ANCA-stimulated neutrophils release BLyS and promote B cell survival: a clinically relevant cellular process.

OBJECTIVES: To determine a role for antineutrophil cytoplasmic antibody (ANCA)-activated neutrophils in promoting B cell survival through the release of B lymphocyte stimulator (BLyS). METHODS: Neutrophil BLyS expression was measured by flow cytometry. Concentrations of BLyS in cell supernatants and donor serum samples were measured by ELISA. Cell survival assays were carried out using an L3055 cell line and viability measured by flow cytometry. RESULTS: Tumour necrosis factor α and formyl-Met-Leu-Phe (fMLP) treatment of non-primed neutrophils and treatment of primed neutrophils with anti-PR3 ANCA IgG resulted in a significant increase in surface expression of BLyS within 30 min which returned to basal levels by 2 h. Supernatants from ANCA-stimulated neutrophils were shown to contain increased levels of BLyS and to promote the survival of the centroblast cell line L3055. Serum BLyS concentrations are increased in patients with active ANCA-associated systemic vasculitis and these levels are increased further following 1-3 months of treatment with rituximab. CONCLUSIONS: ANCA specifically causes the release of BLyS from activated neutrophils which can support B cell survival in vitro. The presence of serum BLyS in active disease and its increase following B cell depletion suggest it is an important factor in disease pathogenesis and may facilitate disease relapse.

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis.

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is an autoimmune disease in which the contributions of genetic, epigenetic and environmental factors to aetiology and pathogenesis are being unravelled. The ANCA immunoglobulin G targeting proteinase 3 and myeloperoxidase affects several neutrophil functions, usually to augment or dysregulate these, promoting a proinflammatory phenotype whereby neutrophils have enhanced capabilities of causing collateral damage to endothelial and other cells. In addition, B cells are intimately involved in pathogenesis as anti-B cell therapies are highly effective, but the manner of this involvement still needs to be delineated. Similarly, the T cell compartment is disturbed in ANCA vasculitis and numerous alterations in T cell subsets have been described, but recognition of a novel CD8(+) T cell transcription signature which can predict likelihood of relapse in ANCA vasculitis indicates that more needs to be learnt about the influence of T cells in the disease process. Finally, the role of the alternative complement pathway and the potential therapeutic value of its neutralization is under active investigation after compelling studies in murine models have demonstrated that C5 and factor-B knock-out mice are protected.

Impact of compound heterozygous complement factor H mutations on development of atypical hemolytic uremic syndrome-A pedigree revisited.

Atypical hemolytic uremic syndrome (aHUS) is associated with mutations in the gene CFH encoding the complement regulator factor H (CFH). We previously reported a family, in which three individuals had partial CFH deficiency but only one was affected by aHUS. We have investigated this family further to show that the partial CFH deficiency is associated with a heterozygous CFH mutation (c.2768T>G, p.Tyr899Asp). We used the polymorphic CFH variant p.His402Tyr to track expression of p.Tyr899Asp, and found that this mutant was expressed in minimal quantities in serum. In the one affected individual we found a second CFH mutation (c.3581G>A, p.Gly1194Asp) on the other allele which was expressed normally. We showed that this mutant, which has been described previously in aHUS, has impaired regulation of cell surface complement activation. The affected individual in this family is therefore a compound heterozygote for two functionally significant CFH mutations. Two individuals (mother and male sib) in the pedigree carried only c.2768T>G, p.Tyr899Asp and one (father) carried only c.3581G>A, p.Gly1194Asp, and all three were asymptomatic. Thus, further investigation of this family has enabled us to clarify the genotype-phenotype correlation.

ANCA-associated vasculitides: advances in pathophysiology and treatment.

Substantial progress has been made over the last two decades in our understanding of the immunopathogenesis of antineutrophil cytoplasmic antibodies (ANCA) associated vasculitides. Compelling evidence from in vitro studies and experimental models in conjunction with clinical trials has confirmed that ANCA directly contribute to the evolution and progression of the disease process. Continuous development in our understanding of the mechanisms that drive the disease may ultimately allow us to tailor the multitude of novel therapies, which are rapidly becoming available, to the requirements of individual patients. In this review we endeavour to provide a brief overview of the recent advances in ANCA-associated vasculitides and outline basic principles for diagnosis and treatment of these complex multisystem diseases.

The role of the endothelium in systemic small vessel vasculitis.

