New insight into the effects of heparinoids on complement inhibition by C1-inhibitor.

Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1-esterase inhibitor (C1-INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1-INH. The inhibitory capacity of C1-INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1-INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1-INH on coagulation were investigated. C1-INH, heparinoids or combinations were analysed in a dose-dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa(®) -kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1-INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1-INH significantly on the CP and LP. For the AP, significant potentiation of C1-INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1-INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1-INH. Therefore, their combined use is a promising and a potentially cost-effective treatment option for complement-mediated diseases.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Combined C4d and CD3 immunostaining predicts immunoglobulin (Ig)A nephropathy progression.

A number of molecules have been shown recently to be involved in the pathogenesis and progression of immunoglobulin (Ig)A nephropathy (IgAN). Among these, we have selected C4d (complement lectin pathway involvement), CD3 (T cell marker, traducing interstitial inflammation), transglutaminase 2 (TGase-2, involved in tissue fibrosis development) and p-extracelluar-regulated kinase (ERK)1/2 (protein kinase intracellular signaling molecule) to perform a panel of immunohistological biomarkers and assess its predictive value for disease progression. Immunohistochemical staining of these biomarkers was performed in paraffin sections from 74 renal biopsy cases with the clinical diagnosis of IgAN. Association between score analysis of these parameters and disease course was assessed through univariate and multivariate analysis, including baseline clinical and histological data. Univariate analysis showed that glomerular C4d, tubulointerstitial TGase2 and CD3 scores were associated with baseline proteinuria and disease progression. Multivariate analysis showed that only baseline estimated glomerular filtration rate (eGFR), C4d and CD3 were associated independently with progressive kidney disease (decline of at least 50% in the eGFR or progression to end-stage renal disease (ESRD) during the follow-up period). Establishing an accurate prediction model for IgAN progression is still a matter of research in clinical nephrology. The complement system, particularly lectin pathway activation, and T cell activation, have been shown previously to be potential modifiers of the disease course. Here we show that the combination of two histological biomarkers (C4d and CD3) can be a powerful predictor of IgAN progression and a potential useful tool for the clinical approach of this disease.

Functional assessment of rat complement pathway activities and quantification of soluble C5b-9 in an experimental model of renal ischemia/reperfusion injury.

There is a growing interest in the monitoring of complement activation, not only in clinical settings but also in experimental models. However, for rodents only a limited number of tools are available to assess complement activity and activation. Here we describe three ELISAs for the measurement of rat classical (CP), MB-lectin (LP) and alternative (AP) pathway activities in serum and plasma. Moreover, we optimised a soluble C5b-9 (sC5b-9) ELISA for the detection of low level complement activation in rat. We determined the conditions for correct sample handling and showed that the assays had low inter- and intra-assay variation. We applied these assays to monitor complement activation in an experimental rat model of renal ischemia/reperfusion injury. We did not observe major complement consumption following reperfusion in CP or LP, and only minor AP consumption at 24h post reperfusion. However, MBL depletion prior to ischemia/reperfusion using a monoclonal antibody, transiently and specifically inhibited 75% of LP activity and ameliorated the AP consumption at 24h. To further assess complement activation during renal IRI, we monitored serum sC5b-9 and found that it was only significantly increased 72h post-reperfusion, but not when rats were pre-treated with anti-MBL or after sham surgery. In conclusion the described assays enable sensitive, reproducible and comprehensive assessment of complement activation in experimental rat models.

Complement mediated renal inflammation induced by donor brain death: role of renal C5a-C5aR interaction.

Kidneys retrieved from brain-dead donors have impaired allograft function after transplantation compared to kidneys from living donors. Donor brain death (BD) triggers inflammatory responses, including both systemic and local complement activation. The mechanism by which systemic activated complement contributes to allograft injury remains to be elucidated. The aim of this study was to investigate systemic C5a release after BD in human donors and direct effects of C5a on human renal tissue. C5a levels were measured in plasma from living and brain-dead donors. Renal C5aR gene and protein expression in living and brain-dead donors was investigated in renal pretransplantation biopsies. The direct effect of C5a on human renal tissue was investigated by stimulating human kidney slices with C5a using a newly developed precision-cut method. Elevated C5a levels were found in plasma from brain-dead donors in concert with induced C5aR expression in donor kidney biopsies. Exposure of precision-cut human kidney slices to C5a induced gene expression of pro-inflammatory cytokines IL-1 beta, IL-6 and IL-8. In conclusion, these findings suggest that systemic generation of C5a mediates renal inflammation in brain-dead donor grafts via tubular C5a-C5aR interaction. This study also introduces a novel in vitro technique to analyze renal cells in their biological environment.

