Julie M. Williams

ANCA induces β2 integrin and CXC chemokine-dependent neutrophil-endothelial cell interactions that mimic those of highly cytokine-activated endothelium

Antineutrophil cytoplasm antibodies (ANCA) activate neutrophils to undergo a series of coordinated interactions, leading to transendothelial migration, eventually culminating in vascular destruction. The molecular events underlying neutrophil recruitment in ANCA‐associated vasculitis need to be defined to enable effective therapeutic manipulation. A flow‐based adhesion ssay was used to investigate the role of β2 integrins (CD11a/CD18 and CD11b/CD18) and chemokine receptors [CXC chemokine receptor (CXCR)1 and CXCR2] in neutrophil migration through the endothelium. Two endothelial models were used: a highly activated model stimulated with 100 U/ml tumor necrosis factor α (TNF‐α) and a minimally activated model stimulated with 2 U/ml TNF‐α and in which ANCA was present as a secondary neutrophil stimulus. CD11a/CD18, CD11b/CD18, and CXCR2 contributed to adhesion and transendothelial migration in both models. However, when the endothelium was minimally activated with TNF‐α, CD11b/CD18 played an important role in stabilizing adhesion induced by ANCA immunoglobulin G (IgG). Analysis of β2 integrins and chemokine receptors demonstrated that ANCA IgG had no effect on expression levels at the neutrophil surface but enabled an active conformational change of CD11b/CD18. Similar molecular mechanisms control neutrophil adhesion and migration through highly or minimally TNF‐α‐activated endothelium. However, the direct ANCA‐mediated neutrophil stimulation is needed to drive migration through minimally activated endothelium, and CD11b/CD18 is recruited for greater stability of adhesion during this process and can undergo an activatory, conformational change in response to ANCA IgG.

Pharmacological comparison of human homomeric 5-HT3A receptors versus heteromeric 5-HT3A/3B receptors

The present study determined the detailed pharmacological profile of heterologously expressed human (h) homomeric 5-HT3A receptors in direct comparison to heteromeric h5-HT3A/3B receptors. The very minor differences in their respective pharmacological profiles indicates that the 5-HT3B receptor subunit alters, predominantly, the biophysical rather than the pharmacological properties of the 5-HT3 receptor.

Confirmation of the genetic association of CTLA4 and PTPN22 with ANCA-associated vasculitis

BackgroundThe genetic contribution to the aetiology of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is not well defined. Across different autoimmune diseases some genes with immunomodulatory roles, such as PTPN22, are frequently associated with multiple diseases, whereas specific HLA associations, such as HLA-B27, tend to be disease restricted. We studied ten candidate loci on the basis of their immunoregulatory role and prior associations with type 1 diabetes (T1D). These included PTPN22, CTLA4 and CD226, which have previously been associated with AAV.MethodsWe genotyped the following 11 SNPs, from 10 loci, in 641 AAV patients using TaqMan genotyping: rs2476601 in PTPN22, rs1990760 in IFIH1, rs3087243 in CTLA4, rs2069763 in IL2, rs10877012 in CYP27B1, rs2292239 in ERBB3, rs3184504 in SH2B3, rs12708716 in CLEC16A, rs1893217 and rs478582 in PTPN2 and rs763361 in CD226. Where possible, we performed a meta-analysis with previous analyses.ResultsBoth CTLA4 rs3087243 and PTPN22 rs2476601 showed association with AAV, P = 6.4 × 10-3 and P = 1.4 × 10-4 respectively. The minor allele (A) of CTLA4 rs3087243 is protective (odds ratio = 0.84), whereas the minor allele (A) of PTPN22 rs2476601 confers susceptibility (odds ratio = 1.40). These results confirmed previously described associations with AAV. After meta-analysis, the PTPN22 rs2476601 association was further strengthened (combined P = 4.2 × 10-7, odds ratio of 1.48 for the A allele). The other 9 SNPs, including rs763361 in CD226, showed no association with AAV.ConclusionOur study of T1D associated SNPs in AAV has confirmed CTLA4 and PTPN22 as susceptibility loci in AAV. These genes encode two key regulators of the immune response and are associated with many autoimmune diseases, including T1D, autoimmune thyroid disease, celiac disease, rheumatoid arthritis, and now AAV.

