Involvement of Fcgamma receptors and beta2 integrins in neutrophil activation by anti-proteinase-3 or anti-myeloperoxidase antibodies.

We previously described the requirement of tumour necrosis factor-alpha (TNF-alpha) and the role of beta2 integrins in the Fc-gamma receptor IIa (FcgammaRIIa)-mediated mechanism of neutrophil activation by antiproteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies. In the present study, we assessed the involvement of FcgammaRIIIb by studying the respiratory burst activation of completely FcgammaRIIIb-deficient neutrophils primed by TNF-alpha and exposed to anti-PR3 or anti-MPO. Activation of the NADPH oxidase occurred normally in these neutrophils, which indicates that engagement of FcgammaRIIIb is not essential in our model. Experiments performed with neutrophils from severe leucocyte adhesion deficiency (LAD) patients confirmed that beta2 integrins play a pivotal role in this activation. We next studied whether adhesion per se, beta2-integrin-mediated adhesion, or beta2-integrin ligation without adhesion is necessary or sufficient for this activation. Anti-PR3 or anti-MPO induced an FcgammaRIIa-dependent burst in TNF-primed neutrophils incubated in wells coated with poly-L-lysine, known to induce beta2-integrin-independent adhesion, but this reaction was still inhibited by blocking CD18 antibodies. In a system with granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, which did not enhance adhesion, we measured a similar activation by anti-PR3 or anti-MPO and inhibition by CD18. We also noticed that treatment with the beta2-integrin-activating CD18 MoAb KIM185 per se is insufficient for neutrophil activation by anti-PR3 or anti-MPO. We therefore conclude that ligation of beta2 integrins rather than adherence per se is essential for this activation, and that TNF-alpha or GM-CSF is needed for priming but not for adherence.

Anti-neutrophil cytoplasmic auto-antibodies (ANCA) in ulcerative colitis (UC): no relationship with disease activity.

The relationship between anti-neutrophil cytoplasmic auto-antibodies (ANCA) and disease activity in inflammatory bowel diseases remains controversial. The aim of this study was to highlight the relationship between ANCA presence or titers and disease activity in ulcerative colitis (UC). Three groups of patients with UC were studied: 1) group A included 39 patients who had not undergone colectomy, 2) group B, 43 patients with subtotal colectomy and ileo-rectal anastomosis, 3) group C, 98 patients with proctocolectomy and ileo-anal anastomosis, including 88% with pouchitis and 12% without pouchitis at the time of the study. Determination of ANCA was performed using the standardized indirect immunofluorescence assay. ANCA were positive in 59%, 65%, and 54% of patients from groups A, B, and C, respectively (NS). No relationship between ANCA presence or titers and UC activity could be detected within groups A and B. In group C, 45 of 86 patients (52%) without pouchitis and 8 of 12 patients (67%) with pouchitis, were ANCA positive (NS). These results do not support a relationship between ANCA and UC activity in this cohort of 180 patients.

Alpha1-antitrypsin phenotypes and anti-neutrophil cytoplasmic auto-antibodies in inflammatory bowel disease.

OBJECTIVES: Alpha1-antitrypsin (alpha1-AT) is encoded by a highly polymorphic gene with over 75 codominantly expressed alleles at the protease inhibitor (Pi) locus classified as normal, deficient, dysfunctional or null. The aim of this study was to determine in patients with inflammatory bowel disease: (i) the prevalence of anti-neutrophil cytoplasmic auto-antibodies (ANCA) and their antigen specificities; (ii) alpha1-AT Pi phenotypes; and (iii) possible associations between ANCA, disease activity and deficient alpha1-AT alleles. DESIGN: Study of 95 consecutive patients with ulcerative colitis (UC) and 63 patients with Crohn's disease (CD). METHODS: Diagnosis and disease activity were determined by clinical, endoscopic and histological criteria. ANCA by indirect immunofluorescence (IIF) and Pi phenotyping by isoelectric focusing were performed for all patients. Positive IIF sera were tested in antigen-specific enzyme-linked immunosorbent assay: proteinase 3 (PR3), myeloperoxidase (MPO), lactoferrin (LF), lysozyme, human leucocyte elastase (HLE), cathepsin G and bactericidal/permeability increasing protein (BPI). RESULTS: Sixty-one patients with UC (64.2%) and only 11 with CD (17.5%) had ANCA (P < 0.001). Antigen specificities were PR3 (7/61), MPO (3/61), LF (6/61), HLE (1/63) and BPI (10/61) in UC, and PR3 (2/11) and BPI (2/11) in CD. Three PiZ alleles were found, matching the prevalence in the normal French control population. No relationship was found between the presence of ANCA, antibody specificity, disease activity and deficient alpha1-AT alleles. CONCLUSION: ANCA are more frequent in UC than CD and do not correlate with disease activity. ANCA and protease/antiprotease imbalance do not appear to modulate the clinical course of inflammatory bowel disease.

