S100A8/A9 promotes parenchymal damage and renal fibrosis in obstructive nephropathy.

Despite advances in our understanding of the mechanisms underlying the progression of chronic kidney disease and the development of fibrosis, only limited efficacious therapies exist. The calcium binding protein S100A8/A9 is a damage-associated molecular pattern which can activate Toll-like receptor (TLR)-4 or receptor for advanced glycation end-products (RAGE). Activation of these receptors is involved in the progression of renal fibrosis; however, the role of S100A8/A9 herein remains unknown. Therefore, we analysed S100A8/A9 expression in patients and mice with obstructive nephropathy and subjected wild-type and S100A9 knock-out mice lacking the heterodimer S100A8/A9 to unilateral ureteral obstruction (UUO). We found profound S100A8/A9 expression in granulocytes that infiltrated human and murine kidney, together with enhanced renal expression over time, following UUO. S100A9 KO mice were protected from UUO-induced renal fibrosis, independently of leucocyte infiltration and inflammation. Loss of S100A8/A9 protected tubular epithelial cells from UUO-induced apoptosis and critical epithelial-mesenchymal transition steps. In-vitro studies revealed S100A8/A9 as a novel mediator of epithelial cell injury through loss of cell polarity, cell cycle arrest and subsequent cell death. In conclusion, we demonstrate that S100A8/A9 mediates renal damage and fibrosis, presumably through loss of tubular epithelial cell contacts and irreversible damage. Suppression of S100A8/A9 could be a therapeutic strategy to halt renal fibrosis in patients with chronic kidney disease.

Renal endothelial protein C receptor expression and shedding during diabetic nephropathy.

UNLABELLED: Essentials Endothelial protein C receptor (EPCR) promotes diabetic nephropathy (DN) outcome improvement. Renal expression and shedding of EPCR were measured in diabetic patients with or without DN. Inhibition of metalloproteinase-driven EPCR shedding restored glomerular endothelium phenotype. EPCR shedding through metalloproteinase ADAM17 contributes to the worsening of DN. SUMMARY: Background Diabetic nephropathy (DN) represents the leading cause of end-stage renal disease. The endothelial protein C receptor (EPCR) and its ligand (activated protein C) have been shown to ameliorate the phenotype of DN in mice. EPCR activity can be regulated by proteolytic cleavage involving ADAMs, yielding a soluble form of EPCR (sEPCR). Objective To characterize the renal expression and shedding of EPCR during DN. Methods EPCR levels were measured in plasma, urine and biopsy samples of diabetic patients with (n = 73) or without (n = 63) DN. ADAM-induced cleavage of EPCR was investigated in vitro with a human glomerular endothelium cell line. Results DN patients showed higher plasma and urinary levels of sEPCR than diabetic controls (112.2 versus 135.2 ng mL(-1) and 94.35 versus 140.6 ng mL(-1) , respectively). Accordingly, glomerular endothelial EPCR expression was markedly reduced in patients with DN, and this was associated with increased glomerular expression of ADAM-17 and ADAM-10. In vitro, EPCR shedding was induced by incubation of glomerular endothelium in high-glucose medium, and this shedding was suppressed by ADAM-17 inhibition or silencing, which led to improved vascular endothelial cadherin (VE-cadherin) expression and reduced mRNA expression of transforming growth factor (TGF)-ß. In addition, EPCR silencing led to minor effects on VE-cadherin but to a significant increase in TGF-ß mRNA expression. Conclusion Inhibition of ADAM-driven glomerular EPCR shedding restored the endothelial phenotype of glomerular endothelium, whereas EPCR silencing led to enhanced expression of TGF-ß, a marker of endothelial-mesenchymal transition. These findings demonstrate that EPCR shedding driven by ADAMs contributes to the worsening of DN.

The urokinase plasminogen activator receptor is crucially involved in host defense during acute pyelonephritis.