Mannan-binding lectin mediates renal ischemia/reperfusion injury independent of complement activation.

Ischemia/reperfusion injury (IRI) remains a major problem in renal transplantation. Clinical studies have identified that high serum levels of Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, are associated with inferior renal allograft survival. Using a rat model, we identified an entirely novel role for MBL in mediating renal IRI. Therapeutic inhibition of MBL was protective against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. Following reperfusion, exposure of tubular epithelial cells to circulation-derived MBL resulted in internalization of MBL followed by the rapid induction of tubular epithelial cell death. Interestingly, this MBL-mediated tubular injury was completely independent of complement activation since attenuation of complement activation was not protective against renal IRI. Our identification that MBL-mediated cell death precedes complement activation strongly suggests that exposure of epithelial cells to MBL immediately following reperfusion is the primary culprit of tubular injury. In addition, also human tubular epithelial cells in vitro were shown to be susceptible to the cytotoxic effect of human MBL. Taken together, these data reveal a crucial role for MBL in the early pathophysiology of renal IRI and identify MBL as a novel therapeutic target in kidney transplantation.

Association of complement C3 gene variants with renal transplant outcome of deceased cardiac dead donor kidneys.

Local renal complement activation by the donor kidney plays an important role in the pathogenesis of renal injury inherent to kidney transplantation. Contradictory results were reported about the protective effects of the donor C3F allotype on renal allograft outcome. We investigated the influence of the donor C3F allotype on renal transplant outcome, taking all different donor types into account. C3 allotypes of 1265 donor-recipient pairs were determined and divided into four genotypic groups according to the C3F allotype of the donor and the recipient. The four genotypic groups were analyzed for association with primary nonfunction (PNF), delayed graft function, acute rejection, death-censored graft survival and patient survival. Considering all donor types, multivariable analysis found no association of the donor C3F allotype with renal allograft outcome. Also, for living and deceased brain-dead donors, no association with allograft outcome was found. Post hoc subgroup analysis within deceased cardiac dead (DCD) donors revealed an independent protective association of donor C3F allotype with PNF. This study shows that the donor C3F allotype is not associated with renal allograft outcome after kidney transplantation. Subgroup analysis within DCD donors revealed an independent protective association of the donor C3F allotype with PNF, which is preliminary and warrants further validation.

Enhanced complement activation is part of the unfavourable cardiovascular risk profile in South Asians.

South Asian immigrants in western societies exhibit a high burden of diabetes and subsequent vascular complications. Diabetic vascular complications are associated with vascular inflammation. We hypothesize that enhanced complement activation is involved. Therefore, levels of complement C3 and SC5b-9 - the soluble end product of complement activation - in a group of 200 South Asians were compared with an age- and sex-matched control group of native Caucasians. In addition, the association between complement levels and albuminuria, an indicator of renal damage and a cardiovascular risk marker, was assessed in the diabetic South Asian group. Compared with native Caucasians, South Asians had significantly higher levels of both serum C3 and plasma SC5b-9, even when only non-diabetic South Asians were considered. Diabetic South Asians had significantly higher C3 levels compared with non-diabetic South Asians. In diabetic South Asians, higher levels of SC5b-9 were associated with an increased prevalence of albuminuria (odds ratio 5.4, 95% confidence interval 1.8-15.8). These results suggest that enhanced complement activation is part of the unfavourable cardiovascular risk profile in South Asians.

Anti-cyclic citrullinated peptide antibodies from rheumatoid arthritis patients activate complement via both the classical and alternative pathways.

