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At least 19 recordsLinked to original sources
PubMed2003

IL-4 down-regulates anaphylatoxin receptors in monocytes and dendritic cells and impairs anaphylatoxin-induced migration in vivo.

Anaphylatoxins mobilize leukocytes to the sites of inflammation. In the present study we investigated the impact of GM-CSF, IL-4, and IFN-gamma on anaphylatoxin receptor expression in monocytes and dendritic cells (DC). IL-4 was identified as the strongest down-regulator of the receptors for C5a and C3a in monocytes and monocyte-derived DC (MoDC). To study the impact of IL-4 on anaphylatoxin-induced chemotaxis, an in vivo migration model was established. For this purpose, human monocytes and MoDC were injected i.v. into SCID mice that at the same time received anaphylatoxins into the peritoneal cavity. A peritoneal influx of human monocytes could be demonstrated by 4 h after injections of C5a and C3a. In line with receptor down-regulation, IL-4 treatment inhibited in vivo mobilization of human monocytes and MoDC in response to C5a and C3a. In addition to its effects on human cells, IL-4 reduced C5a receptors in murine bone marrow-derived DC and impaired recruitment of labeled bone marrow-derived DC in syngeneic BALB/c mice to i.p. injected C5a. Overall, these data suggest that inhibition of a rapid anaphylatoxin-induced mobilization of monocytes and DC to inflamed tissues represents an important anti-inflammatory activity of the Th2 cytokine IL-4.

Anaphylaxis↗
PubMed1969

Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum.

A small fragment of C3, called C3a, which has smooth muscle contracting activity, was isolated by three different methods. At pH 8.6, C3a behaved as cation, and using the Archibald method, its mol wt was determined to be 7000. A specific antiserum to C3a showed the fragment to be antigenically distinct from the rest of the C3 molecule, i.e., the C3b portion. The same antiserum and an anti-whole C3 were able to inhibit the biologic activity of C3a. In addition to anaphylatoxin activity, leukocyte chemotactic activity was shown to reside in C3a. Treatment with trypsin caused the cationic fragment to become anionic and abolished the anaphylatoxin but not the chemotactic activity. C3a fragments with identical biologic activity and comparable cationic properties, as determined by acid disc electrophoresis, were obtained by treatment of C3 with C3 convertase, C3 inactivator complex, trypsin, and plasmin. Thrombin produced a similar C3 fragment which was inactive. It was concluded that C3a corresponds to an unusually basic portion of C3 which may be liberated by attack of a variety of enzymes on a highly susceptible region of the native C3 molecule. C3b was cleaved by trypsin and less efficiently by thrombin or plasmin into two antigenically distinct pieces: the larger C3c fragment corresponding to beta(1A) and the smaller C3d fragment to alpha(2D) of aged serum. The c- and the d-fragments were separated and characterized. Isolated C3a rapidly lost its anaphylatoxin activity when treated with small amounts of a partially purified, thermolabile 10S alpha-pseudoglobulin of human serum. The conditions of inactivation suggested an enzymatic reaction. The anaphylatoxin inactivator also destroyed the activity of C5-derived anaphylatoxin and of lysyl bradykinin.

Anaphylaxis↗
PubMed1973

Formation of C3a and C5a anaphylatoxins in whole human serum after inhibition of the anaphylatoxin inactivator.

Two biologically and chemically distinct anaphylatoxins (ATs) could be generated in whole human serum after removal of the AT inactivator (AI) by immune-absorption or after inhibition of AI with 1 M epsilon-aminocaproic acid (EACA). Both human ATs could be generated by treatment of serum with antigen-antibody complexes, which activate the classical complement pathway, and with inulin or yeast, both of which trigger the alternate pathway. The ATs were isolated from serum in active form and characterized as C3a and C5a. Although human C3a had been characterized previously, C5a had not. The molecular weight of human C5a AT was 17,500; its electrophoretic mobility at pH 8.5 was -1.7 x 10(-5) cm(2) V(-1) s(-1). The minimal effective concentration in vitro was 7.5 x 10(-10) M. The minimal effective doses of human C5a in producing a wheal and erythema in the human skin was 1 x 10(-15) mol. The results strongly suggest a biological function for both ATs and indicate that the expression of their activity is controlled by the AI of normal blood plasma.

