Otto Götze

The C‐terminus of complement regulator Factor H mediates target recognition: evidence for a compact conformation of the native protein

The complement inhibitor Factor H has three distinct binding sites for C3b and for heparin, but in solution uses specifically the most C‐terminal domain, i.e. short consensus repeats (SCR) 20 for ligand interaction. Two novel monoclonal antibodies (mABs C14 and C18) that bind to the most C‐terminal domain SCR 20 completely blocked interaction of Factor H with the ligands C3b, C3d, heparin and binding to endothelial cells. In contrast, several mAbs that bind to the N‐terminus and to the middle regions of the molecule showed no or minor inhibitory effects when assayed by enzyme‐linked immunosorbent assay (ELISA) and ligand interaction assays. This paradox between a single functional binding site identified for native Factor H versus multiple interaction sites reported for deletion constructs is explained by a compact conformation of the fluid phase protein with one accessible binding site. On zymosan particles mAbs C14 and C18 blocked alternative pathway activation completely. Thus demonstrating that native Factor H makes the first and initial contact with the C terminus, which is followed by N terminally mediated complement regulation. These results are explained by a conformational hypothetical model: the native Factor H protein has a compact structure and only one binding site accessible. Upon the first contact the protein unfolds and exposes the additional binding sites. This model does explain how Factor H mediates recognition functions during complement control and the clustering of disease associated mutations in patients with haemolytic uraemic syndrome that have been reported in the C‐terminal recognition domain of Factor H.

Activated Human T Lymphocytes Express a Functional C3a Receptor

The C3a molecule is an anaphylatoxin of the C system with a wide spectrum of proinflammatory effects predominantly on cells of myeloid origin. In this study we investigated the expression of the high affinity receptor for C3a (C3aR) in human T lymphocytes using receptor-specific mAb. C3aR expression was detected in CD4+ and CD8+ blood- or skin-derived T cell clones (TCC) from birch pollen-sensitized patients with atopic dermatitis. No significant difference in C3aR expression in CD4+ or CD8+ TCCs could be observed. In contrast to C3a(desArg), C3a led to a transient calcium flux in TCCs expressing the C3aR, whereas C3aR-negative TCCs were unreactive. Circulating T cells from patients suffering from severe inflammatory skin diseases expressed the C3aR, whereas no expression of C3aR could be found in unstimulated T lymphocytes from patients with mild inflammatory skin diseases or from healthy individuals. Type I IFNs, which are potent stimulators of cellular immunity, were identified as up-regulators of C3aR expression in vitro in freshly isolated or cloned T lymphocytes. Moreover, C3aR+ T cells were found at the sites of injection in IFN-β-treated patients with multiple sclerosis. These data provide direct evidence for the expression of C3aR on activated human T lymphocytes; this may point to a biological function of C3a in T cell-dependent diseases.

The C5a receptor is expressed in normal renal proximal tubular but not in normal pulmonary or hepatic epithelial cells

C5a, a 74 amino acid peptide cleaved from the complement protein C5, is an extremely potent anaphylatoxin. Expression of the receptor for the anaphylatoxin C5a (C5aR) has been thought to be restricted to cells of myeloid origin. However, recent evidence suggests that the C5aR is also expressed in hepatocytes as well as in pulmonary epithelial, endothelial and smooth muscle cells. In the present study, we investigated the tissue distribution of C5aR by immunohistochemistry in normal human lung, liver, intestine and kidney using well‐defined monoclonal antibodies (mAbs) directed against the extracellular N‐terminus of the receptor. In all tissues examined, macrophages displayed an abundant expression of C5aR protein. However, in the normal human lung, C5aR expression was not detectable in bronchial and alveolar epithelial cells or in vascular smooth muscle or endothelial cells. In the normal human liver, no C5aR protein was detected in hepatocytes, whereas Kupffer cells strongly expressed the C5aR. In normal human kidney, the C5aR was detectable only in proximal tubular cells. C5aR gene transcription in Kupffer cells and proximal tubular cells was confirmed by in situ hybridization. Thus, our results point to an as yet unknown role of the C5aR in normal renal physiology. In the normal lung and liver, however, previous evidence for the ubiquitous expression of C5aR in epithelial, endothelial and smooth muscle cells
in situ should be re‐evaluated.

