Proteinase 3/myeloblastin as a growth factor in human kidney cells.

BACKGROUND: Numerous data support the possible role of myeloblastin/proteinase 3 (PR3) in growth and differentiation of neutrophil granulocytes and certain monocyte subtypes. However, whether PR3 is expressed in non-myeloid cells remains a matter of debate even though recent studies clearly demonstrated its expression in endothelium, kidney epithelial cells and epithelial tumor cell lines. METHODS: To survey PR3 transcript presence in human tissues, we analyzed different human tissues by dot blot and northern blot using a cloned PR3-cDNA probe. To examine the physiological function of PR3 expression in non-myeloid cells, we constructed different recombinant retroviral vectors containing human PR3-cDNA variants and expressed them in tubular epithelial cells (TEC). Using an MTT-based proliferation assay, we determined the proliferation rate of PR3-transduced kidney cells. RESULTS: The resulting expression pattern clearly indicated that PR3 transcripts are not only present in tissues known to harbor hematopoietic cells, but surprisingly PR3 was highly expressed in fetal organs including the kidney. The proliferation assay revealed that the growth rate of TEC transduced with native PR3 was significantly enhanced relative to non-transduced TEC. CONCLUSIONS: The results supported our theory that PR3 can act as a growth factor in non-hematopoietic cells, analogous to its role in hematopoietic cells. The cells, recombinant vectors and methods described here serve as a basis to investigate PR3 function in cellular differentiation and proliferation, as well as its role in autoimmune diseases.

Correlation of renal tubular epithelial cell-derived interleukin-18 up-regulation with disease activity in MRL-Faslpr mice with autoimmune lupus nephritis.

OBJECTIVE: MRL-Fas(lpr) mice spontaneously develop an autoimmune disease that mimics systemic lupus erythematosus in humans. Infiltrating T cells expressing interferon-gamma (IFNgamma) are responsible for the autoimmune kidney destruction in MRL-Fas(lpr) mice, and interleukin-18 (IL-18) released by mononuclear phagocytes stimulates T cells to produce the IFNgamma. Since MRL-Fas(lpr) T cells are characterized by an overexpression of the IL-18 receptor accessory chain, we sought to determine the impact of IL-18 on the progression of lupus nephritis in MRL-Fas(lpr) mice. METHODS: IL-18 expression in sera and kidney tissues from MRL-Fas(lpr) mice was determined by enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blotting. IL-18 production by primary cultured tubular epithelial cells (TECs) from MRL-Fas(lpr) and BALB/c mice were examined by RT-PCR, ELISA, and Western blotting. The interactions of TEC-derived IL-18 and MRL-Fas(lpr) T cells were studied in coculture assays. IL-18-related effects on TEC viability and adhesion molecule expression were determined by fluorescence-activated cell sorting and cell proliferation assays. RESULTS: Up-regulation of mature IL-18 was restricted to nephritic MRL-Fas(lpr) kidneys and increased in parallel with the severity of lupus nephritis. IL-18 expression was not confined to infiltrating monocytes but was primarily detected in TECs. Similarly, interleukin-1beta-converting enzyme expression, which is required for the processing of precursor IL-18, was localized in TECs. De novo synthesis of IL-18 by MRL-Fas(lpr) TECs was confirmed by RT-PCR and Western blotting. Functional assays revealed that activated TECs induced IFNgamma production in MRL-Fas(lpr) T cells through IL-18. IL-18, in turn, increased apoptotic TEC death and up-regulation of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. CONCLUSION: Taken together, our findings suggest that IL-18-producing TECs may directly be involved in the pathogenesis of lupus nephritis.

Regulation of organic anion transporters in a new rat model of acute and chronic cholangitis resembling human primary sclerosing cholangitis.