Serial measurements of antineutrophil cytoplasmic autoantibodies in patients with systemic vasculitis.

PURPOSE: To assess the value of serial determinations of antineutrophil cytoplasmic autoantibodies (ANCA) for monitoring disease activity in patients with systemic vasculitis. PATIENTS AND METHODS: Forty-three patients with histologically proven vasculitis (21 with Wegener's granulomatosis, 17 with microscopic polyangiitis, and 5 with renal-limited vasculitis) were studied for a median follow-up of 22 months. Disease activity was prospectively assessed and quantified by the Birmingham Vasculitis Activity Score. A total of 347 sera were analyzed for ANCA determination. RESULTS: Relapses occurred in 23 (54%) of 43 patients. Diagnostic category (Wegener's granulomatosis vs micropolyangiitis and renal-limited vasculitis), severity of initial symptoms (mean vasculitis activity score, mean number of organs involved), and ANCA pattern [cytoplasmic-ANCA (c-ANCA) vs perinuclear-ANCA (p-ANCA)] did not significantly differ between relapsers and nonrelapsers. Lung involvement was more frequent at onset among relapsers [16 of 23 (70%) vs 6 of 20 (30%); P = 0.02]. Relapses were slightly, but not significantly, more frequent in patients with Wegener's granulomatosis or a c-ANCA pattern. The percentage of relapsers was greater in patients with persistently positive ANCA than in patients with negative or decreasing ANCA titers (86% vs 20%, P = 0.0001). However, the predictive value of an increase in ANCA titers for the occurrence of a subsequent relapse was only 28% (4 of 14) for c-ANCA, 12% (2 of 17) for anti-proteinase 3-ANCA, and 43% (6 of 14) for anti-myeloperoxidase-ANCA. An increase in ANCA occurred before or during relapse in 33% (10 of 30) of cases for c-ANCA/anti-proteinase 3 antibodies, and 73% (11 of 15) of cases for anti-myeloperoxidase antibodies. CONCLUSION: The persistence of ANCA positivity is strongly associated with relapses. However, an increase in ANCA titers has a poor value for the early prediction of a subsequent relapse and should not be used as a sole parameter for therapeutic intervention. In addition, our results suggest that serial anti-myeloperoxidase determination may be useful as a prognostic marker in patients who are p-ANCA positive.

[Extracapillary glomerulonephritis with anti-myeloperoxidase antibodies in 2 patients with systemic scleroderma treated with penicillamine D]. / Glomérulonéphrite extracapillaire avec anticorps anti-myéloperoxydase chez 2 malades ayant une sclérodermie systémique traitée par D-pénicillamine.

BACKGROUND: Side-effects, including autoimmune disorders, are frequent with D-penicillamine therapy. Proteinuria is observed in 10% of the patients, often secondary to extramembranous glomerulopathy. Necrotizing extracapillary glomerulonephritis is however exceptional. CASE REPORTS: Two patients with systemic sclerodermia were treated with D-penicillamine for 7 and 14 years. Both developed necrotizing extracapillary glomerulonephritis with anti-myeloperoxidase antibodies (anti-MPO), associated with hemorrhagic alveolitis in one case. Partial regression of the renal failure was obtained after withdrawal of D-penicillamine and combination treatment with prednisone and cyclophosphamide. DISCUSSION: Extracapillary glomerulonephritis or a lung-kidney syndrome are frequently associated with anti-MPO antineutrophil cytoplasm antibodies (ANCA). In systemic sclerodermia, the presence of anti-MPO appears to define a group of patients at risk of pauci-immune extracapillary glomerulonephritis or a lung-kidney syndrome. In addition, the presence of ANCA in patients with renal failure would suggest extracapillary glomerulonephritis rather than sclerodermic microangiopathy. Development of extracapillary glomerulonephritis with anti-MPO in patients who are taking D-penicillamine suggests that inductor mechanisms other than D-penicillamine are involved in the pathogenesis of these glomerulopathies.

