Inhibition of neutrophil-mediated production of reactive oxygen species (ROS) by endothelial cells is not impaired in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis patients.

Leucocyte transendothelial migration is strictly regulated to prevent undesired inflammation and collateral damage of endothelial cells by activated neutrophils/monocytes. We hypothesized that in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis (AAV) patients' dysregulation of this process might underlie vascular inflammation. Peripheral blood mononuclear cells (PBMC) and neutrophils from AAV patients (n = 12) and healthy controls (HC, n = 12) were isolated. The influence of human umbilical vein endothelial cells (HUVEC) on neutrophil/monocytes function was tested by N-formyl-methionyl-leucyl-phenyl-alanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-mediated ROS production, degranulation and interleukin (IL)-8 production. In addition, the ability of lipopolysaccharide (LPS)-stimulated PBMC to produce tumour necrosis factor (TNF)-alpha in the presence or absence of HUVEC was tested. HUVEC inhibited ROS production dose-dependently by fMLP-stimulated neutrophils but did not influence degranulation. No differences between neutrophils from HC and AAV were found. However, in only one active patient was degranulation inhibited significantly by HUVEC only before cyclophosphamide treatment, but not 6 weeks later. Co-cultures of HUVEC with LPS-stimulated neutrophils/monocytes increased IL-8 production while TNF-alpha production was inhibited significantly. There was no apparent difference between AAV patients and HC in this respect. Our findings demonstrate that HUVEC are able to inhibit ROS and modulate cytokine production upon stimulation of neutrophils or monocytes. Our data do not support the hypothesis that endothelial cells inhibit ROS production of neutrophils from AAV patients inadequately. Impaired neutrophil degranulation may exist in active patients, but this finding needs to be confirmed.

Imatinib mesylate, a new kid on the block for the treatment of anti-neutrophil cytoplasmic autoantibodies-associated vasculitis?

Persistent T cell activation is a common finding in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitis (AAV) patients. Because imatinib, a selective inhibitor of the ABL, ARG, PDGFR and c-KIT tyrosine kinases, inhibits T cell activation, this study was conducted to evaluate the potential use of imatinib for the treatment AAV patients refractory to conventional therapy. In particular, we investigated the inhibition of T cell activation by this drug and its efficacy on activated T cells from anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitides (AASV) patients. T cell stimulation has been induced by anti-CD3/anti-CD28 antibodies or by phorbol myristate acetate (PMA)/ionomycin. T cell proliferation was analysed by tritiumthymidine incorporation. Cell cycle progression was determined by propidium iodide staining using fluorescence activated cell sorter (FACS) analysis and by RNAse protection assay (RPA). Cytokine levels were assessed by enzyme-linked immunosorbent assay. T cell proliferation was inhibited significantly by imatinib, due most probably to cell cycle arrest in the G1-phase. This was paralleled by inhibition in the expression of cyclin-dependent kinases 1 and 2 mRNA. The expression of CD25 in naive and memory T cells was decreased significantly by imatinib in activated T cells. Similarly, conversion from naive to memory T cells after T cell activation was impaired by imatinib. Imatinib did not influence interleukin-2 and tumour necrosis factor-alpha production but increased interferon-gamma production. These observed effects of imatinib were similar in T cells from AASV patients and from healthy individuals. Imatinib might be an alternative therapeutical option for AASV patients refractory to conventional therapy.

In vivo effects of cyclic administration of 15-deoxyspergualin on leucocyte function in patients with Wegener's granulomatosis.