Adsorption↗
PubMed1995

Expression of functional receptors for human C5a anaphylatoxin (CD88) on the human hepatocellular carcinoma cell line HepG2. Stimulation of acute-phase protein-specific mRNA and protein synthesis by human C5a anaphylatoxin.

Acute inflammation is characterized by increased production of acute phase proteins in the liver. The induction of the hepatocytic response is primarily mediated through soluble cytokines such as IL-1, IL-6, TNF-alpha, and transforming growth factor beta, which bind to specific cell surface receptors and regulate gene expression of acute-phase proteins. Hepatoma cell lines, such as HepG2, represent a model system for studying acute-phase protein synthesis. HepG2 is induced to produce a variety of acute-phase proteins, including alpha 1-antitrypsin, alpha 1-antichymotrypsin, fibrinogen, alpha 1-acid glycoprotein, and haptoglobin, upon stimulation with cytokines. Analysis of HepG2 by reverse transcriptase PCR indicated that this cell line synthesized mRNA specific for the human C5a receptor (CD88). Flow cytometric analysis of HepG2 cells indicated that these cells bound anti-CD88 Ab, thus confirming our RT-PCR data by demonstrating that these cells also express the C5a receptor. Because C5a has been shown to be a potent mediator of inflammation and HepG2 cells express CD88, we assessed the possibility that C5a was capable of stimulating acute-phase protein synthesis by HepG2 cells. The results indicate that binding of human C5a to CD88 on HepG2 cells resulted in an increased production of alpha 1-antitrypsin- and alpha 1-antichymotrypsin-specific mRNA as assayed by RT-PCR. Analysis of culture supernatants derived from C5a-stimulated HepG2 cells showed an increased production of alpha 1-antitrypsin as measured by solid-phase ELISA. alpha 1-antitrypsin production by HepG2 cells was a direct result of C5a stimulation as evidenced by the fact that anti-C5a receptor Ab inhibited the response. These results suggest that C5a may be an important mediator of APP production in the regulation of the inflammatory response.

Acute-Phase Proteins↗
PubMed1984

Structure and function of the anaphylatoxins.

Chemical and physical characterization of the anaphylatoxin molecules have provided a reasonably clear description of the architecture of these bioactive proteins. The primary structures of C3a, C4a, and C5a from man and from a number of animal species have been elucidated, and it is apparent that the three anaphylatoxins are genetically related. The anaphylatoxin protein chains very in length from 74 to 78 residues and no fewer than 30% of the residues are homologous when comparing C3a, C4a, and C5a within or between species. Synthetic peptide studies have been instrumental in identifying molecular features essential for the function of anaphylatoxins. Information gleaned from the structure-function studies with synthetic analogue peptides of the anaphylatoxins define putative "active sites" in these effector molecules. Linear sequences at the carboxy-terminus of C3a and C4a fulfill all of the criteria of an "active site," in that synthetic peptides of an identical sequence can mimic the biologic actions of the natural factors. In the case of human C3a, a crystallographic analysis has been performed and a three-dimensional structure was elucidated at the 3.2 A level. The crystalline structure of C3a provides valuable new information regarding the alpha helical regions and identifies the arrangement of intra-chain disulfide linkages. Taken together, the structural data now accumulated for anaphylatoxins permit molecular modelling of these proteins, designates favored conformational arrangements of the native structures, and specifically localizes the effector sites. Furthermore, elements at the essential active site have been defined with such precision that models are proposed detailing the exact nature of ligand interactions between anaphylatoxins and specific cellular receptors. Biologic characterization of the anaphylatoxins continues at a rapid pace and each advance provides a clearer view of the role of these humoral mediators in host defense. A variety of responses to anaphylatoxins are known to occur at the cellular level and are mediated in a hormone-like fashion. Diversity of action for these factors at the tissue level is readily explained by the numerous cell types stimulated by the anaphylatoxins. Cellular responses to the anaphylatoxins are perhaps the most easily defined and studied; however, tissue and systemic effects more accurately reflect the physiologic role of anaphylatoxins. Considerable progress has been made in understanding the mechanisms whereby anaphylatoxins mediate two major tissue effects, namely enhancement of vascular permeability and induction of smooth muscle contraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1996

Inhibition of anaphylatoxin C3a- and C5a- but not nerve stimulation- or Noradrenaline-dependent increase in glucose output and reduction of flow in Kupffer cell-depleted perfused rat livers.