Evaluation of C3a receptor expression on human leucocytes by the use of novel monoclonal antibodies

Varying results have been published in the past regarding the reactivity of different leucocyte subpopulations, including neutrophils, monocytes and B lymphocytes, to the anaphylatoxin C3a and its degradation product C3a(desArg). To better characterize the cellular distribution of C3a receptor (C3aR) expression, monoclonal antibodies against two different epitopes on the third extracellular domain of the human C3aR were generated. Quantification of C3aR as compared with C5aR densities was performed on peripheral blood leucocytes by quantitative indirect immunofluorescence. Eosinophils and basophils expressed similar numbers of C3aR and C5aR molecules/cell. On eosinophils 10 700±4500 (mean±SD) C3aR and 14 700±4100 C5aR were found, whereas basophils carried 8100±2100 C3aR and 13 500±3800 C5aR. Monocytes expressed approximately six times more C5aR than C3aR molecules on their surface (6000±2500 C3aR versus 34 100±9300 C5aR molecules) whereas on neutrophils, the expression of C5aR was more than 20 times higher than the expression of C3aR (3100±1000 C3aR versus 63 500±12 200 C5aR). No C3aR expression was detectable on peripheral blood‐derived B lymphocytes and on tonsillar B cells before and after stimulation with interleukin‐2/Staphylococcus aureus Cowan strain I. Our findings correspond well with the paucity of data on C3a‐induced functional activities in monocytes and neutrophils and suggest that eosinophilic and basophilic granulocytes represent the primary effector cells in the peripheral blood which can be stimulated by C3a.

Formation of C5a during cardiopulmonary bypass: Inhibition by precoating with heparin☆

A novel enzyme immunoassay based on direct detection of C5a by a monoclonal antibody (C17/5) specific for a neoepitope exposed in C5a/C5adesArg was used to measure in vivo and in vitro C5a formation during cardiopulmonary bypass. In vivo, we observed a significant threefold to fourfold increase in patient plasma C5a/C5adesArg levels from baseline values (5.6; 1.6 to 12.9 ng/mL) (median and range) up to 42 hours postoperatively (17.5; 6.5 to 46.0 ng/mL) when two different uncoated cardiopulmonary bypass circuits were used. Coating of the extracorporeal circuit with end-point-attached heparin completely abolished C5a formation in vitro during circulation of blood through the circuit for 120 minutes. The C5a concentration (median and range) was 3.2 (2.6 to 15.9) ng/mL at the start and 3.1 (2.7 to 15.0) ng/mL at the end of the experiment. In the uncoated setups the corresponding C5a concentrations were 10.1 (6.2 to 17.5) and 19.7 (13.1 to 24.3) ng/mL. Finally, heparin-coated cardiopulmonary bypass circuits were examined in vivo. C5a levels did not increase significantly during the cardiopulmonary bypass period in the heparin-coated group in contrast to the uncoated group, but the postoperative increase in C5a levels was similar in the two groups. We conclude that heparin coating improves biocompatibility by completely abolishing C5a formation in vitro. The discrepancy between the in vitro and the in vivo findings is probably related to the complicated biological turnover of C5a.

Expression of the anaphylatoxin C5a receptor in non-myeloid cells

C5, a 74 amino acid peptide cleaved from the complement protein C5, represents the most potent anaphylatoxin and possesses inflammatory as well as immunomodulatory activities. In the past, expression of the receptor for the anaphylatoxin C5a (C5aR) has been thought to be restricted to cells of myeloid origin. However, recent evidence suggests that the C5aR is constitutively expressed in non-myeloid cells including epithelial, endothelial and smooth muscle cells in the human liver and lung. These findings are contrasted by results from our laboratory which demonstrated that in the normal human liver and lung C5aR expression is detectable exclusively in macrophages and macrophage-derived cells (Kupffer cells). Interestingly, we found evidence that C5aR expression may be inducible in epithelial cells as C5aR mRNA was observed in vivo in human keratinocytes of the inflamed but not of the normal skin. Herein we review the work of our laboratory and others on the expression of the C5aR in various human non-myeloid cells types. A better understanding of the expression patterns of this important anaphylatoxin receptor may provide new insights in the pathophysiological role of C5a in vivo.