The endothelium is the back-drop against which the effects set in train by interactions between Anti-Neutrophil Cytoplasm Antibodies (ANCA) and neutrophils are played out. This review considers the mechanisms of the endothelial cell injury that may result but also questions the impact of endothelial heterogeneity and endothelial cell activation in facilitation of vasculitic lesions, as well as the potential roles for endothelial-dependent anti-inflammatory mechanisms in controlling inflammation.

Mannose-binding lectin gene polymorphisms in a cohort study of ANCA-associated small vessel vasculitis.

OBJECTIVE: To investigate whether single nucleotide polymorphisms (SNPs) within the mannose-binding lectin (MBL) gene are associated with small vessel vasculitis (SVV) and are a risk factor for intercurrent infection, as described previously in other autoimmune diseases. METHODS: Six SNPs in the MBL promoter and coding region were genotyped by sequence-specific polymerase chain reaction or restriction fragment length polymorphism assay in 170 white Caucasians with SVV and 372 ethnically matched controls in a case-control association study. Serum MBL levels were measured by ELISA. The genotype and protein concentrations were correlated to clinical details retrieved from hospital records. RESULTS: No differences in allelic and genotypic frequencies were detected between patients with SVV and control subjects. MBL deficiency did not increase the susceptibility to infection (P = 0.6, Fisher's exact test) or the duration of hospital stay. CONCLUSION: Our data suggest that MBL polymorphisms are not associated with SVV and do not influence the incidence of concomitant infections. These results raise doubts about the usefulness of MBL polymorphisms as a predictive marker for infection in SVV.

Endothelium and regulatory cell interactions.

Endothelial cells are injury targets in vasculitis and other diseases. However, their abilities to regulate their own fate are becoming increasingly recognized and may influence their susceptibility to injury in different vascular beds.

Mediation of endothelial cell damage by serine proteases, but not superoxide, released from antineutrophil cytoplasmic antibody-stimulated neutrophils.

OBJECTIVE: To evaluate potential mediators of endothelial cell injury in systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs), we investigated the factors controlling the neutrophil respiratory burst and endothelial release of von Willebrand factor (vWF) during neutrophil-endothelial cell interactions. METHODS: Superoxide release from neutrophils binding to purified P-selectin or to tumor necrosis factor-activated endothelial cells was measured under flow or static conditions using the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. Neutrophils were activated with fMLP, normal IgG, or ANCA IgG. Enzyme-linked immunosorbent assay was used to measure vWF. Serine protease activity was measured enzymatically. RESULTS: ANCA IgG or fMLP induced superoxide release when perfused over neutrophils that were rolling over P-selectin, but not those that were binding to endothelial cells. In static assays, endothelial cells inhibited superoxide production by neutrophils. Adenosine inhibited the respiratory burst, and, in cocultures, adenosine deaminase overcame the inhibitory effects of endothelial cells. Serine proteases were released during activated neutrophil-endothelial cell coculture. There was enhanced release of vWF during activated neutrophil-endothelial cell coculture; this was not inhibited by diphenyleneiodonium or by SOD plus catalase, but was inhibited by diisopropylfluorophosphate. CONCLUSION: Endothelial cells inhibit superoxide generation by fMLP and ANCA-activated neutrophils. The release of vWF occurs during coculture and is sensitive to serine protease, but not NADPH oxidase inhibition. Serine proteases may play a more important role than reactive oxygen species as mediators of endothelial injury during ANCA-associated systemic vasculitis.

IL-18 is upregulated in the kidney and primes neutrophil responsiveness in ANCA-associated vasculitis.

In antineutrophil cytoplasm autoantibody (ANCA)-associated systemic vasculitis (ASV), autoantibody-induced neutrophil activation is believed to cause organ damage. In vitro, tumor necrosis factor alpha (TNFalpha) primes neutrophils for ANCA stimulation and TNFalpha blockade has been successfully used to treat ASV. Nonetheless, irreversible organ damage can still occur, suggesting that other cytokines may circumvent TNFalpha blockade. We report that interleukin (IL)-18 deposition, as assessed by immunoperoxidase staining, is increased in renal biopsies from ASV patients. Immunofluorescence microscopy demonstrated that podocytes are the predominant glomerular IL-18-positive cell type, whereas in the interstitium, myofibroblasts, distal tubular epithelium, and infiltrating macrophages stained for IL-18. In vitro, IL-18 primed superoxide production by ANCA-activated neutrophils comparably to TNFalpha. IL-18-primed, ANCA-induced superoxide production was unaffected by anti-TNFalpha antibody, which abrogated TNFalpha priming. Furthermore, TNFalpha and IL-18 phosphorylated neutrophil p38 mitogen-activated protein kinase (MAPK), but IL-18-mediated p38 MAPK phosphorylation was unaffected by anti-TNFalpha antibody. The p38 MAPK inhibitor, SB20358, reduced IL-18-primed, ANCA-induced superoxide production in a concentration-dependent manner. ANCA-induced superoxide release was also sensitive to the Leukotriene B4 (LTB4) inhibitor MK-886. IL-18 priming was not associated with increased ANCA antigen expression on isolated neutrophils. We conclude that IL-18 is likely to be important for neutrophil recruitment and priming in ASV. Therapies targeting single priming agents may have limited efficacy in controlling disease.