In isolated in situ perfused rat livers, infusion of anaphylatoxins C3a and C5a, activation peptides of the complement system, as well as stimulation of sympathetic hepatic nerves have been shown to increase hepatic glucose output and to reduce hepatic flow. These effects were mediated via an at least partially prostanoid-dependent intercellular signalling chain between nonparenchymal cells and hepatocytes. Kupffer cells have been implicated as the source of prostanoids in the anaphylatoxin-dependent signalling chain and Ito cells in the nerve stimulation-dependent signalling chain, because anaphylatoxins and noradrenaline increased prostanoid synthesis in isolated Kupffer and Ito cells, respectively. To further corroborate this hypothesis, anaphylatoxins were infused and hepatic nerves were stimulated in perfused rat livers in which Kupffer cells had been largely depleted by treatment of the animals with gadolinium chloride. Native human anaphylatoxin C3a (nhC3a) and recombinant rat anaphylatoxin C5a (rrC5a) increased prostanoid formation as well as glucose output and reduced flow in perfused rat liver. In Kupffer cell-depleted rat livers, the nhC3a- and rrC5a-mediated prostanoid formation was nearly abolished, and the increase in glucose output and the reduction of flow were reduced to between 30% and 50% (area under the curve [AUC]) of control livers. In contrast, stimulation of hepatic nerves increased glucose output and reduced flow to a similar extent in Kupffer cell-depleted livers as in control livers. These results indicate that Kupffer cells were not involved in the prostanoid-mediated nerve stimulation-dependent increase in glucose output and reduction of flow. Kupffer cells seemed, however, to be at least one major source of the anaphylatoxin-mediated prostanoid formation and, consequently, stimulation of glucose release and flow reduction in perfused liver. Because the metabolic and hemodynamic anaphylatoxin effects were not completely blocked in livers of gadolinium-treated animals, either Kupffer cells may not have been entirely eliminated, or yet another nonparenchymal cell type and mediator might be involved in the anaphylatoxin-elicited intercellular communication between nonparenchymal cells and hepatocytes.

Anaphylatoxins↗
PubMed1980

Pulmonary injury induced by C3a and C5a anaphylatoxins.

Homogeneous anaphylatoxins C3a (human or porcine), C5a (porcine), and the porcine classic anaphylatoxin, a mixture of C5a and C5a des Arg, isolated from complement-activated serum, were shown to induce acute pulmonary injury in the guinea pig following intrabonchial instillation. The gross physiologic response to these factors is characterized by respiratory distress with rapid, shallow breathing. Administration of 8--17 micrograms/kg of porcine classic anaphylatoxin proved lethal in 50% of the animals treated. The acute response (less than 20 minutes after instillation) of pulmonary tissue to insult by the anaphylatoxins is characterized by constriction of the smooth muscle walls in both bronchioles and pulmonary arteries and by focal atelectasis. Aggregates of platelets and leukocytes in pulmonary vessels and in other organs such as the chambers of the heart were commonly observed after intrabronchial administration of the anaphylatoxins. Although C3a was never lethal in guinea pigs even when doses as high as 500 micrograms/kg were administered by the intrabronchial route, this anaphylatoxin did induce the same pattern of acute pulmonary injury as C5a. In vitro experiments employing guinea pig platelets indicated that these cells aggregate in the presence of 10(-10) M porcine C5a but are not affected by C3a (human or porcine) even at levels up to 10(-6) M. Hence, platelet aggregation as observed in vivo may be directly affected by C5a, but in the case of C3a, secondary mediators must be involved. Anaphylatoxin preparations were also shown to induce contraction of guinea pig lung strips in vitro: this effect was not inhibited by antihistamines at concentrations that blocked contraction to exogenous histamine. The in vivo response to anaphylatoxin could be blocked with high doses of the antihistamine chlorpheniramine but not by corresponding doses of diphenhydramine.