OBJECTIVE: It has been suggested that anti-citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). To exert their pathologic effects, ACPAs must recruit immune effector mechanisms such as activation of the complement system. Mouse models of RA have shown that, surprisingly, arthritogenic antibodies activate the alternative pathway of complement rather than the expected classical pathway. This study was undertaken to investigate whether human anti-cyclic citrullinated peptide (anti-CCP) antibodies activate the complement system in vitro and, if so, which pathways of complement activation are used. METHODS: We set up novel assays to analyze complement activation by anti-CCP antibodies, using cyclic citrullinated peptide-coated plates, specific buffers, and normal and complement-deficient sera as a source of complement. RESULTS: Anti-CCP antibodies activated complement in a dose-dependent manner via the classical pathway of complement, and, surprisingly, via the alternative pathway of complement. The lectin pathway was not activated by anti-CCP antibodies. Complement activation proceeded in vitro up to the formation of the membrane attack complex, indicating that all activation steps, including the release of C5a, took place. CONCLUSION: Our findings indicate that anti-CCP antibodies activate the complement system in vitro via the classical and alternative pathways but not via the lectin pathway. These findings are relevant for the design of interventions aimed at inhibition of complement-mediated damage in RA.

In situ deposition of complement in human acute brain ischaemia.

Experimental animal models indicate that complement contributes to tissue damage during brain ischaemia and stroke, but limited data are available for a role of the complement in human stroke. We, therefore, evaluated whether acute ischaemia leads to complement activation in human brain. Indirect immunohistochemical staining was performed on paraffin-embedded, formalin-fixed human brain from 10 patients and 10 controls. Complement components C1q, C3c and C4d were detected in all ischaemic lesions, suggesting activation via the classical pathway. C9, C-reactive protein and IgM were detected in necrotic zones. Marked CD59 and weak CD55 expression were found in normal brains, but these complement regulators were virtually absent in ischaemic lesions. Modest amounts of mannose-binding lectin (MBL), MBL-associated serine protease-2 and factor B were found in both ischaemic lesions and controls. These data suggest that increased deposition of complement components combined with decreased expression of complement regulators is a possible mechanism of tissue damage during ischaemia in human brain.

Diagnostic efficacy and prognostic value of serum procalcitonin concentration in patients with suspected sepsis.

BACKGROUND: Procalcitonin is released in response to bacterial infection and it is not released in Inflammatory and viral diseases. OBJECTIVE: To show the diagnostic efficacy and prognostic value of procalcitonin for sepsis. METHODS: A consecutive series of 103 patients with suspected sepsis were admitted to the intensive care unit over a 2-year period. During the first 24 hours of the admission procalcitonin, C-reactive protein, and complement proteins were determined. The diagnostic efficacy was tested with predictive values, likelihood ratios, receiver operating characteristic curves, and multiple logistic regression. The association of procalcitonin with mortality was assessed by the Multivariate Cox proportional hazards model. RESULTS: Procalcitonin had a better positive likelihood ratio than C-reactive protein -2.2 (95% confidence interval: 1.3-3.7) versus 1.1 (95% confidence interval: 0.9-1.2). Sequential Organ Failure Assessment yielded the highest discriminative value, with an area under the curve of 0.82 (95% confidence interval: 0.73-0.92), followed by procalcitonin (0.81; 95% confidence interval: 0.72-0.89). Multivariate regression analysis showed procalcitonin (adjusted odds ratio: 3.8; 95% confidence interval: 1.2-11.8) and Sequential Organ Failure Assessment score (adjusted odds ratio: 5.3; 95% confidence interval: 1.4-19.9) as the only variables independently associated with infection. Multivariate Cox regression analysis revealed that procalcitonin was not independently associated with mortality. CONCLUSIONS: The diagnostic accuracy of procalcitonin was higher than C-reactive protein and complement proteins. Procalcitonin in combination with Sequential Organ Failure Assessment was useful to diagnose infection. C-reactive protein, Sequential Organ Failure Assessment score, age, and gender showed to be helpful to improve the prediction of mortality risk, but not procalcitonin.

The complement system in the peripheral nerve: friend or foe?

The complement (C) system plays a central role in innate immunity and bridges innate and adaptive immune responses. A fine balance of C activation and regulation mediates the elimination of invading pathogens and the protection of the host from excessive C deposition on healthy tissues. If this delicate balance is disrupted, the C system may cause injury and contribute to the pathogenesis of various diseases, including neuropathies. Here we review evidence indicating that C factors and regulators are locally synthesized in the peripheral nerve and we discuss the evidence supporting the protective or detrimental role of C activation in health, injury and disease of the peripheral nerve.

