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Tissue factor (TF) is a transmembrane protein, which is essential for initiation of the coagulation cascade. TF has been reported to play an important role in the progression of endotoxin (lipopolysaccharide, LPS)-mediated endotoxemia, being induced in numerous tissues, such as kidney, spleen and lung. We developed and validated a rabbit anti-murine TF peptide antiserum to localize TF protein in a murine sepsis model. TF protein distribution was compared to localization of TF mRNA and fibrin deposits, the ultimate resultant of procoagulant TF activity. Evident LPS mediated TF mRNA induction was observed in the tubular area at the cortico-medullar junction in the kidney, and TF activity was increased after 6 hours of endotoxemia. In the spleen, however, TF mRNA was induced in the interfollicular region upon LPS injection, corresponding to increased TF protein in the same area. The clusters of TF-protein positive cells in the spleen are predominantly granulocytes, but no TF mRNA expression was observed within these cells. Based on these observations and the presence of TF-protein positive granulocytes after splenectomy, we hypothesize that granulocytes take-up TF for transport to other locations in order to initiate fibrin formation or to induce pro-inflammatory gene expression upon interaction with factor VIIa.

Background: The use of mouse models for the study of thrombotic disorders has gained increasing importance. Methods for measurement of coagulation activation in mice are, however, scarce. The primary aim of this study was to develop a specific mouse thrombin-antithrombin (TAT) ELISA for measurement of coagulation activation and to compare it with two commercially available assays for human TAT complexes. In addition, we aimed to improve methods for mouse plasma anticoagulation and preparation., Methods and Results: First, for the measurement of TAT-complexes in plasma a mouse specific TAT-ELISA was developed using rabbit polyclonal antibodies raised against mouse thrombin and rat antithrombin, respectively. This ELISA detected an increase in TAT levels in a mouse model of endotoxemia. Two commercial human TAT ELISAs appeared to be less specific for mouse thrombin-rat antithrombin complexes. Second, to prevent clotting of mouse blood sodium citrate was either mixed with blood during collection in a syringe or was injected intravenously immediately prior to blood collection. Intravenous sodium citrate completely inhibited blood coagulation resulting in plasma with consistently low TAT levels. Sodium citrate mixed with blood during collection resulted in increased TAT levels in 4 out of 16 plasma samples. Third, heparinase was added to plasma samples after in vivo injection of different heparin doses to test its neutralizing effect. Heparinase neutralized up to a 20 U of heparin/mouse and resulted in accurate APTT and factor VIII determinations., Conclusion: These procedures and reagents for plasma preparation and coagulation testing will improve studies on thrombotic disorders in mice.

Background: Tissue factor (TF) is the key initiator of the coagulation cascade. Recent evidence suggests that TF plays a role in renal fibrin formation and renal failure in experimental kidney disease. We hypothesized that hyperglycemia is an important stimulus of TF expression in the kidney., Methods: Mice were injected with streptozotocin (STZ) (200 mg/kg) or with control buffer. Three or 10 weeks after injection, fibrin, thrombin and TF staining and TF activity were evaluated in the kidney. The effect of hyperglycemia on TF expression and secretion by tubular epithelial cells was measured in vitro., Results: Kidneys of STZ-treated mice showed a marked increase in thrombin staining (3.0 +/- 0.5 vs. 1.2 +/- 0.11) (p = 0.002) and an increase in TF clotting activity 10 weeks after STZ injection (33.9 +/- 1.3 vs. 25.4 +/- 1.0 s) (p < 0.0001). Increased glomerular fibrin deposition was present in 3 out of 6 diabetic mice. Tubular cells incubated with D-glucose (25 mmol/l) for 48 h displayed increased cellular TF (p = 0.05). However, soluble TF levels and TF activity in supernatant of cells incubated with D- or L-glucose were not different., Conclusions: These data suggest that hyperglycemia is a causal factor in procoagulant activity associated with diabetic nephropathy., (Copyright 2005 S. Karger AG, Basel.)

Objective: To review the dual characteristics of disseminated intravascular coagulation (DIC), as both a contributor to multiple organ failure as well as a symptom of severe underlying disease associated with systemic vascular changes., Data Sources: Published literature data and unpublished results from the authors., Data Summary: Clinical and experimental studies strongly suggest that DIC contributes to multiple organ failure and death in patients with severe systemic disorders such as sepsis. DIC is evoked by systemic cytokine activity, and the inflammatory response aggravates vascular permeability, inflammation, and cell damage in tissues. In addition to intravascular fibrin formation, thrombin and fibrin generation in tissues is also an important aspect of DIC. An example of DIC at the organ level is adult respiratory distress syndrome, where fibrin in the lung is a characteristic feature. Intravascular fibrin formation and occlusion may elicit a hypoxic response with induction of hypoxia related transcription factors. The resulting ischemic preconditioning may offer protective effects to the involved organ(s)., Conclusions: Overall, the beneficial or harmful effects of activated coagulation and fibrin formation for organ pathology and recovery from DIC remain to be explored. This may be a critical element in the assessment of ischemia-reperfusion effects of specific anticoagulant therapy.

Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean +/- SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 +/- 0.02. A progressive and significant (P <.0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 +/- 0.1, +1 hour: 1.3 +/- 0.9, +2 hours: 4.1 +/- 0.9), peaking at +3 hours (10 +/- 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge. (Blood. 2000;96:554-559)

A PVG rat with total deficiency of C6 and partial deficiency of C2 (PVG/c-), and a syngeneic control strain (PVG/c+), were used to study the production of extrahepatically synthesized complement. Livers of complement deficient rats were transplanted in sufficient rats (Tx-L). The C6 and C2 levels in Tx-L rats declined within 2 days to 25% and 30%, respectively, and remained stable for more than 6 weeks. To investigate the contribution of C6 synthesis by the liver, C6 sufficient livers were grafted in deficient rats (Tx + 1). After an initial increase, with maximum C6 levels of 119% at 10 days following transplantation, the C6 levels decreased gradually and C6 was no longer detectable 28 days after transplantation. This decline in C6 levels was dependent on antibody production against C6. No significant change in the C3, C4, factor H and factor B levels was observed. Expression of C6 mRNA in the grafted PVG/c+ sufficient liver was comparable to the expression of C6 mRNA in control PVG/c+ livers while C6 mRNA expression in the transplanted PVG/ c- liver and the control PVG/c- liver was lower. In conclusion, it was demonstrated in vivo that not only C6 but also C2 is synthesized extrahepatically in PVG/c rats.

Viruses may be eliminated by the host immune system via complement-mediated lysis of infected cells. Previously, we have demonstrated that the synthesis of complement factor B by renal mesangial cells (MC) is enhanced by interferon-alpha (IFN-alpha), -beta and -gamma. In the present study we investigate the effect of human cytomegalovirus (HCMV) infection on the production of complement factors by MC. The production of factor B, C2, C4 and factor H by mock-infected MC was 0.2 +/- 0.4, 3.9 +/- 6.8, 1.7 +/- 0.8 and 149 +/- 36 ng/10(6) cells per 72 h, respectively. In HCMV-infected MC cultures an induction of both factor B and C2 protein synthesis was observed up to 2.2 +/- 1.1 and 156 +/- 74 ng/10(6) cells per 72 h, respectively. The synthesis of C4 and factor H of 2.9 +/- 2.0 and 146 +/- 31 ng/10(6) cells, respectively, was not altered significantly. By Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis it was demonstrated that factor B and C2 mRNA expression were up-regulated in HCMV-infected cell cultures, whereas the levels of C4 and factor H mRNA were not changed. When MC cultures were inoculated with heat- or UV-inactivated HCMV no enhancement of factor B mRNA expression was observed. The enhanced expression was not blocked by phosphono acetic acid (PAA), suggesting that expression of the HCMV immediate early or early genes is sufficient to induce complement synthesis. We conclude that infection of MC cultures with HCMV selectively induces complement C2 and factor B production, probably mediated by interferons.

Deposition of immune complexes (ICX), with or without complement, occurs in various forms of glomerulonephritis. It has been reported that upregulation of complement C3 mRNA expression is found in kidneys of patients with ICX glomerulonephritis. In vitro studies have indicated that mesangial cells (MC) synthesize C3. Furthermore, MC express Fc gammaRIII receptors. This study investigates whether ICX alter C3 and factor H production by MC. MC were cultured in medium alone or in medium with insoluble heat-aggregated rat IgG (AIgG) or with insoluble ICX. Basal production of C3 and factor H was 10 +/- 1 ng/10(6) and 605 +/- 15 ng/10(6) cells, respectively. The presence of 400 microg/ml AIgG or ICX resulted in upregulation of C3 production to 999 +/- 15 ng/10(6) and 510 +/- 1 ng/10(6) cells, respectively, whereas no significant change in factor H production was observed. The upregulation of C3 was inhibitable by cycloheximide, suggesting that de novo protein synthesis was required. By reverse transcription PCR and Northern blot analysis, it was demonstrated that C3 and interleukin-6 mRNA expression was upregulated in MC after incubation with AIgG. No detectable change in factor H mRNA expression was seen. In conclusion, it is shown that incubation of MC with AIgG or ICX not only results in upregulated production of inflammatory mediators such as cytokines, but also leads to an upregulation of C3 synthesis. Therefore, it is hypothesized that ICX deposited within the mesangium may enhance the local production of C3 via interaction with Fc receptors on MC.