Generation of a selective 5-HT3B subunit-recognising polyclonal antibody; identification of immunoreactive cells in rat hippocampus.

The present study generated a polyclonal antibody (AP86/3) that recognises a peptide sequence of the h5-HT(3B) receptor subunit. Western blot analysis of homogenates prepared from cell lines expressing either homomeric (h5-HT(3A)) or heteromeric (h5-HT(3A/3B)) receptors, as well as immunocytochemical studies with the same cell lines, indicated that AP86/3 recognised, selectively, the 5-HT(3B) subunit. Immunohistochemical labelling was also apparent in cells in the rat hippocampus that displayed the distribution and morphology of interneurones.

Translating basic science into patient therapy for ANCA-associated small vessel vasculitis

ANCA (anti-neutrophil cytoplasm antibody)-associated small vessel vasculitis is an inflammatory condition associated with the production of autoantibodies to neutrophil cytoplasmic components. The disorder results in destruction of the microvasculature, infiltration of neutrophils into tissues, which is followed later by mononuclear cells, leading to injury and the formation of granulomatous lesions. Initiators for the disease are undetermined but a pro-inflammatory environment is required. Other influencing factors may include environmental triggers, genetic propensity or infectious agents. The primary cellular event in the condition involves the neutrophils, which are likely to be responsible for the majority of tissue injury. Binding of the autoantibody to neutrophils initiates cell activation via a complex intracellular signalling cascade, culminating in the release of pro-inflammatory mediators, proteolytic enzymes and reactive oxygen species. Adhesion of neutrophils to endothelial cells is observed in vitro and more investigations in this area may explain the focussing of the disease to certain vessels/tissues. Current treatment regimens have substantial toxicity. Although newer developments are an improvement there is still a pressing need for more targeted therapies, which could be provided by extrapolating information emerging from basic scientific research.

Anti–Endothelial Cell Antibodies in Vasculitis

Anti–endothelial cell antibodies have been described in association with small vessel systemic vasculitides since the late 1980s. Opinions have waxed and waned about their importance. An early study from this group suggested they were present in 59% of 168 samples from patients with Wegener’s

Effect of verocytotoxins (Shiga-like toxins) on human neutrophils in vitro

Neutrophil activation occurs in diarrhoea-associated HUS and correlates with disease severity, implying a role in pathogenesis. Verocytotoxin (Shiga-like toxin) has been shown to stimulate endothelium to release chemokines and express leukocyte adhesion molecules that would lead to indirect neutrophil-endothelial interaction. A direct action of verocytotoxin (VT) on neutrophils has been proposed, although in vitro studies of this are controversial. In this report we examine the effect of verocytotoxin-1 (Shiga-like toxin-1) (VT1) and verocytotoxin-2 (VT2) on human neutrophils in vitro with regard to priming, the release of superoxide and elastase, and chemotaxis. Neutrophils were incubated with VT1 or VT2 and superoxide and elastase release was measured over 120 and 45 minutes respectively. Priming was investigated by pre-treating the neutrophils with VT1 or VT2, exposing them to formyl-met-leu-phe (fMLP) or phorbol myristic acid (PMA) and measuring superoxide release. Neutrophil chemotaxis towards fMLP was assessed with and without pre-incubation with VT1 and VT2. We found that neither of the toxins induced superoxide or elastase release. Priming with VT1 significantly reduced superoxide release when neutrophils were stimulated with fMLP or PMA. VT2 priming gave a reduced superoxide release with PMA but not fMLP. Heat-inactivation of the toxins gave similar results. Pre-treatment of neutrophils with VT1 or VT2 did not affect chemotaxis towards fMLP after a 2-hour incubation period. In conclusion, VT1 and VT2 do not activate primed neutrophils in vitro. Nor do they affect chemotaxis towards fMLP. They may impair neutrophil priming.