Depressed activation of the lectin pathway of complement in hereditary angioedema.

The possibility of simultaneous measurement of the classical pathway (CP), mannan-binding lectin (MBL)--lectin pathway (LP) and alternative pathway (AP) of complement activation by the recently developed Wielisa method allowed us to investigate the in vivo significance of the C1-inhibitor (C1INH) in three complement activation pathways. Functional activity of the CP, LP and AP were measured in the sera of 68 adult patients with hereditary angioedema (HAE) and 64 healthy controls. In addition, the level of C1q, MBL, MBL-associated serine protease-2 (MASP-2), C4-, C3- and C1INH was measured by standard laboratory methods. MBL-2 genotypes were determined by polymerase chain reaction. Besides the complement alterations (low CP and C1INH activity, low C4-, C1INH concentrations), which characterize HAE, the level of MASP-2 was also lower (P = 0.0001) in patients compared with controls. Depressed LP activity was found in patients compared with controls (P = 0.0008) in homozygous carriers of the normal MBL genotype (A/A), but not in carriers of variant genotypes (A/O, O/O). Activity of CP correlated with LP in patients (Spearman's r = 0.64; P < 0.0001), but no significant correlation was found in the control group and no correlation with AP was observed. In contrast, the activity of CP and AP correlated (Spearman's r = 0.47; P < 0.0001) in healthy controls, but there was no significant correlation in the HAE patients. We conclude that the activation of LP might also occur in subjects with C1INH deficiency, which is reflected by the low MASP-2 and C4 levels.

Secretory immunoglobulin A (IgA) responses in IgA nephropathy patients after mucosal immunization, as part of a polymeric IgA response.

Secretory immunoglobulin A (SIgA), although generated at mucosal surfaces, is also found in low concentrations in the circulation. Recently, SIgA was demonstrated in mesangial deposits of patients with immunoglobulin A nephropathy (IgAN), suggesting a role in the pathogenesis. This finding is in line with the belief that high molecular weight (HMW) immunoglobulin A (IgA) is deposited in the kidney. However, there is little information on the size distribution of antigen-specific IgA in circulation upon mucosal challenge. In this study we measured antigen-specific IgA, including SIgA, in serum following challenge of IgAN patients and controls via intranasal vaccination with a neoantigen, cholera toxin subunit B (CTB). We size-fractionated serum and nasal washes to study the size distribution of total IgA, SIgA and CTB-specific IgA. Finally, we compared the size distribution of antigen-specific IgA after mucosal immunization with the distribution upon systemic immunization. A significant induction of antigen-specific SIgA was detectable in serum of both patients with IgAN and controls after mucosal immunization with CTB. Independent of the route of immunization, in both groups the antigen-specific IgA response was predominantly in the polymeric IgA fractions. This is in contrast to total IgA levels in serum that are predominantly monomeric. We conclude that mucosal challenge results in antigen-specific SIgA in the circulation, and that the antigen-specific IgA response in both IgAN patients and in controls is of predominantly HMW in nature. No differences between IgAN patients and controls were detected, suggesting that the size distribution of antigen-specific IgA in the circulation is not disturbed specifically in IgAN patients.

Mannose-binding lectin levels during pregnancy: a longitudinal study.

BACKGROUND: Pregnancy is associated with changes in the immune system. Although previous studies have focussed mainly on adaptive immunity, there are indications that components of innate immunity, such as mannose-binding lectin (MBL), are associated with pregnancy outcome. Although this would suggest that pregnancy also involves adaptations in innate immunity, there are few studies in this area. Therefore, we aimed to determine whether MBL concentrations and the following steps in complement pathway activation are influenced by pregnancy. METHODS: MBL and Ficolin-2 concentrations, MBL-MBL-associated serine protease (MASP) complex activity, MBL pathway activity and classical complement pathway activity were determined by enzyme-linked immunosorbent assay (ELISA) in sera from pregnant women (n=32) during each trimester and post-partum. MBL genotyping was performed by PCR. RESULTS: During pregnancy, MBL concentrations increased to 140% [interquartile range (IQR) 116-181%, P < 0.0001]. This increase was already present at 12 weeks of pregnancy and was most pronounced in the high-production AA-genotype. Directly Post-partum MBL concentrations dropped to 57% of baseline (IQR 44-66%, P < 0.0001). Variations in MBL levels were reflected by similar changes in the following steps of complement activation, r > 0.93 (P < 0.01). Ficolin-2 levels and classical complement pathway activity were not similarly influenced by pregnancy. CONCLUSIONS: Pregnancy and the post-partum period profoundly influence MBL serum concentration and MBL complement pathway activity.

