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PubMed1987

Solution conformation of carboxy-terminal fragments of the third component of human complement C3: proton nuclear magnetic resonance study of C3a, des-Arg-C3a, and C3a Arg69.

A proton nuclear magnetic resonance (NMR) study is reported of the solution conformation of human C3a, that is released on activation of C3, the third component of complement. The intact C3a was used along with des-Arg-C3a, which is formed on cleavage of Arg-77 at the C terminal of C3a, and C3a Arg69, which is a 69-residue fragment produced on tryptic digestion of C3. A method of carboxypeptidase digestion/difference spectroscopy (Endo & Arata (1985) Biochemistry 24, 1561-1568) was extensively used for the spectral assignments of Ile-43, Ile-60, Leu-63, Tyr-15, and Tyr-59. On the basis of the results of nuclear Overhauser effect (NOE) measurements, we discuss the solution conformation of the C3a molecule. It has been concluded that removal of Arg-77, which is essential for expression of the biological activity of C3a, does not induce any significant change in the solution conformation of the C3a molecule. The C3a molecule is known to consist of a core region that comprises segment Tyr-15-Tyr-59. We conclude that in solution the C terminal segment sticks out of the core and takes on a helix-like conformation. Possible roles of the core region and the N terminal segment in maintaining the conformation of the C terminal segment are briefly discussed.

Amino Acid Sequence↗
PubMed1997

Synthetically modified cellulose: an alternative to synthetic membranes for use in haemodialysis?

Renal replacement therapy relies predominantly on the use of cellulose-based membranes. Such membranes have a biocompatibility profile which is inferior to membranes manufactured from synthetic polymers. Synthetically modified cellulose (SMC) is a new, low-flux haemodialysis membrane in which hydroxyl groups have been replaced with benzyl groups. The biocompatibility profile characterized by changes in white cell and platelet counts and the activation of complement components (C3a, C5a and C5b-9) have been studied in vivo and compared with those of cellulose acetate, unmodified cellulose (Cuprophan ) and low-flux polysulphone (Fresenius Polysulfone) in the same group of patients. For SMC, the white cell count at 15 min declined to 65.6% of pretreatment level, compared with 63.8% for the cellulose acetate, 79.6% for low-flux polysulphone and 28.1% for Cuprophan, thereafter returning to pretreatment levels. Both modified cellulose membranes were superior to unmodified cellulose (P = 0.001); the differences between the modified cellulose membranes were not significant statistically. The changes induced by all three cellulose-based membranes exceeded those for low-flux polysulphone (P = 0.001). Associated with the neutropenia was a reduction in platelet count, but this was independent of membrane type. The mean time-averaged concentrations of C3a(des Arg) over 150 min were 1168 ng ml(-1) (SMC), 1030 ng ml(-1) (cellulose acetate), 1297 ng ml(-1) (Cuprophan) and 790 ng ml(-1) (low-flux polysulphone). Equivalent values for C5a(des Arg) were 6.12 (SMC), 2.98 (cellulose acetate), 11.03 (Cuprophan) and 1.33 ng ml(-1) (low-flux polysulphone). C5b-9 values were 385 (SMC), 386 (cellulose acetate), 177 (Cuprophan) and 185 ng ml(-1) (low-flux polysulphone). For each of the complement components the differences between the membranes were significant [P = 0.0009 (C3a(des Arg)), P = 0.0001 (c5a(des Arg) and C5b-9)]. The levels of C5b-9 generated during dialysis also showed a significant positive correlation compared to C5a for all membranes considered as a single group (Pearson's correlation coefficient = 0.870, P = 0.0001). It is concluded that the modification of the cellobiosic unit is a promising approach to improve the biocompatibility profile of cellulose-based membranes. The two different methods of modification lead to similar improvements in biocompatibility compared with unmodified cellulose, but as yet do not match that of low-flux polysulphone.

Biocompatible Materials↗
PubMed1993

Expression of biologically active C3a as fusion proteins.

