total IgG levels. B cells expressing high levels of CD38 spontaneously produced IgG class anti‐dsDNA in vitro. Persistence of activated B cells during periods with clinically quiescent disease in SLE seems to underly hypergammaglobulinaemia but not anti‐dsDNA production. Prior to clinical disease activity, further activation of T and B cells occurs, which is paralleled by rises of anti‐dsDNA but not of total IgG. This suggests that the production of anti‐dsDNA is a T cell‐dependent antigen‐driven process, which is independent of the polyclonal activation of the immune system inherent to the disease.

Mycophenolate mofetil prevents a clinical relapse in patients with systemic lupus erythematosus at risk

Background: Systemic lupus erythematosus (SLE) is characterised by the presence of antibodies to double stranded DNA (dsDNA), which are involved in the pathogenesis of SLE. Previous studies showed that at least two thirds of patients develop a clinical relapse within six months after a significant rise in the anti-dsDNA level, and most relapses were prevented by the administration of corticosteroids at the time of the rise. Objective: To determine whether mofetil mycophenolate (MMF) can prevent a clinical relapse without the side effects associated with corticosteroids. Methods: 36 patients with SLE were examined monthly to determine whether a rise in anti-dsDNA level had occurred. A rise was defined as an increase of 25% of the level of the previous sample of at least 15 IU/ml within a four month period. After a rise patients were treated with MMF 2000 mg daily for six months. Patients were monitored monthly for the occurrence of a clinical relapse and to assess the serological activity and state of activation of CD4+, CD8+, and CD19+ lymphocyte subsets. Results: Anti-dsDNA rose in 10 patients. Treatment with MMF was started in all these patients, and after six months no clinical relapse had occurred. Side effects were minimal. Antibodies to dsDNA decreased during the treatment (p<0.001), associated with a decrease in the state of activation of CD19+ lymphocytes. No changes were found in the state of activation of CD4+ or CD8+ lymphocyte subsets. Conclusion: Administration of MMF after a rise in antibodies to dsDNA is well tolerated, decreases anti-dsDNA and B cell activation, and seems to prevent the occurrence of a clinical relapse in patients with SLE.

Changes in plasma levels of interleukin-2 receptor in relation to disease exacerbations and levels of anti-dsDNA and complement in systemic lupus erythematosus.

Interleukin‐2 receptor (IL‐2R) is expressed and released predominantly by activated T cells. In order to investigate whether disease exacerbations of systemic lupus erythematosus (SLE) are preceded by T cell activation, we prospectively measured levels of IL‐2R once a month, from 6 months prior to exacerbations until 1 month afterwards. To assess the temporal relation between T cell activation and B cell activation, we measured, in addition, levels of anti‐dsDNA, complement C3JC4, and total IgG. During a mean follow‐up period of 23 months, 40 exacerbations occurred in 21 out of the 71 participating patients. For the present study one exacerbation per patient was evaluated. During exacerbation levels of IL‐2R were increased in 18 out of the 21 cases and correlated with levels of anti‐dsDNA (P < 0.02). C3 (P < 0.02), and C4 (P < 0.01), but not with the score of the disease activity index. Levels of IL‐2R rose prior to the excerbation (P < 0.02) and fell afterwards following treatment (P < 0.05). Even in the absence of disease activity or during minor disease symptoms IL‐2R levels were higher (P < 0.01) than in healthy controls. Sixteen out of the 21 exacerbations (76%) were preceded by a significant increase in IL‐2R. Changes in levels of anti‐dsDNA and complement C3JC4 tended to precede changes in levels of IL‐2R. We conclude that increased levels of IL‐2R, compatible with T cell activation, are present in SLE already during inactive disease. These levels further increased prior to exacerbations of disease. As such, IL‐2R is an indicator of disease activity in SLE. Serial measurement of IL‐2R is a sensitive test for predicting disease exacerbations of SLE.

Clinical associations of antiribonucleoprotein antibodies in patients with systemic lupus erythematosus

The authors undertook a cross-sectional study to investigate the clinical associations of antiribonucleoprotein (anti-RNP) antibodies in 49 patients with systemic lupus erythematosus (SLE) without other concomitant connective tissue disorders. The traditional counterimmunoelectrophoresis (CIE) and the immunoblotting (IB) technique were compared. Clinically, special attention was given to the identification of sclerodermalike features. All patients completed a detailed questionnaire, physical examination, and additional investigations including pulmonary function tests, chest roentgenogram, radionuclide transit studies of the esophagus, and nailfold capillary microscopy. Pulmonary function testing and radionuclide transit studies of the esophagus were very sensitive for the detection of (subclinical) pulmonary and esophageal involvement, respectively. Within the relatively homogeneous SLE population, a subset was recognized that was characterized clinically by the presence of sclerodermalike features such as Raynauds phenomenon, sclerodactyly, interstitial changes on chest roentgenogram, and decreased numbers of nailfold capillary loops, and serologically by the presence of anti-RNP antibodies. IB was somewhat more sensitive than CIE for the detection of anti-RNP (anti-Sm/anti-nRNP) antibodies but did not identify other clinical associations. Thus, anti-RNP antibodies in SLE are associated with scleroderma-associated features. For clinical practice, CIE is the technique recommended for their detection.

Antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis: defined specificities may be associated with distinct clinical features

PURPOSE The clinical significance of antineutrophil cytoplasmic autoantibodies (ANCA) in primary sclerosing cholangitis has not been established. We investigated whether analysis of the antigenic specificities of ANCA is useful for delineating clinical subsets of the disease. METHODS Sixty-nine patients with primary sclerosing cholangitis were studied. The presence of ANCA was analyzed by indirect immunofluorescence. Antibodies directed against specific antigens--proteinase 3, myeloperoxidase, elastase, bactericidal/permeability-increasing protein, cathepsin G, and lactoferrin--were identified by enzyme-linked immunosorbent assay. RESULTS ANCA were detected by indirect immunofluorescence in 46 (67%) patients. In antigen-specific enzyme-linked immunosorbent assays, 37 (55%) of the 69 patients had antibodies to one or more antigens: 32 (46%) had antibodies to bactericidal/permeability-increasing protein, 16 (23%) had antibodies to cathepsin G, and 15 (22%) had antibodies to lactoferrin. Only 3 patients had antibodies to proteinase 3. Antibodies to myeloperoxidase or elastase were not detected. Twenty (29%) patients had antibodies to different antigens simultaneously. ANCA as detected by indirect immunofluorescence were not significantly associated with the presence of cirrhosis nor with the coexistence of inflammatory bowel disease. However, antibodies to bactericidal/permeability-increasing protein and cathepsin G were both associated with the presence of cirrhosis, and antibodies to lactoferrin were more frequently detected in patients with primary sclerosing cholangitis in conjunction with ulcerative colitis than in those without inflammatory bowel disease. CONCLUSION Defined specificities of ANCA in primary sclerosing cholangitis may be related to particular clinical features of the disease.