Anaphylatoxins↗
PubMed1975

Purification and partial characterization of human and porcine C3a anaphylatoxin.

C3a anaphylatoxin is a protein fragment generated enzymatically in serum during activation of the third component of complement (C3). A four-step procedure is described for the purification of human and porcine C3a anaphylatoxins from their respective sera after activation with inulin. Because serum carboxypeptidase rapidly inactivates C3a, the inhibitor epsilon-aminocaproic acid (EACA) was added during C3 activation, thus permitting isolation of fully active C3a anaphylatoxins directly from serum. A 2000-fold purification of C3a was achieved with an average 30% recovery assuming total conversion of C3 during treatment of serum with inulin. Human C3a anaphylatoxin obtained through the action of the C3 activating enzyme of the "alternate" pathway appeared nearly identical with the C3a obtained from isolated C3 after treatment with the C4,2 enzyme of the "classical" pathway or with trypsin. Comparisons were made between various properties of human and porcine C3a anaphylatoxins. The molecular weights differed only slightly. Electrophoresis on a cellulose acetate strip at pH 8.6 indicated a difference of approximately one net charge between human and porcine C3a, with the human anaphylatoxin exhibiting the more basic behavior. Although the amino acid compositions are similar, significant differences exist. The most marked difference was the total absence of threonine residues in porcine C3a. The NH2-terminal sequences of 20 amino acid residues were examined; homology existed for 16 of the 20 positions. Although partial analysis of the primary structure of human and porcine C3a indicates approximately 80% homology, no immunological cross-reactivity between the anaphylatoxins could be detected with antisera produced to either human or porcine C3a. In spite of the structural differences, the biological activities of porcine and human C3a were essentially identical. Smooth muscle contraction, increase in vascular permeability, and release of histamine from mast cells were similarly induced by equal amounts of anaphylatoxin from either human or porcine origin. Porcine C3a, like human C3a, was shown to contain a COOH-terminal arginyl residue essential for smooth muscle contraction and for induction of histamine release from mast cells. The sequence adjacent to the COOH-terminal arginine was Leu-Ala-Arg-COOH for both humans and porcine C3a. Current evidence suggests common mechanisms exist for the generation of C3a in various animal species and that the two known C3 activating enzymes in serum exhibit trypsin-like specificity.

Amino Acid Sequence↗
PubMed1984

Radioimmunoassay for anaphylatoxins: a sensitive method for determining complement activation products in biological fluids.

Activation of the blood complement system generates bioactive fragments called anaphylatoxins. The three anaphylatoxins C3a, C4a, and C5a are released during "classical pathway" activation while only C3a and C5a are released when the "alternative pathway" of complement is activated. Radioimmunoassays were designed to individually detect and quantitate the activation fragments C3a, C4a, and C5a in biological fluids without interference from the precursor molecules C3, C4, and C5. Kinetics of complement activation in fresh human serum exposed to the activators zymosan, heat-aggregated immunoglobulin, or cobra venom factor were monitored using the radioimmunoassay technique. For the first time, activation of components C3, C4, and C5 was followed simultaneously in a single serum sample. Analysis of the patterns and extent of anaphylatoxin formation during activation in serum may be used to screen for deficiencies or defects in the complement cascade. Levels of the anaphylatoxins in freshly drawn serum were much higher than levels detected in EDTA-plasma. Detection limits of anaphylatoxins in plasma are governed by background levels of 152 +/- 69, 155 +/- 33, and 5.4 +/- 6.6 ng/ml for C3a, C4a, and C5a, respectively. Detection of low-level complement activation in patient's blood, urine, or synovial fluid, using anaphylatoxin formation as an indicator, may prove useful in signaling numerous forms of inflammatory reactions. The demonstration of anaphylatoxins in clinical samples is being recognized as a valuable diagnostic tool in monitoring the onset of immune disease.