Complement C6 plays an important role in the effector phase of complement-mediated cell lysis. Recently, a PVG/c rat strain deficient in haemolytic C6 activity was discovered. In the present study we show that these rats lack both antigenic and functional C6, and that repetitive immunization of these rats with PVG/c+ serum results in generation of specific anti-rat C6 antibodies. The observed absence of rat C6 was further investigated at the genomic and transcriptional level using a 492-bp cDNA of rat C6, cloned from a rat liver cDNA library using full length human C6 as a probe. Northern blot analysis revealed the presence of C6 mRNA in livers of both PVG/c- and PVG/c+ rats, corresponding to a size of approximately 3.3 kb, although the level of C6 mRNA expression was approximately 100-fold less in PVG/c- rats. In addition, using rat C6-specific primers, positive signals were obtained in kidneys of both rat strains by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Southern blot analysis of digested genomic DNA did not reveal evidence for large C6 gene deletions. We conclude that the lack of C6 protein in the PVG/c- rat strain is not due to a (large) C6 gene deletion, but presumably is caused by an unstable mRNA or a point mutation in the C6 gene resulting in an aberrant transcription of the C6 gene. Alternatively, a gene coding for a product involved in C6 biosynthesis that acts in trans may carry a mutation.

Infiltration of the glomerular mesangium by monocytes and macrophages is a central pathologic feature in various forms of glomerulonephritis. Dependent on the presence and activity of local survival factors, monocytes may undergo apoptosis. Therefore, we looked for the interaction between cultured human mesangial cells (HMC) and intact, necrotic or apoptotic monocytic cells with different stages of programmed cell death (U937 cells and blood-derived human monocytes) and the possible evoked secretory responses of HMC. Interleukin-6 (IL-6) synthesis of HMC after a two hour co-culture with late apoptotic U937 cells was significantly increased (505 +/- 55 pg/ml) as compared to intact U937 cells (349 +/- 27 pg/ml; HMC alone, 319 +/- 62 pg/ml), and was further elevated after 20 hours (815 +/- 108 pg/ml). U937 cells alone, after incubation in HMC-conditioned medium or after coincubation with HMC, did not produce any detectable IL-6. A high mesangial IL-6 synthesis in response to apoptotic U937 cells was dependent on the cellular contact between HMC and U937 and could not be mimicked by apoptotic U937 culture supernatants. Radiolabeling studies indicated that HMC bound (16.6 +/- 2.4%) and ingested (12.5 +/- 1.9%) apoptotic U937 cells to a much higher amount as compared to intact U937 (5.3 +/- 2.0% binding; 5.0 +/- 1.1% phagocytosis). Binding and ingestion of monocytic cells undergoing apoptosis was confirmed by morphologic studies using electron microscopy. Incubation of HMC with a blocker of the CD36/ vitronectin receptor (VnR) dependent recognition mechanism of phagocytes for apoptotic leukocytes (RGDS peptide) did not alter binding, phagocytosis or IL-6 synthesis of HMC in response to apoptotic U937. Phospho-L-serine as an antagonist of the phosphatidylserine (PS) mediated recognition pathway for apoptotic cell disposal was able to reduce binding and IL-6 production by HMC but not phagocytosis. Thus, binding of apoptotic monocytic cells by HMC rather than ingestion may be the prerequisite for a stimulated secretory response. To elucidate whether binding and phagocytosis of particles in general might stimulate HMC to produce IL-6, we looked for mesangial IL-6 production after binding and ingestion of opsonized zymosan particles. In this case, IL-6 synthesis was markedly down-regulated. Furthermore, HMC proliferated after zymosan treatment, whereas after apoptotic cell uptake the mesangial cell number remained constant. In conclusion, apoptotic monocytic cells provoked an enhanced mesangial IL-6 synthesis by a PS-dependent recognition mechanism. This secretory response may have secondary implications for humoral or cellular processes within the mesangium.

In order to study the contribution of extrahepatic C6 to anti-Thy1.1 nephritis, C6 deficient PVG/c- livers were grafted in C6 sufficient PVG/c+ rats (Tx-L). Infusion of anti-Thy1.1 antibodies in Tx-L and PVG/c+ rats resulted in generation of C5b-9 complexes and subsequent glomerular injury, while infusion of anti-Thy1.1 antibodies in PVG/c- rats revealed no detectable C6 deposition. Because C6 mRNA was expressed in both liver and kidney tissue of PVG/c+ rats, we assessed whether production of C6 in the kidney alone was sufficient for glomerular injury. One kidney of a PVG/c- rat was replaced with a PVG/c+ kidney (Tx + K) followed by administration of anti-Thy1.1 antibodies. C6 deposits were detectable neither in PVG/c+ kidneys nor in PVG/c- kidneys of Tx + K rats, indicating that C6 production in PVG/c+ kidneys alone is not sufficient to contribute to renal injury. That C6 production had occurred was suggested by the presence of equal amounts C6 mRNA in control PVG/c+ kidneys and in grafted PVG/c+ kidneys of Tx + K rats. C6 mRNA expression in kidney tissue of PVG/c+ rats is presumably derived from peritubular sites. In conclusion, we have demonstrated that extrahepatic, but not renal synthesis of, C6 is sufficient to contribute to glomerular injury during anti-Thy1.1 nephritis.