We selected three kinds of plasmids for expression of C3a as fusion proteins. The proteins were purified by affinity chromatography using the respective specific resins, and their activities were measured by guinea pig platelet aggregation. We showed that polyhistidine (polyHis)-C3a fusion protein was able to exhibit 30% of the activity of natural C3a. However, glutathione S-transferase (GST)-C3a fusion protein exhibited only 10% of such activity, and no activity was measured for maltose binding protein (MBP)-C3a fusion protein. The purified polyHis-C3a fusion protein was attached to the Ni-NTA agarose column in an attempt to isolate the C3a receptor from guinea pig platelets. The C3a binding protein isolated from digitonin-solubilized guinea pig platelet membrane was approximately 50 kDa on SDS-polyacrylamide gel. This is the first report of C3a fusion protein production with biological activity.

Adenosine Triphosphate↗
PubMed1997

Blood- and skin-derived monocytes/macrophages respond to C3a but not to C3a(desArg) with a transient release of calcium via a pertussis toxin-sensitive signal transduction pathway.

Controversial results have been published in the past regarding the functional reactivity of monocytes (Mo) and macrophages (M phi) to the anaphylatoxin C3a and its degradation product C3a(desArg). In this study we performed binding and calcium mobilization experiments with recombinant human C3a (rC3a) and rC3a(desArg). Blood Mo displayed non-inhibitable binding of FITC-labeled rC3a (rC3aFITC) but responded to rC3a with a transient release of the intracellular calcium concentration ([Ca2+]i), whereas rC3a(desArg) was completely inactive. In contrast, binding of rC3aFITC to eosinophilic granulocytes and the mast cell line HMC-1 which have been shown previously to express C3a binding sites could be blocked by a monoclonal anti-C3a antibody. The rC3a-induced [Ca2+]i release in blood Mo was pertussis toxin (PTX)-sensitive suggesting the involvement of G-proteins in the signal transduction pathway. Skin-derived Mo/M phi reacted similarly to blood Mo as no specific binding of rC3aFITC to these cells could be demonstrated, whereas an intracellular release of calcium ions in response to the anaphylatoxin was observed. Homologous desensitization to rC3a but not heterologous desensitization to rC5a was detected in further experiments. The functional effect of C3a, but not the unspecific binding of rC3aFITC to blood Mo and skin-derived Mo/M phi could be blocked by the monoclonal anti-C3a antibody. These results suggest the expression of the recently cloned G-protein-coupled receptor for C3a on human blood Mo and skin-derived Mo/M phi. However, the total number of specific C3a binding sites on these cells is distinctly lower as compared to eosinophilic granulocytes and cells of the mast cell line HMC-1. The small number of C3a receptors on Mo/M phi may be masked by a pronounced non-inhibitable binding of rC3aFITC. This binding, however, may contribute to the recently described biological effects of C3a(desArg) on Mo.

Arginine↗
PubMed1999

Complement and contact activation related to surfactant response in respiratory distress syndrome.

The activation of inflammation and coagulation cascades is part of the pathogenesis of adult respiratory distress syndrome (RDS). Previous studies have demonstrated contact activation in preterm infants with RDS, whereas no concordant results have been found with complement activation. In this study, both systems were investigated in preterm infants with severe RDS and related to surfactant response. Thirty preterm newborns with severe respiratory distress (FiO2 > 0.5), but with no evidence of infection or fetal acidosis, were studied. Eighteen healthy preterm newborns of similar gestational age and birth weight served as controls. The study group was divided into two subgroups, according to their response to a porcine natural surfactant 6 h after administration: responders (FiO2 reduction > 50%) and poor responders (FiO2 reduction < or = 50%). C1q, C4, factor B, C3a, C5a, complement, and C1-inhibitor activity, as well as factor XIIa, were determined in blood samples, drawn 24 h after birth. Except for C1-inhibitor concentration and C1-inhibitor activity, all parameters for infants with severe RDS were different from controls. Complement precursor proteins were lower, and activated split products of the complement and contact system were higher. Infants with a poor response after application of surfactant showed higher amounts of C3a, C5a, and factor XIIa but lower C1q and C4 levels compared with infants with a good response to surfactant. Activation of the complement and the contact system was demonstrated in all respiratory distress patients. This activation was more pronounced in poor responders to exogenous surfactant.