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.

Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, approximately 20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.

Anti-neutrophil cytoplasm antibody IgG subclasses in Wegener's granulomatosis: a possible pathogenic role for the IgG4 subclass.

A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.

Malignancy is increased in ANCA-associated vasculitis.

OBJECTIVE: In the light of previous reports of an association between malignancy and renal vasculitis, we aimed to investigate the association of malignancy in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, either Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA), and compare it with the general population and disease control groups comprising patients with systemic lupus erythematosus (SLE) or Henoch-Schonlein purpura (HSP). METHODS: A retrospective review of 200 consecutive patients with WG or MPA was performed. Malignancies preceding or concurrent with vasculitis were recorded and the incidence of malignancy was compared with those in a population of 129 patients with HSP, 333 patients with SLE and a normal population in the West Midlands of the UK. RESULTS: Twenty patients had a diagnosis of malignancy, 14 had MPA and six had WG. Patients with ANCA-associated vasculitis had an increased risk of malignancy compared with HSP patients, of whom six patients had malignancy (relative risk 0.85, confidence interval 0.69-1.05; P = 0.034), or SLE patients, of whom five patients had malignancy (relative risk 0.31, 95% confidence interval 0.14-0.7; P<0.0001). The rate of malignancy compared with an age-matched control group was increased in patients with ANCA-associated vasculitis and HSP (ANCA-associated vasculitis, relative risk 6.02, 95% confidence interval 3.72-9.74; HSP, relative risk 5.25, 95% confidence interval 2.4-11.5). The presence of ANCA was not predictive of malignancy. CONCLUSION: In conclusion, patients with ANCA-associated vasculitis have an increased risk of preceding or concurrent malignancy.

Production of antineutrophil cytoplasm antibodies derived from circulating B cells in patients with systemic vasculitis.

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. In certain systemic vasculitides, autoimmunity plays an important role with autoantibodies developing towards neutrophils, which are termed antineutrophil cytoplasm antibodies (ANCA). There is a growing body of evidence that T cells may contribute to the pathogenesis of ANCA-associated vasculitides. A system was set up to determine whether B cells require T cell help to produce antibodies in a peripheral blood lymphocyte (PBL) culture system enriched for B cells and dendritic cells (DC). As a control, tetanus toxoid (TT) antibody production was detected from individuals not recently immunized with tetanus vaccine when stimulated with TT antigen. Proteinase 3 (PR3) and myeloperoxidase (MPO) antibodies were produced from B cell and DC enriched cultures prior to the addition of antigen in some ANCA-positive patients with high ANCA titres, but not from patients with low ANCA titres or controls. PBMC from individuals recently immunized with tetanus vaccine were also maximally stimulated in that addition of antibody did not enhance antibody production. We conclude that this system supports a role for T cell help in the production of TT antibodies in individuals not immunized recently with tetanus vaccine. However, in patients with ANCA-associated vasculitis and controls recently immunized with tetanus vaccine, circulating B cells are apparently spontaneously producing autoantibody, possibly reflecting a system already maximally driven in vivo, and therefore masking underlying potential T cell-B cell collaboration. Such B cells may be less responsive to regulatory stimuli in vivo.

Fractalkine expression on human renal tubular epithelial cells: potential role in mononuclear cell adhesion.