Anaphylatoxin C5a receptor mRNA is strongly expressed in Kupffer and stellate cells and weakly in sinusoidal endothelial cells but not in hepatocytes of normal rat liver

Anaphylatoxins (C5a and C3a), which are generated during complement activation, have recently been shown to increase glucose output from hepatocytes (HC) in perfused rat liver. They did not act directly on HC but indirectly by prostanoid release from non‐parenchymal cells (NPC), probably Kupffer cells (KC). In order to corroborate this mechanism, the distribution of anaphylatoxin receptors in the different cell types of rat liver was determined by quantitative RT‐PCR with primers specific for the rat C5a receptor (rC5aR) using RNA isolated from KC, sinusoidal endothelial cells (SEC), hepatic stellate cells (HSC) and HC. In line with functional data, C5aR mRNA was detected in freshly isolated NPC but not in HC of rat liver. Mainly KC but also HSC clearly expressed C5aR mRNA, while SEC did so only weakly. KC expressed up to 10‐fold more C5aR mRNA than HSC and these in turn up to 10‐fold more than SEC. These results support the proposed indirect action of anaphylatoxins on HC.

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6‐deficient and one combined C6‐/C7‐deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro‐organisms whose destruction requires TCC formation.

Detection of anaphylatoxin receptors on CD83+ dendritic cells derived from human skin

Dendritic cells (DC) are recruited to sites of inflammation for the initiation of immune responses. As the anaphylatoxins C5a and C3a are important mediators of inflammation, we investigated the expression of their receptors (C3aR and C5aR) on human DC. DC were isolated from human skin or generated from purified blood monocytes and were identified by their expression of CD1a or CD83. Freshly isolated or cultured dermal CD1a+ and CD83+ DC bound anti‐C5aR and anti‐C3aR monoclonal antibodies (mAbs), as detected by flow cytometry. C5a induced calcium fluxes in dermal CD1a+ and CD83+ DC, which could be inhibited by C17/5, an anti‐C5a mAb. C3a did not induce calcium fluxes in these cells. Anaphylatoxin receptor expression was down‐regulated on dermal DC by adding tumour necrosis factor‐α (TNF‐α) to the culture medium. On CD1a+ CD83− cells generated from isolated blood monocytes by culture with 6·25 ng/ml of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and 125 U/ml of interleukin‐4 (IL‐4), expression of both C5aR and C3aR was observed. In these cells, both C5a and C3a induced calcium fluxes. After addition of TNF‐α to the culture medium, the majority of the CD1a+ cells expressed CD83+. These cells – expressing a phenotype of ‘mature DC’– down‐regulated the expression of the anaphylatoxin receptors and lost their reactivity to the respective ligands. Our results demonstrate the expression of the anaphylatoxin receptors C5aR and C3aR on human skin‐derived DC and blood‐derived cells expressing the DC‐associated membrane molecule, CD1a. Furthermore, the expression of anaphylatoxin receptors on CD83+ dermal DC is indicative of an intermediate stage of maturation of these cells, which was not observed on in vitro‐differentiated CD83+ cells.

Quantitation of components of the alternative pathway of complement (APC) by enzyme-linked immunosorbent assays

Sensitive enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies have been developed to specifically detect components of the alternative pathway of complement in human blood plasma. Normal values of the factor B split products Ba (1.01 +/- 0.30 micrograms/ml, mean +/- SD), Bb (0.65 +/- 0.23 micrograms/ml), of the C3-fragments C3b/iC3b/C3dg (17.9 +/- 5.7 micrograms/ml), native factor B (238 +/- 48 micrograms/ml), factor D (1.05 +/- 0.27 micrograms/ml), and factor H (702 +/- 292 micrograms/ml) were determined in the EDTA-plasma of healthy probands (n = 55). The simultaneous quantitation of the main cleavage products and of control proteins in the plasma samples permits precise analysis of the activation of the alternative pathway of complement in various disease states. In addition, we describe a method for the specific depletion of factor B prior to fragment-specific assays utilizing monoclonal antibodies conjugated to paramagnetic beads. The latter should permit the quantitation of other complement split products.