The urokinase plasminogen activator receptor (uPAR) is expressed at the cell surface of inflammatory cells and plays an important role in neutrophil migration. To investigate the in vivo role of uPAR during urinary tract infection, acute pyelonephritis was induced in uPAR-/- and wild-type (WT) mice by intravesical inoculation with 1 x 10(9) colony-forming units (CFU) of uropathogenic Escherichia coli. Mice were killed after 24 and 48 h, after which bacterial outgrowth and cytokine levels in kidney homogenates were determined. Influx of neutrophils was quantified by myeloperoxidase-enzyme-linked immunosorbent assay. uPAR-/- kidneys had significantly higher numbers of E. coli CFU, accompanied by higher levels of interleukin-1beta (IL-1beta), IL-6, keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-alpha (TNF-alpha). However, the number of infiltrating neutrophils was similar in uPAR-/- and WT mice at both time points, suggesting that uPAR-/- neutrophils have a lower ability to eliminate E. coli. To further investigate this, neutrophil oxidative burst and phagocytosis was measured. The generation of reactive oxygen species upon stimulation with E. coli was not diminished in uPAR-/- neutrophils compared with WT. Interestingly, uPAR-/- neutrophils displayed significantly impaired phagocytosis of E. coli organisms compared with WT neutrophils. We conclude that uPAR is crucially involved in host defense through phagocytosis during E. coli induced acute pyelonephritis.

Dendritic cells overexpressing Fas-ligand induce pulmonary vasculitis in mice.

Dendritic cells (DC) genetically engineered to express Fas (CD95) ligand (FasL-DC) have been proposed as immunotherapeutic tools to induce tolerance to allografts. However, we and others recently showed that FasL-DC elicit a vigorous inflammatory response involving granulocytes and can promote Th1-type CD4+ and cytotoxic CD8+ T lymphocytes. This prompted us to evaluate the pathology induced by intravenous injection of FasL-DC in mice. We observed that FasL-DC obtained after retroviral gene transfer of bone marrow precursors derived from Fas-deficient C57Bl/6 mice induce massive pulmonary inflammation and pleuritis one day after a single intravenous injection in C57Bl/6 mice. Two months later, all mice presented granulomatous vasculitis of small to medium sized vessels, alveolar haemorrhage and pleuritis. In these lesions, apoptotic bodies were found in large number. Anti-neutrophilic cytoplasmic and anti-myeloperoxidase autoantibodies were not detected. This study documents that intravenous injection of FasL-DC causes severe lung granulomatous vasculitis. This new animal model for vasculitis is inducible, highly reproducible and shares many features with human Wegener granulomatosis. This model may be an appropriate tool to further investigate the pathogenesis of vasculitis and test new therapeutic strategies. Moreover, our findings highlight the potential severe complications of FasL-DC-based immunotherapy.

Release of urokinase plasminogen activator receptor during urosepsis and endotoxemia.

BACKGROUND: The urokinase receptor (uPAR; CD87) is a multifunctional molecule involved in fibrinolysis, in proteolysis, in renal tubular functions, and in migration and adhesion of inflammatory cells to the site of infection. METHODS: To gain insight into systemic and local release of uPAR and into its regulation during urosepsis, which is one of the leading causes of chronic renal failure, uPAR was measured in urine and plasma of healthy human controls (N = 20), patients with culture-proven urosepsis (N = 30), and healthy human volunteers intravenously injected with endotoxin (N = 7). RESULTS: Patients had elevated uPAR levels in both plasma and urine. Three hours after endotoxin challenge in volunteers, there was also a significant increase of uPAR in plasma and in urine. The urine/plasma ratio for uPAR was highly elevated during urosepsis and experimental endotoxemia, suggesting local production in the kidney. Accordingly, damaged tubuli strongly expressed uPAR during pyelonephritis. Moreover, tubular epithelial cells produced uPAR in vitro, and this secretion was strongly up-regulated after stimulation with interleukin-1 beta or tumor necrosis factor-alpha. CONCLUSIONS: We found that uPAR is released systemically and in the urinary tract during urosepsis and experimental endotoxemia. This systemic and renal production of uPAR during pyelonephritis may play a central role in eliminating the infection and protecting renal function.