Fractalkine (CX3CL1) is a transmembrane molecule with a CX3C chemokine domain attached to an extracellular mucin stalk which can induce both adhesion and migration of leucocytes. Mononuclear cell infiltration at renal tubular sites and associated tubular epithelial cell damage are key events during acute renal inflammation following renal allograft transplantation. Using northern and Western blot analysis, we have demonstrated the expression of fractalkine message and protein by renal tubular epithelial cells in vitro. The expression was up-regulated by TNF-alpha, a key proinflammatory cytokine in acute rejection. Investigation of surface expression of fractalkine on cultured proximal tubular epithelial cells revealed only a subpopulation of positively staining cells. Immunohistochemistry revealed that only a proportion of tubules in renal allograft biopsies showed induction of fractalkine expression. Studies using a static model of adhesion demonstrated CX3CR1/fractalkine interactions accounted for 26% of monocytic THP-1 cell and 17% of peripheral blood natural killer cell adhesion to tubular epithelial cells, suggesting that fractalkine may have a functional role in leucocyte adhesion and retention, at selected tubular sites in acute renal inflammation. Thus, fractalkine blockade strategies could reduce mononuclear cell mediated tubular damage and improve graft survival following kidney transplantation.

Hypogalactosylation of serum IgG in patients with ANCA-associated systemic vasculitis.

The triad of small vessel vasculitides (SVV) comprise Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CS). All three are associated with presence of circulating IgG antineutrophil cytoplasm antibodies (ANCA) which target autoantigens contained, primarily, within neutrophil azurophilic granules. The widely accepted model of pathogenesis suggests that ANCA activate cytokine-primed neutrophils within the microvasculature, leading to by-stander damage to endothelial cells, and rapid escalation of inflammation with recruitment of mononuclear cells. Activation may be initiated, in vitro, by the coligation of the PR3 or MPO antigen, translocated to the cell surface, and FcgammaRIIa/FcgammaRIIIb receptors. This suggests that the IgG subclass profile of ANCA and, possibly, its glycosylation status could influence the inflammatory mechanisms activated. The glycosylation status of total IgG isolated from the sera of patients with WG (13), MPA (6) and CSS (1) was determined by analysis of the released oligosaccharides. A deficit in IgG galactosylation is demonstrated for all patient samples, compared to controls. The mean percentage values for the agalactosylated (G0) oligosaccharides were 57% (SD +/- 9.71), 47% (SD +/- 4.25) and 28% (SD +/- 4.09) for WG, MPO and control samples, respectively. The G0 levels for polyclonal IgG isolated from the sera of both WG and MPA patients were significantly increased compared to controls (P < 0.0001). The major glycoform present therefore is agalactosylated (G0) IgG. In previous studies the G0 glycoform of IgG has been shown to bind and activate mannan binding lectin, and hence to activate the complement cascade, and to facilitate mannose receptor binding and the uptake of IgG complexes by macrophages and dendritic cells. Both of these activities could impact on the processing and presentation of self-antigens in autoimmune disease.

Adjuvant treatment of patients with antineutrophil cytoplasmic antibody-associated vasculitis with vitamins E and C reduces superoxide production by neutrophils.

OBJECTIVES: Neutrophils when activated generate a respiratory burst which has been implicated in the pathogenesis of primary systemic vasculitis. Neutrophils from patients with vasculitis have a greater respiratory burst than normal healthy donors. The aim of this study was to assess the effects of antioxidant treatment (vitamins E and C) on the generation of a respiratory burst from neutrophils isolated from patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. METHODS: Neutrophils were isolated from patients with systemic vasculitis and healthy donors. Spontaneous superoxide generation was measured by the reduction of ferricytochrome c. The patients were treated with antioxidants, vitamins E and C, and spontaneous superoxide generation, vitamin C and total antioxidant capacity were measured before and after treatment. RESULTS: The treatment of the patients with antioxidants resulted in a reduction in spontaneous superoxide generation (pre-treatment 8.41+/-0.7 nmol/10(6) cells; post-treatment 5.64+/-0.6 nmol/10(6) cells; P<0.05). There was no significant difference in the superoxide generation from normal controls who did not receive treatment, measured prior to commencement of the study and 10 days later (first reading 4.81+/-0.5 nmol/10(6) cells; second reading 5.32+/-0.4 nmol/10(6) cells; P>0.05). Total antioxidant capacity increased significantly following treatment with vitamins C and E (555.4+/-142 vs. 668.6+/-186 micromol/l trolox equivalent; P=0.01) as did vitamin C concentrations (56.5+/-27 vs. 137.7+/-64 micromol/l; P=0.002). CONCLUSIONS: In this preliminary study, the treatment of patients with antioxidants, vitamins E and C, reduced neutrophil generation of superoxide and suggests that antioxidants may have an important role as adjuvant therapy. The evidence presented should form the basis of a larger randomized placebo-controlled trial of vitamins E and C as adjuvant therapy in patients with ANCA-associated systemic vasculitis.