Inhibition of complement component C3 reduces vein graft atherosclerosis in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND: Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. METHODS AND RESULTS: To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. CONCLUSIONS: The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.

The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

BACKGROUND: Thrombomodulin (TM) is predominantly a vascular endothelial cell plasma membrane glycoprotein that, via distinct structural domains, interacts with multiple ligands, thereby modulating coagulation, fibrinolysis, complement activation, inflammation and cell proliferation. We previously reported that by mediating signals that interfere with mitogen-activated protein kinase and nuclear factor kappaB pathways, the amino-terminal C-type lectin-like domain of TM has direct anti-inflammatory properties. METHODS: In the current study, we use murine models of acute inflammatory arthritis and biochemical approaches to assess the mechanism by which the lectin-like domain of TM modifies disease progression. RESULTS: Mice lacking the lectin-like domain of TM (TM(LeD/LeD)mice) develop inflammatory arthritis that is more rapid in onset and more severe than that developed in their wildtype counterparts. In two models of arthritis, treatment of mice with recombinant soluble lectin-like domain of TM significantly suppresses clinical evidence of disease and diminishes monocyte/macrophage infiltration into the synovium, with weaker expression of the pro-inflammatory high mobility group box chromosomal protein 1. While thrombin-TM mediated activation of thrombin activatable fibrinolysis inhibitor inactivates complement factors C3a and C5a, we show that TM has a second independent mechanism to regulate complement: the lectin-like domain of TM directly interferes with complement activation via the classical and lectin pathways. CONCLUSIONS: These data extend previous insights into the mechanisms by which TM modulates innate immunity, and highlight its potential as a therapeutic target for inflammatory diseases.

The role of complement in autoimmune renal disease.

The predominance of renal involvement in autoimmune diseases can most likely be assigned to the specialised function of the kidneys filtrating over 120 ml plasma per minute. Complement activation by autoantibodies directed against planted antigens or antigens already present in renal tissue in the subendothelial and mesangial regions provoke an inflammatory response ultimately resulting in renal damage. New data also suggest complement involvement in the pathogenesis of renal disease caused by subepithelial immune complex deposition. On the other hand complement itself can also be a target of an autoimmune responses causing renal damage as seen in SLE. The results on intervention of complement activation in clinical practise are awaited.

A pathogenic role for secretory IgA in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by deposits of IgA in the renal mesangium. It is thought that deposits of IgA mainly involve high molecular weight (HMW) IgA1. However, there is limited information on the exact composition of HMW IgA in these deposits. In this study, we investigated the presence of secretory IgA (SIgA) in human serum and in the glomerular deposits of a patient with IgAN. Furthermore, we analyzed the interaction of SIgA with mesangial cells. With enzyme-linked immunosorbent assay, SIgA concentrations in the serum of IgAN patients and healthy controls were measured. Both patients and controls had circulating SIgA that was restricted to the HMW fractions. Patients tended to have higher levels of SIgA, but this difference was not significant. However, in patients with IgAN, high serum SIgA concentrations were associated with hematuria. Binding of size-fractionated purified serum IgA and SIgA to mesangial cells was investigated with flow cytometry. These studies showed stronger binding of SIgA to primary mesangial cells compared to binding of serum IgA. Importantly, after isolation and elution of glomeruli from a nephrectomized transplanted kidney from a patient with recurrent IgAN, we demonstrated a 120-fold accumulation of SIgA compared to IgA1 in the eluate. In conclusion, we have demonstrated that SIgA strongly binds to human mesangial cells, and is present in significant amounts in serum. Furthermore, we showed that SIgA is accumulated in the glomeruli of an IgAN patient. These data suggest an important role for SIgA in the pathogenesis of IgAN.