Differential expression of T-cell adhesion molecules and LFA-1-dependent intercellular adhesion in HgCl2-induced autoimmunity and immune suppression.

Exposure of Brown Norway (BN) rats to HgCl2 induces Th2-mediated systemic autoimmunity. In contrast, in Lewis rats, HgCl2 induces immune suppression, mediated by CD8+ T cells. HgCl2 was previously found to enhance expression of LFA-1, ICAM-1 and CD134 (OX40) on T cells in BN rats. In the present study, T cells from Lewis rats were studied at day 4 after injection of HgCl2. CD8+ T lymphoblasts were significantly increased, which were predominantly CD45RC(hi), and which showed enhanced LFA-1 expression. Furthermore, CD4+CD45RC(hi) T cells showed increased numbers of ICAM-1+ cells, whereas expression of CD134 and CD26 was relatively decreased in CD4+ T lymphoblasts. Ex vivo experiments demonstrated that HgCl2-exposure of BN rats, but not of Lewis rats, significantly enhances PMA [phorbol 12-myristate 13-acetate]-induced lymphocyte aggregation, mediated by LFA-1 and ICAM-1. In conclusion, HgCl2-injected Lewis rats show early signs of T-lymphocyte activation, predominantly on CD8+ cells. Strain-dependent effects of HgCl2 on cell adhesion molecules and expression of CD134 may play an important role in development of either autoimmunity or immune suppression.

Glomerular extracellular matrix components and integrins.

It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular visceral epithelial and mesangial cells, using molecular probes and antibodies that have recently become available. Special attention was paid to laminin isoforms and to splice variants of the integrin subunits alpha 3 and alpha 6. Results were compared to the in vivo expression in human fetal, newborn and adult kidneys. The mesangial cells were found to produce laminin-1, nidogen and two as yet unidentified laminin isoforms with putative alpha chains of about 395 (alpha x) and of 375 kDa (alpha y), tentatively described before as bovine kidney laminin. Furthermore, they expressed the integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3A beta 1, alpha 5 beta 1, alpha v beta 3, alpha v beta 5, and small amounts of alpha 6A beta 1 and alpha 6B beta 1. The glomerular visceral epithelial cells produced the two new laminin isoforms mentioned above, laminin-5, but no laminin-1 or nidogen. The integrins alpha 2 beta 1, alpha 3A beta 1, alpha 6A beta 4, alpha 6B beta 4 and the integrin subunit alpha v were found to be expressed. We show that during nephrogenesis, the laminin alpha 1 chain disappears and is replaced by another alpha chain, possibly one of the two as yet unidentified alpha chains mentioned above. The laminin beta 1 chain is replaced by the beta 2 chain somewhat later in glomerular development. In general, the integrins found to be expressed in glomeruli of adult kidney were consistent with those found in cultured glomerular visceral epithelial and mesangial cells. No splice variant switch of the integrin alpha 3 or alpha 6 subunits could be demonstrated during nephrogenesis. Our results suggest an important role for the mesangial cell in providing nidogen as a crucial component of the supramolecular structure of the glomerular basement membrane. Furthermore our results indicate that laminin alpha x beta 2 gamma 1 and alpha y beta 2 gamma 1 isoforms are important in the glomerulus of adult kidney and that the integrin alpha 3A beta 1 is the main integrin receptor for laminin isoforms on glomerular visceral epithelial and mesangial cells, both in vitro and in vivo.

Differential regulation of expression of the MHC class II molecules RT1.B and RT1.D on rat B lymphocytes: effects of interleukin-4, interleukin-13 and interferon-gamma.