Complement Activation↗
PubMed1997

The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

Animals↗
PubMed1991

Active recombinant C3a of human anaphylatoxin produced in Escherichia coli.

DNA sequence coding for the complete human C3a with 77 amino acids was divided into three portions, synthesized separately and constructed for expression in Escherichia coli. High expression of the recombinant C3a was achieved by an expression system using T7 polymerase. Purified recombinant C3a showed the same activities of ileum contraction and platelet aggregation of guinea pig as C3a purified from human serum.

Amino Acid Sequence↗
PubMed1981

Synthetic C3a analogs as specific inhibitors of C3a activity.

Various C3a-related C-terminal synthetic oligopeptides were investigated for their ability to induce a release of serotonin from guinea pig platelets. The results confirm earlier findings that expression of biological C3a activity requires the four to five C-terminal amino acids of the C3a primary structure and underlines the essential role of the C-terminal arginine. Besides their ability to induce a specific release reaction, these peptides--after short incubation with the platelets--lead to a specific desensitization of the cells for C3a or C3a-related stimuli. Expression of this inhibitory activity required concentrations of the peptides more than 100-fold lower than those that were necessary to induce secretion. The possibility of using C3a analogs as specific inhibitors for C3a offers a valuable tool for in vivo studies of biological C3a activity.

Amino Acid Sequence↗
PubMed1989

[Atopic dermatitis. II. The status of complement proteins and the pathogenetic role of anaphylatoxins C4a, C3a and C5a].

Eighty-eight patients with atopic dermatitis have been examined for the complementary proteins C3 (C3c), C3act., C4, C1inact. by radial immunodiffusion; in 36 of these patients anaphylatoxins C3a des Arg, C5a des Arg, and C4a des Arg have been radioimmunoassayed. Increased levels of C4a des Arg have been revealed in 50% of the examinees. C3a des Arg levels did not differ significantly from the reference values, and C5a des Arg level has been within the normal range in all the patients. C4a des Arg has been related to the severity of the skin inflammation and did not depend on the serum IgE content. C3 (C3c), C3act., C1inact. have been elevated and C4 reduced. These findings evidence activation of the complementary system in atopic dermatitis and indicate the role of anaphylatoxin C4a des Arg in the maintenance of the inflammatory reactions.

Adolescent↗
PubMed1988

Terminal complement complexes and anaphylatoxins in septic and ischemic patients.

Terminal complement complex (TCC) and anaphylatoxin formation in 18 patients with sepsis and 20 patients with acute limb ischemia were studied before the start of treatment and seven days later. The septic or ischemic patients had elevated levels of plasma TCC before start of therapy. In successfully treated patients these concentrations were within the normal range one week later. Similarly, the plasma anaphylatoxin level was increased before therapy and returned to the normal range within seven days. Escherichia coli incubated in vitro in fresh human serum at body temperature started formation of TCC in a dose-related manner. As complement will induce cellular lysis via TCC and edema via anaphylatoxins, anemia and impaired respiration in these patients may be influenced by increased concentrations of terminal complement complexes and of C3a and C5a.

Aged↗
PubMed1998

The human mast cell line HMC-1 binds and responds to C3a but not C3a(desArg).