Anti-Saccharomyces cerevisiae mannan antibodies combined with antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease: prevalence and diagnostic role.

BACKGROUND: Perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) are a well recognised marker for ulcerative colitis. Antibodies to oligomannosidic epitopes of the yeast Saccharomyces cerevisiae (ASCA) are a new marker associated with Crohn's disease. AIMS: To assess the value of detecting pANCA and/or ASCA for the diagnosis of ulcerative colitis and Crohn's disease. METHODS: Serum samples were obtained from 100 patients with Crohn's disease, 101 patients with ulcerative colitis, 27 patients with other miscellaneous diarrhoeal illnesses, and 163 healthy controls. Determination of pANCA and ASCA was performed using the standardised indirect immunofluorescence technique and an ELISA, respectively. RESULTS: The combination of a positive pANCA test and a negative ASCA test yielded a sensitivity, specificity, and positive predictive value of 57%, 97%, and 92.5% respectively for ulcerative colitis. The combination of a positive ASCA test and a negative pANCA test yielded a sensitivity, specificity, and positive predictive value of 49%, 97%, and 96% respectively for Crohn's disease. Among patients with miscellaneous non-inflammatory bowel disorders, three were ASCA positive and two were pANCA positive. One control was ASCA positive. The presence of ASCA in patients with Crohn's disease was associated with small bowel involvement. CONCLUSION: ASCA and pANCA are strongly associated with Crohn's disease and ulcerative colitis, respectively. Combination of both tests could help the diagnosis of inflammatory bowel disease.

Immunoglobulin G subclass distribution of anti-endothelial cell antibodies (AECA) in patients with ulcerative colitis or Crohn's disease.

Anti-endothelial cell antibodies have been described in sera from patients with inflammatory bowel disease. The aim of this study was to determine, by ELISA, the IgG subclass distribution of anti-endothelial cell antibodies, in patients with ulcerative colitis (N = 28) or Crohn's disease (N = 82) as compared with blood donors (N = 95). Thirty-six percent of ulcerative colitis and 23% of Crohn's disease patients were positive for at least one of the IgG anti-endothelial cell subclasses. Interestingly, the pattern of IgG anti-endothelial cell subclass observed in the two inflammatory bowel diseases differs. In Crohn's disease, the IgG1 anti-endothelial cell antibody level was significantly increased (P < 0.05) while IgG2 and IgG4 anti-endothelial cell antibody levels were decreased (P < 0.0001 and P < 0.01, respectively) as compared to ulcerative colitis patients. The immunoglobulin G3 anti-endothelial cell antibody level was decreased in both ulcerative colitis and Crohn's disease patients as compared to healthy blood donors. No relationship was detected between disease activity of ulcerative colitis or Crohn's disease patients and anti-endothelial cell IgG subclasses. Finally, the disparity of IgG anti-endothelial cell subclass distribution in these two inflammatory bowel diseases suggests that the ability to activate effector mechanisms is not identical, and hence, deals with the concept of distinctive pathogenetic mechanisms in these two diseases.

Are HLA-DR or TAP genes genetic markers of severity in ulcerative colitis?