15-Deoxyspergualin (DSG) is an alternative treatment modality for Wegener's granulomatosis (WG) patients refractory to conventional treatment. Nevertheless, it is unclear how DSG modulates disease activity in these patients. This study was conducted to investigate which parameters of adaptive and acquired immunity were influenced during two subsequent cycles of DSG treatment. Emphasis was put upon T cell and monocyte activation, neutrophil function and surface expression of proteinase-3 (PR-3). Anti-CD3/anti-CD28 and interleukin (IL)-15/IL-7-mediated T cell proliferation were assessed by fluorescence activated cell sorter (FACS) analysis using carboxyfluorescein succinimidyl ester (CSFE) labelling. Interferon (IFN)-gamma and IL-10 production were determined in the supernatants of these cultures by enzyme-linked immunosorbent assay. Monocyte activation was assessed in lipopolysaccharide (LPS)-stimulated whole blood, using tumour necrosis factor (TNF)-alpha as read-out. Neutrophil function was determined by measuring oxidative burst, chemotaxis and phagocytosis. T cell activation markers and PR3 expression were measured by FACS. All parameters were determined directly before and after each DSG cycle. Anti-CD3/anti-CD28-mediated T cell proliferation was reduced directly after DSG treatment. Directly before a subsequent cycle of DSG was started, T cell proliferation was increased. Similar findings were observed for IFN-gamma and IL-10 production by T cells. DSG did not influence IL-15/IL-7-mediated T cell proliferation. LPS-mediated TNF-alpha production was also impaired directly after DSG treatment. No influence on T cell activation markers, neutrophil function and surface PR-3 expression was observed in peripheral blood of these patients. Our data demonstrate that DSG influences T cell and monocyte activation in a reversible fashion. Although DSG causes neutropenia in these patients, it does not influence neutrophil function.

The role of T lymphocytes in ANCA associated vasculitis: neglected or revisited?

"Antineutrophil cytoplasmic antibodies (ANCA) are pathogenic--Oh yes they are!" is the title of a recent review [Falk et al. 2002], discussing the current evidence on the pathogenic role of ANCA in vasculitis. But what about T lymphocytes? Do these cells also contribute to disease manifestation and if so to what extend? T-cells most likely play a role in delivering proper signals to autoreactive B cells for the production of ANCA, but, in the efferent arm of the immune response the involvement of T cells is less obvious and controversially discussed. Numerous studies provide evidence that peripheral T-cell phenotypes are dramatically changed in ANCA associated vasculitis (AAV) patients. How these changes relate to disease manifestation is still a matter of discussion. In an attempt to provide a better understanding of how T cells might play a role in AAV, the present paper will review recent data presented at the 12th international vasculitis and ANCA workshop.

Abnormalities of CD4 T cell subpopulations in ANCA-associated vasculitis.

In patients with ANCA-associated vasculitis (AAV), CD25 expression is increased on circulating T cells. Although in animal experiments the role of CD4(+) CD25(+) T-regulatory-cells (T(reg)) in protection against autoimmunity is well established, the role of these cells in AAV is unknown. To investigate the hypothesis that an increased expression of CD25 on T cells is related to persistent T cell activation and not to disturbances in T(reg) cells in AAV (34 patients, six of them after renal transplantation), we investigated CD25 expression in different subpopulations of CD4(+) cells and FOXP3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). In addition, T cell proliferation and cytokine secretion after stimulation with anti-CD3 and anti-CD28 and intracellular cytokine production after stimulation with phorbol myristate acetate (PMA)-ionomycin was determined. Controls were non-vasculitic renal transplant patients (n = 9) and healthy controls (HC) (n = 13). In AAV the total number of lymphocytes, CD4(+) lymphocytes and the percentage of naive T cells are lower than in HC and RTX. An increased percentage of CD25(+) cells was found in AAV and AAV/RTX, irrespective of disease activity, but not in HC or RTX. This was confined to the naive (CD4(+) CD45RB(high)) population only. FOXP3 mRNA expression in CD4(+) T cells did not differ between AAV patients and healthy controls. In vitro T cell proliferation was enhanced in AAV patients compared to HC (P < 0.01). PBMC of AAV patients produced significantly less interleukin (IL)-10 and interferon (IFN)-gamma after anti-CD3/CD28 stimulation. The percentage of IL-10 and IL-12, but not IFN-gamma, IL-4 or tumour necrosis factor (TNF)-alpha-producing cells was significantly higher in patients compared to HC. These findings were confined to the memory population of CD4(+) cells. We conclude that AAV patients are lymphopenic and have low numbers of CD4(+) T cells, which seem to be in a persistent state of activation.