Susceptibility to induction of both T helper 1- (Th1) and Th2-mediated autoimmunity is multifactorial and involves genetic linkage to the major histocompatibility complex (MHC) class II haplotype. Brown Norway (BN) rats exposed to mercuric chloride develop a Th2-dependent systemic autoimmunity, whereas Lewis rats, which are highly susceptible to Th1-mediated autoimmunity, develop immune suppression after mercuric chloride exposure. Exposure to mercuric chloride is known to enhance B-lymphocyte expression of the MHC class II molecule RT1.B, predominantly in BN rats. We demonstrate that, in contrast, expression of RT1.D was unmodified on these B cells, whereas both RT1.B and RT1.D were up-regulated on epithelial cells. Regulation of B-cell MHC class II isotype expression was further studied in vitro, using BN rat lymph node (LN) cells. Interleukin-4 (IL-4) strongly enhanced B-cell expression of RT1.B (2.8-fold), whereas RT1.D expression was only slightly, although significantly, modified (1.2-fold). B cells from Lewis rats showed a similar IL-4-induced enhancement of RT1.B expression (2.5-fold), whereas, in contrast, RT1.D expression was unmodified. Exposure of LN cells from BN rats to interferon-gamma induced a moderate increase of B-cell MHC class II expression, predominantly of RT1.B. Strong and rapid enhancement of B-cell RT1.D expression was observed after stimulation by phorbol 12-myristate 13-acetate and ionomycin. Rat IL-13 did not modify B-cell MHC class II expression; however, it induced typical morphological changes in peritoneal macrophages. These experiments demonstrate isotype-specific and strain-dependent regulation of MHC class II expression on rat B lymphocytes, which may be of pathophysiological relevance for the strain-dependent susceptibility for Th1- or Th2-mediated autoimmunity.

Thiol levels in CD134-defined subsets of rat T lymphocytes: possible implications for HgCl2-induced immune dysregulation.

CD134 (OX40), a member of the tumour necrosis factor receptor family, is expressed on activated T cells and mediates T and B cell costimulation. Its expression is increased after exposure to the thiol-binding compound HgCl2 in BN rats, but not in Lewis rats, in association with induction of a T cell-dependent systemic autoimmune syndrome only in BN rats. Intracellular thiols are involved in regulation of activation and death in T lymphocytes. Therefore, we examined intracellular thiol levels in CD134-defined T cell subsets from BN and Lewis rats. Levels of total thiols and glutathione (GSH) were significantly higher in CD134+CD4+ cells than in CD134+CD4+ cells in both strains. In Lewis rats, total thiol levels in CD4+CD134+ cells, but not in CD4+CD134+ cells, were higher than in BN rats. In contrast, BN rats showed higher GSH levels in CD4+CD134+ cells, but not in CD4+CD134+ cells. In vitro exposure to HgCl2 decreased intracellular thiol levels, predominantly in CD4+CD134+ cells. Furthermore, HgCl2-induced enrichment of CD134+ viable cells was inversely correlated to HgCl2-induced cell death. Strain-dependent differences in thiol levels in CD134-defined subsets of CD4+ lymphocytes and subset-specific modification of thiol levels may contribute to differential lymphocyte activation by oxidizing chemicals.

Enhanced T lymphocyte expression of LFA-1, ICAM-1, and the TNF receptor family member OX40 in HgCl2-induced systemic autoimmunity.

Injection of mercuric chloride into Brown Norway (BN) rats induces a T lymphocyte-dependent autoimmune syndrome. In order to investigate whether modification of adhesion and costimulatory molecules on T lymphocytes may be involved in early T lymphocyte activation by HgCl2, the authors analysed expression of these molecules in peripheral lymph node cells from BN rats at day 4 after injection of HgCl2. Tri-colour flow cytometry was performed for expression analysis within CD45RC-defined subsets of CD4+ and CD8+ cells. Compared to control rats, HgCl2-exposed rats showed increased numbers of lymphocytes, especially of T lymphocyte blast cells. The levels of LFA-1 expression as well as the fractions of ICAM-1 + cells were significantly increased in all CD45RC-defined subsets of CD4+ and CD8+ cells. Within the CD4 + CD45RC10 T lymphocyte population, HgCl2-injected rats showed a highly significant increase in the number of cells expressing OX40, which is a member of the TNF receptor family. Moreover, only CD4 + CD45RC10 blast cells of HgCl2-exposed rats showed decreased expression of CD43, increased expression of CD49d and decreased numbers of CD26 + cells. The results indicate that induction of autoimmunity by HgCl2 in BN rats is associated with altered expression of T lymphocyte costimulatory molecules, predominantly on CD4+ CD45RC10 cells, which may be caused by a direct effect of HgCl2 on these cells, and may precipitate further activation of T and B lymphocytes by HgCl2.