The pathogeny of ulcerative colitis (UC) is not yet elucidated, but some arguments suggest the implication of genetic factors. Among the candidate genes, those encoding for HLA class II genotypes have been extensively studied in UC; however, discordant data may be imputable to heterogeneity, characterized by immunological markers such as atypical ANCA (p-ANCA), or to inclusion of more or less intractable UC. The aim of our study is to evaluate the interest of HLA class II and TAP genetic markers to identify different clinical forms of UC, according to p-ANCA status. Unrelated patients with a history of UC (n = 91) and healthy control subjects with no personal or family history of inflammatory bowel diseases (IBD) (n = 200) were included. HLA-DRB1*03 was less frequent in UC patients than in healthy controls (8% vs 28%, PC < 0.03). No association was found with any TAP genotypes. Moreover, there was no association with the HLA-DR2 specificity, either in the entire group of UC patients (38% vs 28%) or in the p-ANCA-positive subgroup of patients (30%). The most consistent finding in the present study is that some genetic markers may characterize intractability in UC patients. HLA-DR2 was associated with poor prognosis, regardless of p-ANCA status. In HLA-DR2 and non-HLA-DR2 groups, colectomy was done in 55% and 27% of patients, respectively, (PC < 0.05). Furthermore, in non-HLA-DR2 patients, p-ANCA could be of interest to characterize those with more severe prognosis. Our results confirm the interest of genetic studies to define UC genetic susceptibility, taking into account intractability of the disease. They do not support the hypothesis that p-ANCA is a subclinical marker of genetic susceptibility to UC.

Distinct production of autoantibodies to nuclear components in ulcerative colitis and in Crohn's disease.

Despite extensive research, the pathogenesis of inflammatory bowel disease (IBD) is still unclear. Immunological disorders have been described in patients with both Crohn's disease (CD) and ulcerative colitis (UC). In this work serum samples collected from 58 patients with CD and 55 patients with UC were tested in ELISA against a panel of nuclear and cytoplasmic proteins and peptides in order to determine whether specific autoantibodies are produced in these patients. Low levels of IgG antibodies to histones H1, H2A, H2B, H3, and H4, to Hsp-70 and ubiquitin stress proteins, Ro/SSA and La/SSB proteins and myosin were detected in some of these sera. In contrast, the following antibodies of IgG isotype could be much more frequently demonstrated: antibodies to ubiquitinated H2A (U-H2A) peptide T4 (51.7% in CD; 18.2% in UC), antibodies to the zinc-finger peptide F2 of poly-(ADP-ribose polymer)ase (PARP) involved in DNA repair (58.6% in CD; 25.5% in UC) and actin antibodies (43.1% in CD; 7.3% in UC). In a follow-up study of 12 patients with CD and UC (75 additional samples), we found IgG antibodies to several histone peptides occurring essentially in the serum of patients with CD. Although we found no obvious correlation between the presence or level of these various antibodies and C-reactive protein, or the location of the disease, in a number (but not all) of patients, we observed a strikingly good relationship between antibodies to histone peptides, U-H2A peptide T4, and PARP peptide F2 and the Crohn's disease activity index. The mechanism of induction of these antibodies still remains obscure.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-endothelial cell antibodies in sera from patients with inflammatory bowel disease.

IgG anti-endothelial cell antibodies (AECA) have been described in sera from patients with vasculitis and other immune disorders such as systemic lupus erythematosus. Presence of AECA may be relevant to the hypothesis that Crohn's disease (CD) is a form of intestinal vasculitis. The aim of this study was to search for IgG AECA among 141 patients with CD, 94 patients with ulcerative colitis (UC) and 71 healthy blood donors and to assess the relationship between AECA and demographic or disease data. The cut-off point was defined from the mean OD values + 2 SD obtained from healthy blood donors. Seventeen percent of sera from patients with CD were positive for IgG AECA, whereas 24.5% of sera from patients with UC were positive. Among disease data, only a significant relationship between presence of IgG AECA and CD activity was noticed. These results might reinforce the hypothesis that intestinal vascular injury may be an important event in CD. However, detection of AECA in an almost similar percentage of patients with UC is more suggestive of an immune response to hidden endothelial self-antigen exposed after endothelial cell damage or a further marker of disturbed immunoregulation in inflammatory bowel disease.

Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies.

