Christopher J. Brooks

IL‐1β production by human polymorphonuclear leucocytes stimulated by anti‐neutrophil cytoplasmic autoantibodies: relevance to systemic vasculitis

Neutrophils accumulate in the acute blood vessel lesions of patients with autoimmune systemic vasculitis. They have been shown previously to produce the cytokine IL‐1β in response to stimulation with TNF. This study demonstrates that neutrophils can be stimulated by anti‐neutrophil cytoplasmic antibodies (ANCA), which are present in patients with systemic vasculitis, to express mRNA and protein for IL‐1β. Both human ANCA and MoAbs to a variety of autoantigens recognized by ANCA, including proteinase 3, myeloperoxidase, bactericidal/permeability increasing protein and elastase, are effective. This response can be inhibited by actinomycin and cycloheximide, suggesting a requirement for de novo protein synthesis. IL‐1β production can be inhibited by pooled human intravenous immunoglobulins but not by FK506 or cyclosporin A. These data suggest that ANCA in patients with active vasculitis may stimulate neutrophils to produce cytokines. It is hypothesized that cytokine production from neutrophils that accumulate in significant numbers in vasculitic lesions contribute to and augment the local inflammatory response by the activation of vascular endothelial cells and infiltrating leucocytes.

T lymphocyte responses to anti-neutrophil cytoplasmic autoantibody (ANCA) antigens are present in patients with ANCA-associated systemic vasculitis and persist during disease remission

ANCA with specificity for myeloperoxidase (MPO) and proteinase 3 (PR3) are present in patients with systemic vasculitis. The aim of this work was to determine whether such patients have T cell responses to these antigens and whether these responses are related to disease activity. Peripheral blood lymphocytes from 45 patients and 19 controls were cultured with ANCA antigens and proliferation measured. The antigens used were heat‐inactivated (HI) MPO, HI PR3, native (non‐HI) PR3, HI whole α‐granules, and 25 overlapping peptides covering the entire PR3 sequence. Significant responses to both whole PR3 preparations were seen from patient and control groups, and to the α‐granules from the patient group. Patients responded at all stages of disease: active, remitting, treated or untreated. Only two patients responded significantly to MPO. Responses were significantly higher with the patient group than the control group to all four whole ANCA antigens. Responses to those PR3 peptides containing epitopes known to be recognized by ANCA were detected from one patient. Thus, these studies demonstrate that T cells from vasculitis patients can proliferate to PR3 and occasionally to associated ANCA antigens. Further, responses may persist even after disease remission has been achieved.

Endothelial cells and renal epithelial cells do not express the Wegener's autoantigen, proteinase 3

Proteinase 3 (PR3) is the major antigen for autoantibodies (C‐ANCA) against cytoplasmic components of neutrophils which are strongly associated with Wegeners granulomatosis (WG). Recent data that PR3 may be expressed by renal tubular epithelial cells and endothelial cells suggest potential for a direct pathogenic effect against these cells by C‐ANCA or cytoxic T lymphocytes. Using a semi‐quantitative polymerase chain reaction (PCR), ELISA and indirect immunofluorescence staining we studied endothelial and epithelial cell PR3 expression. By PCR, no PR3 expression was found in human umbilical vein endothelial cells (HUVEC) either untreated, or when treated with interferon‐gamma (IFN‐γ) (200 U/ml, 6h, 24h), IL‐1 (20U/ml, 6h), tumour neurosis factor‐alpha. (TNF‐α) (200U/ml. 0. 1.2, 4, 6h) or IFN‐γ+ TNF‐a (6h); iliac vein and artery endothelial cells did not express PR3 either. In contrast. PR3 was detected in HL60 cells and neutrophils by PCR. expression being confirmed by sequence analysis. Three PR3 MoAbs showed no binding to unstimulated or TNF‐α‐stimulated HUVEC either by ELISA or by indirect immunofluorescence staining. The epithelial cell line A549 expressed PR3 when assayed by PCR. However, three renal epithelial cell lines (two tubular and one glomerular) showed little or no PR3 expression by PCR or ELISA. These studies fail to demonstrate evidence for PR3 expression by endothelial cells, even when using the highly sensitive PCR assay. Whilst PR3 expression by A549 cells is intriguing, the relevance of this in the pathology of WG is doubtful considering the negligible expression by renal epithelial cell lines.

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‐α, 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.

CELL ADHESION MOLECULE EXPRESSION WITHIN HUMAN GLOMERULAR AND KIDNEY ORGAN CULTURE

A glomerular and kidney organ culture method was developed to study cytokine inducibility of adhesion molecules and MHC antigen expression. Glomerular cells showed constitutive expression of intercellular cell adhesion molecule‐1 (ICAM‐1) and MHC class I and II antigens, but not vascular cell adhesion molecule‐1 (VCAM‐1), E‐selectin, or P‐selectin. Expression of E‐selectin on glomerular endothelial cells (ECs) was induced by interleukin‐1 (IL‐1), tumour necrosis factor (TNF), interferon‐γ (IFN‐γ) and granulocyte/macrophage‐colony stimulating factor (GM‐CSF); this induction was inhibited by transforming growth factor beta (TGFβ) and by IL‐4. P‐selectin expression was never seen within glomeruli. VCAM‐1 was constitutively expressed by Bowmans capsule and proximal tubules and was weakly induced on glomerular ECs by TNF and IL‐4 in combination. Glomerular endothelial ICAM‐1 expression was increased by IL‐1, TNF, IFN‐γ, and GM‐CSF, while TGFβ inhibited induction by TNF and IL‐1. Expression of MHC class I and II antigens by glomerular ECs was constitutive; further upregulation of MHC class II by IFN‐γ was observed. These studies suggest that leukocyte–endothelial cell interactions that occur within the kidney follow broadly similar principles as are proposed to occur elsewhere in the body but, in addition, there are subtle differences that reflect local conditions.

Human vascular endothelial cells do not induce anergy in allogeneic CD4+ T cells unless costimulation is prevented.

Human vascular endothelial cells expressing MHC class II molecules have previously been shown to stimulate the proliferation of allogeneic CD4+ human T lymphocytes. Here we show that allogeneic CD4+ T cells from individual A (TA) respond to class II+ endothelial cells from individual B (EB) by inducing interleukin (IL)-2 mRNA, detectable by semiquantitative polymerase chain reaction, within 12 hr. Responding T cells (TA) that are harvested after 12 hr, rested for 3 days, and then re-exposed to the same class II+ EB stimulators can again respond by proliferation that is equivalent in degree to that observed with third-party class II+ endothelial cells (EC) as stimulators and a little greater than that observed in the primary responses. Incorporation of antibodies to LFA-3, an endothelial costimulatory molecule for T cells, or to both IL-2 and IL-2 receptor (R) during the first-round stimulation prevented the subsequent second-round proliferation of TA to class II+ EB but not to class II+ EC. This nonresponsiveness induced by anti-LFA-3 or anti-IL-2/IL-2R could be overcome by the incorporation of cyclosporine during the first-round stimulation or by incorporation of IL-2 during the second-round stimulation. These observations suggest that class II+ endothelial cells within allografts will not induce anergy in host CD4+ T cells unless costimulation is blocked or the ability of CD4+ T cells to respond by proliferation is prevented; even then the response may be modified by prevailing cyclosporine or IL-2 levels.