Controversial results have been published in the past regarding the functional reactivity of different cell types to the anaphylatoxin C3a and its degradation product C3a(desArg). To understand better the effects of C3a and C3a(desArg) on human mast cells, the authors performed binding experiments and calcium mobilization studies on the human mast cell line HMC-1 which has been shown previously to express C3a binding sites. For this purpose, functionally active, recombinant C3a (rC3a) was constructed with an 11 amino acid peptide attached to the N-terminus of the molecule. Using a monoclonal antibody (MoAb) against this tag, binding of rC3a to HMC-1 cells could be demonstrated by flow cytometry. Its binding was specific as it could be blocked with serum-derived C3a. In contrast, no binding of rC3a(desArg) to HMC-1 cells was detectable. Recombinant C3a led to a transient mobilization of intracellular calcium [Ca2+]i in HMC-1 which was inhibitable by the C3a-specific MoAb K13/16. No increase of [Ca2+]i was detected when the cells were treated with C3a(desArg). The authors found C3a receptor (C3aR)-specific mRNA in HMC-1 cells indicating that this receptor represents the binding site for C3a on these cells. These results demonstrate a specific binding for C3a but not for C3a(desArg) on cells of the human mast cell line HMC-1. As a consequence, functional activity was restricted to C3a with C3a(desArg) being completely inactive. Therefore, the data strongly suggest that the recently cloned high affinity C3aR which is assumed to represent the binding site for the anaphylatoxin on HMC-1 cells is unresponsive to C3a(desArg).

Animals↗
PubMed1999

C3a(desArg) does not bind to and signal through the human C3a receptor.

Contradictory results have been published in the past regarding the functional responses of different cell types to the anaphylatoxin C3a and its natural catabolite C3a(desArg). To elucidate the interaction of the C3a receptor (C3aR) with its ligand(s) we studied the binding of human recombinant C3a (rC3a) and rC3a(desArg) to RBL-2H3 transfectants which express the C3aR. As the addition of 11 aminoterminal amino acids did not alter the functional activity of the recombinant C3a as compared to serum-derived C3a the specific binding of rC3a and rC3a(desArg) to the transfectants could be determined by flow cytometry using a monoclonal antibody (mab) against their N-terminal histidine tag. Recombinant C3a bound to the C3aR with a half maximal concentration of about 3 nM whereas no evidence for a binding of rC3a(desArg) could be obtained. Furthermore, rC3a(desArg) did not signal through the C3aR. Neither the release of lysosomal N-acetyl-beta-D-glucosaminidase nor the directional migration of C3aR-expressing RBL-2H3 transfectants could be detected in response to rC3a(desArg) whereas rC3a was highly active in both assays. Our data demonstrate a defined ligand specificity of the C3aR for the anaphylatoxin C3a. Its natural catabolite C3a(desArg), however, does not signal through the C3aR. Modulating effects of C3a(desArg) on the synthesis of cytokines in human monocytes and B lymphocytes may therefore be induced by receptor-independent mechanisms while their in vivo relevance remains as yet undefined.

Acetylglucosaminidase↗
PubMed1996

Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells.

A cDNA clone encoding the human C3a anaphylatoxin receptor (C3aR) was isolated from a pcDNAI/Amp expression library prepared from U-937 cells which had been differentiated with dibutyryl cAMP to a macrophage-like phenotype. The cDNA clone contained an insert of 4.3 kbp and was able to confer to transfected human HEK-293 cells the capacity to bind specifically iodinated human C3a. Chinese hamster ovary cells co-transfected with this cDNA clone and a G-protein alpha subunit (G alpha-16) became functionally responsive to C3a and a C3a analog synthetic peptide, as measured by increased phosphoinositide hydrolysis. As inferred from the cDNA sequence, the clone encodes a 482-residue polypeptide with seven hydrophobic membrane-spanning helices and a high homology to the human C5a and formyl-Met-Leu-Phe receptors. Uniquely among the family of G-protein coupled receptors, the C3aR contains an exceptionally large second extracellular loop of approximately 175 residues. Northern hybridizations revealed an approximately 2.3-kb transcript as the major and an additional approximately 3.9 kb-transcript as a minor transcription product of the C3aR. The C3aR appears to be widely expressed in different lymphoid tissues, as shown by Northern hybridizations, providing evidence for a central role of the C3a anaphylatoxin in inflammatory processes.

Amino Acid Sequence↗
PubMed1999

Inhibition of IgE-mediated triggering of mast cells by complement-derived peptides interacting with the Fc epsilon RI.