Antineutrophil cytoplasmic autoantibodies (ANCA) have been described in sera of patients with several forms of systemic vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. The two main targets of ANCA in vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). ANCA are capable of activating neutrophils primed by tumor necrosis factor-alpha (TNF-alpha) in vitro, which may be relevant for the induction of the vascular inflammation observed in vivo. Recently, it has been suggested that engagement of Fc gamma receptor IIa (Fc gamma RIIa) on the neutrophils is involved in the activation by ANCA. In the present study, we show that activation of the neutrophil respiratory burst by anti-PR3 and anti-MPO is strongly enhanced after TNF priming and lost on removal of the Fc parts of the antibodies. Similar results were obtained when the neutrophils were activated with antibodies against known membrane antigens without major changes in the expression of the target antigens. The TNF-induced enhancement of the neutrophil activation was not observed when adherence of the cells was prevented by continuous stirring of the suspension or by the addition of CD18 antibodies before TNF exposure. Hence, our results indicate that engagement of both Fc gamma RIIa and beta 2 integrins is instrumental in neutrophil activation induced by ANCA.

[Sensitivity and specificity of antineutrophil cytoplasmic antibodies in systemic vasculitis]. / Sensibilité et spécificité des anticorps anticytoplasme des neutrophiles (c-ANCA) au cours des vascularites systémiques.

The diagnostic value of c-ANCA as a specific marker of systemic vasculitis (particularly Wegener's granulomatosis) is well established. The prognostic value of c-ANCA for determining disease activity is controversial. We have prospectively studied in ten patients with systemic vasculitis over a mean period of 34 months (extreme 2-61 months). All patients had c-ANCA at the moment of the diagnosis: four patients had high titer of c-ANCA all over the period study; three clinical and biological exacerbations of the disease was observed without variation of the c-ANCA titer. In four patients c-ANCA disappeared within 6 months after the beginning of the treatment correlated with disease activity. Sometimes a rise of c-ANCA titer was observed with or without disease activity. In one case c-ANCA titer had a serrated evolution. The sensitivity and the specificity of the c-ANCA for disease activity in the ten studied patients were respectively 1 and 0.28. In patients with systemic vasculitis and c-ANCA at the time of the diagnosis, variation in c-ANCA titer alone is of limited prognostic value for predicting disease course.

Atypical autoantigen targets of perinuclear antineutrophil cytoplasm antibodies (P-ANCA): specificity and clinical associations.

Atypical antineutrophil cytoplasm antibodies (A-ANCA) are defined here as ANCA detected by IIF and not directed against the predominant ANCA antigens, proteinase 3 (PR3) and myeloperoxidase (MPO). A-ANCA are found in a variety of clinical conditions, namely rheumatoid arthritis, inflammatory bowel diseases, chronic hepatic diseases and several infections including HIV infection. They are directed against a variety of still ill-defined neutrophil antigens and most frequently yield a perinuclear pattern (P-ANCA) of binding by indirect immunofluorescence on ethanol fixed neutrophils. This paper reviews the literature on A-ANCA and our recent data suggesting that, among others, cathepsin G is one of the predominant antigen targets of A-ANCA. From a clinical point of view, the distinction between MPO-ANCA and A-ANCA is not possible by indirect immunofluorescence (IIF). The determination of ANCA antigens by specific ELISA is therefore necessary to differentiate P-ANCA with MPO specificity from those with undefined specificity. This is of importance because the clinical value of MPO-ANCA is clearly established while the presence of A-ANCA is difficult to interpret given their occurrence in a large variety of clinical conditions.

Antineutrophil cytoplasmic auto-antibodies in sera from patients with ulcerative colitis after proctocolectomy with ileo-anal anastomosis.

Presence of antineutrophil cytoplasmic auto-antibodies (ANCA) in ulcerative colitis could be an epiphenomenon related to colonic inflammation and/or may reflect a primitive disturbance of immune regulation. In this regard, study of ANCA status after the whole colorectal mucosa has been removed could favor one of these two hypothesis. We compared the prevalence of ANCA in a first group of 70 patients with non operated UC and in a second group of 32 patients with UC having had a proctocolectomy with ileoanal anastomosis. Perinuclear ANCA (p-ANCA) were found in 34/70 (49%) of the first group as compared to 11/32 (34%) in the second group (NS). Our results further support that the presence of ANCA in UC reflects an immune disturbance not linked to the presence of the target organ.