Autoantibodies to the laminin P1 fragment in HgCl2-induced membranous glomerulopathy.

Exposure to mercuric chloride induces the development of a membranous glomerulopathy with high proteinuria in DZB rats, in which immunoglobulin (Ig)G1 and IgG2a bound in the glomeruli were previously found to react with laminin of the EHS tumor and several unidentified glomerular basement membrane components. Monoclonal antibodies were prepared by fusing cervical and mandibular lymph node cells from a HgCl2-treated DZB rat with a nonsecreting mouse myeloma. Monoclonal antibodies were screened for reactivity with collagenase-digested glomerular basement membrane and kidney sections; upon subcloning, eight stable hybridomas were obtained, named MEC1 to MEC8. MEC2 (IgG1, kappa), MEC3 (IgM, kappa), and MEC5 (IgG1, kappa), as well as the polyclonal glomerular eluate, reacted preferentially with the P1 fragment of the laminin-1 (alpha 1 beta 1 gamma 1) isoform. MEC8 (IgM, kappa) reacted with the P1 and the E4 fragment of laminin. Both MEC6 (IgM, kappa) and MEC8 bound to actin and to various other, unidentified cellular antigens, indicating that MEC6 and MEC8 are polyreactive antibodies. MEC7 (IgM, kappa) bound to a cytoskeleton-linked cell membrane antigen, present on various epithelial cells and between heart muscle fibers and associated with small peripheral, intramuscular nerves. Several of the MEC monoclonal antibodies bound in vivo along the glomerular capillary wall. Although discrete electron-dense subepithelial immune aggregates were not detected and proteinuria was not induced, MEC3 localization changed from a continuous pattern into a fine granular pattern along the glomerular basement membrane, and focally along the TBM, upon passive transfer into naive DZB rats. These findings suggest a pathogenetic role for the P1 fragment of laminin either in the induction phase of HgCl2-induced membranous glomerulopathy as an immunogen or in the effector phase as a target antigen.

Mercuric chloride-induced programmed cell death of a murine T cell hybridoma. I. Effect of the proto-oncogene Bcl-2.

Mercuric chloride (HgCl2) as well as several drugs can induce T cell activation leading to systemic immune-mediated diseases in genetically susceptible individuals or rodents. T cell hybridomas represent a well-characterized model system for in vivo mechanisms of various stimuli-induced cell death. The cellular response to HgCl2 was examined using various T cell lines and particularly the murine T cell hybridoma 2B4.11. Exposure to HgCl2 induced both necrosis and apoptosis in a dose- and time-dependent way as demonstrated by DNA fragmentation analysis, flow cytometry of the whole cells and of isolated nuclei, and morphological examination. HgCl2-induced cell death was partly inhibited by cycloheximide. The expression of human Bcl-2 in 2B4.11 cells after transfection significantly prevented HgCl2-induced cell death but did not affect the susceptibility to apoptosis induced by an anti-CD3 epsilon mAb. Subcytotoxic doses of HgCl2 enhanced metabolic activity of Bcl-2 transfectants in contrast with mock-transfected cell line. Thus, we conclude that apoptosis is part of the cell death process induced by HgCl2 and that the ability of Bcl-2 to prevent the death of one particular cell line is stimulus-dependent suggesting the existence of different pathways leading to cell death.