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiology. Although the primary defect affects cholangiocytes, cholestatic injury of hepatocytes may promote further liver damage. Since down-regulation of hepatocellular organic anion transporters is implicated in the molecular pathogenesis of cholestasis, expression of these transporters was determined in a novel rat model, which closely resembles human PSC. METHODS: Hepatic protein and mRNA expression of basolateral (Ntcp, Oatp1, 2 and 4) and canalicular (Mrp2, Bsep) organic anion transporters were analyzed 1, 4 and 12 weeks after induction of experimental PSC by 2,4,6-trinitrobenzenesulfonic acid (TNBS). RESULTS: Specific down-regulation of basolateral and canalicular transport systems except Oatp4 and Bsep proteins occurred during the acute phase of inflammation. In chronic cholangitis 12 weeks after TNBS Mrp2 protein and mRNA remained down-regulated by 40-50% of controls (P<0.05). In addition Oatp1 protein was also reduced by 40+/-13% (P<0.05), whereas all other transporters returned to control values. CONCLUSIONS: In chronic cholangitis only canalicular Mrp2 expression remained down-regulated. This might represent the first injury to hepatocytes in chronic cholangitis as an extension of liver injury from the level of cholangiocytes to hepatocytes in PSC.

Proteinase-3 as the major autoantigen of c-ANCA is strongly expressed in lung tissue of patients with Wegener's granulomatosis.

Proteinase-3 (PR-3) is a neutral serine proteinase present in azurophil granules of human polymorphonuclear leukocytes and serves as the major target antigen of antineutrophil cytoplasmic antibodies with a cytoplasmic staining pattern (c-ANCA) in Wegener's granulomatosis (WG). The WG disease appears as severe vasculitis in different organs (e.g. kidney, nose and lung). Little is known about the expression and distribution of PR-3 in the lung. We found that PR-3 is expressed in normal lung tissue and is upregulated in lung tissue of patients with WG. Interestingly, the parenchymal cells (pneumocytes type I and II) and macrophages, and not the neutrophils, express PR-3 most strongly and may contribute to lung damage in patients with WG via direct interaction with antineutrophil cytoplasmic antobodies (ANCA). These findings suggest that the PR-3 expression in parenchymal cells of lung tissue could be at least one missing link in the etiopathogenesis of pulmonary pathology in ANCA-associated disease.

Interaction of antibodies to proteinase 3 (classic anti-neutrophil cytoplasmic antibody) with human renal tubular epithelial cells: impact on signaling events and inflammatory mediator generation.

Among the anti-neutrophil cytoplasmic Abs (ANCA), those targeting proteinase 3 (PR3) have a high sensitivity and specificity for Wegener's granulomatosis (WG). A pathogenetic role for these autoantibodies has been proposed due to their capacity of activating neutrophils in vitro. Recently, PR3 was also detected in human renal tubular epithelial cells (TEC). In the present study, the effect of murine monoclonal anti-PR3 Abs (anti-PR3) and purified c-ANCA targeting PR3 from WG serum on isolated human renal tubular cell signaling and inflammatory mediator release was characterized. Priming of TEC with TNF-alpha resulted in surface expression of PR3, as quantified in immunofluorescence studies and by flow cytometry. Moreover, PR3 was immunoprecipitated on surface-labeled TEC. Primed TEC responded to anti-PR3 with a dose- and time-dependent activation of phosphoinositide hydrolysis, resulting in a remarkable accumulation of inositolphosphates. Control IgG was entirely ineffective, whereas PR3-ANCA reproduced the phosphoinositide response. The signaling response was accompanied by a pronounced release of superoxidanion into the cell supernatant. Moreover, large amounts of PGE(2) and, to a lesser extent, of thromboxane B(2), the stable metabolite of TxA(2), were secreted from anti-PR3-stimulated TEC. In parallel, a rise in intracellular cAMP levels was observed, which was blocked by the cyclooxygenase inhibitor indomethacin. We conclude that anti-PR3 Abs directly target renal TECs, thereby provoking pronounced activation of the phosphoinositide-related signal transduction pathway. Associated metabolic events such as the release of reactive oxygen species and lipid mediators may directly contribute to the development of renal lesions and loss of kidney function in WG.