Anaphylatoxins↗
PubMed1983

Potentiation of the anaphylatoxins in vivo using an inhibitor of serum carboxypeptidase N (SCPN). I. Lethality and pathologic effects on pulmonary tissue.

Carboxypeptidase N (EC 3.4.12.7) (SCPN) is a plasma enzyme that efficiently inactivates the anaphylatoxins C3a and C4a and significantly reduces C5a spasmogenic activity by removing the C-terminal arginyl residue from each of these factors. The arginine analog DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (SCPN-INH) is a potent competitive inhibitor of SCPN with a Ki for this carboxypeptidase in serum of 2 x 10(-9) M. Therefore, we have used the SCPN inhibitor to potentiate biologic activity of the anaphylatoxins in vivo. Infusion via the carotid artery of about 40 mg of SCPN-INH into each of 8 adult guinea pigs inactivated the SCPN for at least 3 hours and caused no measurable toxic effects. When cobra venom factor (CVF) is infused into guinea pigs, it activates the alternative pathway of complement, thereby generating the anaphylatoxins C3a and C5a. Ordinarily, infusion of CVF is nonlethal, because the generated anaphylatoxins are rapidly converted to C3a des Arg and C5a des Arg by SCPN. However, CVF (200 micrograms) plus SCPN-INH delivered intravenously in 5 animals induced a lethal reaction in less than 5 minutes. The authors conclude that the lethal effect is due largely to the anaphylatoxins. Histologic sections of the lungs from treated animals show dramatic structural changes consistent with peripheral small airway constriction, bronchial constriction, and vasoconstriction of small muscular arteries. Also, cell aggregates are present in blood vessels. Other histologic changes include severe congestion, pulmonary edema, and an interstitial infiltrate of mononuclear cells. Large doses of chlorpheniramine prevent this lethal reaction. Lethality is apparently attributable to asphyxia and is dependent on the level of CVF administered: eg, 100 micrograms CVF was not lethal in 4 animals given SCPN inhibitor, although signs of respiratory distress were observed. On histologic examination of lungs from guinea pigs given CVF and SCPN-INH, the features are similar to those described when anaphylatoxins are instilled into guinea pig lungs. Intravenous application of purified C3a plus SCPN-INH also proved lethal in 3 of the 6 animals challenged. This is the first evidence that the C3a anaphylatoxin can elicit a lethal response.

Anaphylatoxins↗
PubMed1994

Adsorption of anaphylatoxins and platelet-specific proteins by filtration of platelet concentrates with a polyester leukocyte reduction filter.

Anaphylatoxins generated during storage of platelet concentrates (PCs) may potentially have side effects on platelet transfusion. We evaluated the anaphylatoxin-scavenging abilities of white blood cell reduction filters. Among the commercially available filters for PCs, one made with polyester fiber (PL50) dramatically adsorbed C3a and C4a anaphylatoxins to the respective mean level of 1,721-208 ng/ml and 1,240-141 ng/ml in 3-day-old PCs. C3a and C4a were measured as the native and des Arg form of each complement by radioimmunoassay. C3a and C4a anaphylatoxins in the supernatant plasma fraction from 3-day-old PC again decreased from 1,136 to 114 ng/ml and from 1,086 to 65 ng/ml, respectively. The filter also adsorbed 85% of platelet factor 4 (PF4) and 31% of beta-thromboglobulin (beta-TG), which had been released from platelets into the plasma during storage. The plasma levels of adhesive proteins such as fibronectin, fibrinogen, and von Willebrand factor, and plasma lactate dehydrogenase activity did not decrease after filtration. Another polyester filter (PL5A), on the other hand, significantly increased C3a and C4a levels with filtration. In addition, there was no PF4 adsorption ability during the filtration. The filters for red cells (RC50, BPF4, and R500A) had no anaphylatoxin adsorption capabilities. The observed specific adsorption of anaphylatoxins might be attributed to the electrostatic force between the positively charged anaphylatoxins with high pI and the possibly negatively charged filter membranes. Since PF4 and beta-TG have positively charged moieties in the C-terminal position, the same adsorption mechanism might operate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