Increased expression of adhesion receptors in both lesional and non-lesional psoriatic skin.

Adhesion receptors and their ligands play a vital role in the immune system. We studied the expression of different adhesion receptors, using single- and double-staining immunohistochemical techniques, in both lesional and non-lesional skin specimens from seven psoriasis patients and in skin biopsy specimens from eight normal healthy controls. Our results showed an overall increased expression of several adhesion receptors in both lesional and non-lesional psoriatic skin. We consistently found an increased expression in particular of ICAM-1 and E-selectin on endothelial cells, and ICAM-1 on T cells and Langerhans cells. In contrast, a weak expression of VCAM-1 was found on endothelial cells and mononuclear cells in lesional psoriatic skin specimens alone. Interestingly, LFA-1 was also expressed on Langerhans cells, with a greater frequency in skin from lesional than from non-lesional sites, but was never expressed in skin from normal healthy individuals. Furthermore, significantly increased numbers of Langerhans cells and T cells with a positive reactivity for MAb HECA-452 were found in both lesional and non-lesional psoriatic skin. We hypothesize that the enhanced expression of adhesion receptors on migrating immunocompetent cells and endothelial cells of psoriatic skin in general facilitates the increased influx of activated T lymphocytes and other immunocomponent cells into the skin, and thus underscores the generalized character of the disease.

Endothelial cell marker PAL-E reactivity in brain tumor, developing brain, and brain disease.

BACKGROUND: The endothelial cell marker PAL-E is not reactive to vessels in the normal brain. The present study concerns the PAL-E reactivity in brain tumors in contrast to normal brain and nonneoplastic brain disease. METHODS: A total of 122 specimens were examined: brain tumors (n = 94), nonneoplastic brain disease (n = 19), normal brain (n = 8), and fetal brain (n = 1). Standard immunohistochemical procedures using a panel of endothelial cell markers were applied to detect vessels reactive to PAL-E. RESULTS: PAL-E reactivity to endothelial cells was found in all cases of glioblastoma multiforme, in 75% of the cases of anaplastic astrocytoma, and in 46% of the cases of astrocytoma. Furthermore, PAL-E reactivity was present in diseases with a developmental etiology, such as primitive tumors and congenital vascular malformations. The developing human brain (6-weeks' gestation age) and special sites of the mature brain, sites without blood-brain barrier, showed a strong reactivity, which indicates a relation with the status of blood-brain barrier development. CONCLUSIONS: PAL-E is the only marker out of a panel of endothelial cell markers that shows no reactivity to endothelial cells in the normal brain with an intact blood-brain barrier. In primary and metastatic brain tumors, PAL-E is reactive to endothelial cells, except for 25% of anaplastic astrocytoma and 54% of astrocytoma. PAL-E reactivity in brain tumors most likely is related to angiogenesis and to blood-tumor barrier properties not present in the normal blood-brain barrier.

Neuronophagia in the motor cortex in amyotrophic lateral sclerosis.

This study was designed to identify which phagocytic cells in the cerebral cortex of amyotrophic lateral sclerosis (ALS) patients are involved in the process of neuronophagia. For this purpose a number of single and double immunocytochemical stains were carried out on five ALS cases which were selected on the basis of the presence of degenerative and phagocytic phenomena in the cerebral cortex. The cortical degenerative process is mainly present in the third and fifth layers and is not restricted to the fifth layer which contains the cell bodies of the Betz cells. The present study indicates that a number of cells are involved in the process of phagocytosis in ALS. Resident macrophages (from microglial or perivascular origin) and astrocytes seem to play an immunologically-mediated role in the disappearance of neurons. Some of the cells involved in the degenerative process, i.e. rounded macrophages and microglia, expressed major histocompatibility class II antigen. The phagocytic cells in neuronophagia were phenotypically identical to perivascular macrophages and not to microglia. Therefore, the process of phagocytosis of neurons appears to be primarily the task of the perivascularly located macrophage.