Mucosal type mast cells, in contrast to the serosal type ones, do not respond to cationic agents, or to the complement-derived peptides C3a and C5a. Earlier we have found that while C3a does not activate the rat mucosal type mast cells (line RBL-2H3), it strongly inhibits the IgE-mediated triggering of these cells, by interfering with the Fc epsilon RI-initiated signaling pathway. In the present study we further investigated the mechanism of this process. It is shown, that C3a interacts with the beta-chain of the Fc epsilon RI complex. Binding of the complement peptide to the cells apparently causes a decrease in the proximity of the IgE-binding Fc epsilon RI. Investigating certain sequences of C3a we found that the inhibition is caused by the C-terminal sequences of the complement-peptide, ranging from positions 56 to 77 and also by a shorter sequence, ranging from positions 56 to 64. The inhibitory effect of these peptides was observed both in the case of RBL-2H3 cells and mouse bone marrow derived mast cells.

Amino Acid Sequence↗
PubMed1985

Fast deactivation of guinea-pig isolated ileum to C5adesArg: a possible cyclic AMP-dependent mechanism.

The fast component of deactivation of guinea-pig isolated ileum to the spasmogenic action of the complement peptide C5adesArg was further differentiated from the slow component which had been previously analysed (Damerau et al., 1985a, b). Fast deactivation differs from the slow component in the following characteristics: (a) it is unspecific in that it is also induced by C3a, another complement peptide, (b) it depends on the spasmogenic effect of the peptides, and (c) it does not occur at 16 degrees C. In contrast to the slow component, in which the deactivation is thought to be caused by blockade of C5a receptors, fast deactivation seems to be due to a transient increase of intracellular cyclic AMP evoked by C5adesArg and C3a; it is prevented by GDP beta S (5 X 10(-4) M) which blocks activation of adenylate cyclase, and prolonged by agents which sustain cyclic AMP elevations, namely 5 X 10(-4) M theophylline and 5 X 10(-4) M) GTP gamma S.

Animals↗
PubMed2000

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-beta-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.

Antibodies, Monoclonal↗
PubMed1993

Estimation of local vital reactions in severely burned tissues of guinea pig skin using C3a or C3a desArg as a marker.

We observed changes in levels of anaphylatoxin C3a and/or its desArg (C3a/C3a desArg) peptides in the local site of severely burned skin tissues of guinea pigs using immuno-Western blotting methods. The C3a/C3a desArg peptides, which were probably generated during complement activation, were detected at significant levels from 30 min to 72 h following burn injury in an area limited to 3 mm from the wound edge. Levels of these peptides showed a tendency to be highest in that area 24 h after burn infliction. In postmortem injuries, C3a/C3a desArg peptides could not be detected. These peptides were detected in antemortem wounds up to at least 2 days at 22 degrees C and up to 3 days at 4 degrees C after death, although decreases in levels were found. Lower concentrations of these peptides were also found in postmortem burns in which postmortem hypostasis appeared strongly. These results suggest that, except for injuries of the area with obvious postmortem hypostasis, detection of C3a/C3a desArg can be useful for estimation of vital reactions in many kinds of wounds during inflammation.

Animals↗
PubMed1997

Complement activation of electrogenic ion transport in isolated rat colon.

The complement cascade is an important component in many immune and inflammatory reactions and may contribute to both the diarrhoea and inflammation associated with inflammatory bowel disease. Isolated rat colonic mucosae were voltage clamped in Ussing chambers. Basolateral addition of zymosan-activated whole human serum (ZAS) induced a rapid onset, transient inward short circuit current (SCC). This response was concentration dependent and was significantly attenuated by pre-heating ZAS at 60 degrees C for 30 min. Depletion of complement from normal human serum with cobra venom factor (CVF) significantly lowered SCC responses. Chloride was the primary charge carrying ion as responses to ZAS were abolished in the presence of the loop diuretic bumetanide. The complement component C3a stimulated ion transport but not to the same extent as whole serum. Exogenous C5 was without effect. The cyclooxygenase inhibitor piroxicam significantly attenuated the response to ZAS. These findings support the possibility that complement activation may contribute to the pathophysiology of secretory diarrhoea since activation of electrogenic chloride secretion converts intestinal epithelia to a state of net fluid secretion.

Animals↗