Various studies have shown that complement components are synthesized by renal cells and that mRNA for a number of complement components is detectable in renal tissue. The present study shows that complement proteins are present both in fetal and adult human kidneys. The localization of the complement components was compared with the localization of other proteins for which specific expression in defined renal cell types is known from the literature. In adult human kidneys C3, factor B and factor H were detected in the mesangial area by immunohistochemistry, whereas C2 and C4 were present in the proximal tubuli. In fetal kidneys C3 and factor B were expressed in glomeruli of kidneys of 11 weeks of gestation. In kidneys of 13 to 19 weeks of gestation no staining for C3 was found in the glomerulus, whereas for factor B glomerular staining was found in all fetal kidneys examined. Factor B was also detected in fetal tubuli and in the interstitium. Factor H was expressed in fetal tubuli starting at 13 weeks of gestation. For both C3 and C2 weak tubular staining was found in all fetal kidneys investigated. C4 could not be detected in any of the fetal kidneys. While not all the complement proteins investigated were detectable by immunohistochemistry, by RT-PCR analysis, mRNA expression for C3, factor B, factor H, C2 and C4 was found in all adult and fetal renal tissue. The finding of mRNA for the complement components in the fetal and the adult kidneys indicates that local synthesis of complement occurs both in the adult and in the fetal kidney. Next to the in situ expression of complement components in fetal kidneys the synthesis of complement proteins in vitro by fetal renal cells was investigated. Four different primary mesangial cell lines were shown to synthesize all complement proteins investigated. Although a specific role for complement during the development of the kidney is not known, it is possible that certain complement components may play a role during renal differentiation.

Local production of complement within normal or diseased kidneys could be of importance during local inflammatory reactions. In the present study, we demonstrate that human MCs are able to synthesize the MHC class-III-encoded complement proteins factor B and C4 in vitro. This synthesis is strongly upregulated following stimulation with cytokine-containing supernatants of activated peripheral blood mononuclear cells. All primary cell lines tested so far are able to synthesize factor B and C4 after stimulation. To determine more specifically whether defined cytokines are able to enhance factor B and C4 complement production, MCs were stimulated with IL-1 alpha, IFN-gamma, and TNF-alpha. Factor B synthesis was increased in a dose-dependent fashion by IL-1 alpha, TNF-alpha, and IFN-gamma, whereas C4 synthesis was only upregulated by IFN-gamma. Furthermore, factor B synthesis was upregulated after stimulation with IFN-alpha, -beta, and -gamma and C4 synthesis only by IFN-gamma. The synthesis of factor B and C4 was inhibited by cycloheximide, suggesting de novo protein synthesis. The cytoplasmic localization of both components was shown by immunofluorescence studies. Northern and dot blot analysis revealed induction of factor B and C4 mRNA after stimulation with cytokines.

Previous reports have shown production of complement components C4, C2 and factor B by renal tissue. We have shown recently that human proximal tubular epithelial cells (PTEC) synthesize C3 in vitro, and that IL-2 enhances this production. In the present study we demonstrate that human mesangial cells (MC) in culture produce factor H and that supernatants of activated peripheral blood mononuclear cells (T cell growth factor (TCGF)) induce C3 production and enhance factor H synthesis in both a time- and dose-dependent manner. To investigate whether certain defined cytokines from TCGF were responsible for the observed effect, we tested various cytokines for their effect on complement production by MC. It is shown that IL-1 induces C3 synthesis whereas factor H production is up-regulated by IFN-gamma, in both a dose- and time-dependent manner. Antibody blocking experiments revealed that C3 synthesis induced by both TCGF and IL-1 could be blocked with antibodies specific for IL-1, and also that TCGF and IFN-gamma enhanced factor H synthesis could both be blocked with antibodies specific for IFN-gamma. Cycloheximide was able to inhibit C3 and factor H production, suggesting de novo synthesis of the proteins. mRNA-polymerase chain reaction (PCR) analysis revealed mRNA encoding for C3 after stimulation with TCGF and IL-1. Factor H genes are constitutively expressed in cultured mesangial cells and its expression is up-regulated by TCGF and IFN-gamma. Northern blot analysis with specific probes for C3 and factor H revealed bands which support the results obtained by PCR analysis.