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PubMed1994

A physical map of two clusters containing the genes for six proinflammatory receptors.

The genes encoding for six receptors involved in the proinflammatory response lie on different chromosomes. Two receptors for N-formylpeptides (FPR1, FPR2), one homologue of these (FPRL2), and the receptor for complement fragment C5a (C5aR) are encoded by four genes mapped to human chromosome 19. The genes encoding two receptors for Interleukin-8 (IL8RA, IL8RB) have been located on human chromosome 2. In this report we describe the physical linkage between these genes in two different clusters. DNA fragments obtained by digestion with several restriction enzymes were separated by pulsed field gel electrophoresis. Nylon filters were hybridized with probes corresponding to the complete translated sequences of these genes. These probes were obtained from a human neutrophil cDNA-library. The four genes on chromosome 19 are contained in a 200 kilobase (kb) fragment. Both Interleukin-8 receptors are on a 150 kb fragment. The complete translated sequences for these genes were amplified from genomic DNA, indicating that they are contained in a single exon.

Base Sequence↗
PubMed1996

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.

Amino Acid Sequence↗
PubMed1987

Activation of neutrophil chemotaxis by leukotriene B4 and 5-hydroxyeicosatetraenoic acid in chronic inflammatory bowel disease.

Circulating neutrophils were investigated in 15 patients with Crohn's disease (CD), 15 with ulcerative colitis (UC), and 15 healthy volunteers. Dose-response curves for chemotaxis in Boyden chambers were analysed for sensitivity to leukotriene B4 (LTB4), its 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4) catabolites, and 5- and 15-hydroxyeicosatetraenoic acids (HETEs). Positive controls included: complement 5a (C5a), formy-L-methionyl-L-leucyl-L-phenylalanine (f-Met-Leu-Phe), and casein. Control chemotaxis test were performed at concentrations yielding optimal responses in leucocytes of healthy volunteers. Chemotaxis to suboptimal concentrations of LTB4 1.0 and 3.2 nmol/l, and 5-HETE 316 nmol/l, was markedly depressed in patients with chronic inflammatory bowel disease (CIBD). Analyses of individual dose-response curves revealed an underlying decreased sensitivity to LTB4 in 11 out of 30 patients, to 5-HETE in 10 out of 30 patients with a corresponding decrease of median sensitivity to LTB4 and 5-HETE in both CD and UC. Peak responses to LTB4, 5-HETE, f-Met-Leu-Phe, and casein were identical in the three groups tested, whereas the C5a values were significantly depressed in both groups of patients (p less than 0.05). The potency of LTB4 exceeded that of 5-HETE by a factor of approximately 100 whereas 20-OH-LTB4 was nearly as potent as LTB4. 20-COOH-LTB4 and 15-HETE did not activate chemotaxis of human neutrophils. These findings are suggestive of a competitive inhibition of receptors with heterogeneity for LTB4 and 5-HETE.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemotaxis, Leukocyte↗
PubMed1990

The psoriasis-associated antigen, pso p27, participates in the formation of complement activating immune-complexes in psoriatic scale.

The psoriasis-associated antigen, pso p27, and antibodies recognizing this antigen were demonstrated in extracts from psoriatic scales. It was demonstrated that the antigen was present in complexes containing IgG as well as complement factor C1q. The complexes were studied with respect to complement-activating potential. This was measured as the ability of the complexes to induce the generation of the complement factor C5a using rabbit serum as the source of complement. The data obtained showed that the pso p27-containing complexes were able to activate the complement system, indicating that the pso p27 antigen may contribute to the inflammatory process in psoriasis.

Animals↗
PubMed2000

A codominant role of Fc gamma RI/III and C5aR in the reverse Arthus reaction.

Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex disease have led to opposing conclusions. As concluded in IgG FcRgamma-/- mice, manifestation of disease is almost exclusively determined by FcgammaR on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aR-/- mice suggested that, dependent on the tissue site, complement is more important than FcgammaR. In this paper, we demonstrate that, in response to IgG immune complex formation, FcgammaRI/III- and C5aR-mediated pathways are both necessary and only together are they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely independent, suggesting an interaction between FcgammaR and C5aR. Therefore, FcgammaR-mediated responses can be integrated through C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each other.

Animals↗
PubMed1991

IFN-gamma inhibits inflammatory cell recruitment and the evolution of bacterial cell wall-induced arthritis.

Localization of streptococcal cell wall Ag (SCW) in the synovial tissue of treated rats induces an influx of leukocytes and a cell-mediated immune response leading to arthritis and joint destruction. Systemic administration of the T cell product, IFN-gamma (10(6) U/kg/day), suppresses the recruitment of leukocytes into the synovium and effectively inhibits the inflammation and pathology characteristic of SCW-induced arthritis (articular index 10.4 +/- 0.6 for SCW vs 2.0 +/- 0.7 for SCW with IFN-gamma, p less than 0.005). Monocyte-macrophages from animals treated with IFN-gamma exhibited defective chemotactic responses when tested in vitro and furthermore, monocytes cultured with IFN-gamma (25 to 500 U/ml) in vitro had significantly suppressed chemotactic responses to the complement fragment C5a (p less than 0.005). The decreased ability to migrate to C5a was associated with decreased binding of fluorochrome-conjugated C5a indicative of reduced expression of C5a receptors. Based on these data, IFN-gamma that induces monocyte maturation as reflected by increased Ia expression conversely inhibits C5a receptor expression. Although locally elevated IFN-gamma levels may serve to inhibit recruitment away from an inflammatory site, systemic exposure to IFN-gamma appears to inhibit leukocyte recruitment to the inflammatory site by its ability to induce premature maturation and concomitant inability to respond to certain chemotactic ligands. Inasmuch as monocyte recruitment to the synovium is pivotal in the development of SCW-induced polyarthritis, the ability of IFN-gamma to inhibit this event effectively inhibits the synovial pathology.

Animals↗
PubMed1987

Characterization of murine monoclonal antibodies that recognize neutralizing epitopes on human C5a.

We generated a panel of 10 murine monoclonal antibodies (MAbs) that recognize human complement fragment C5a. These MAbs were characterized for their ability to immunoprecipitate 125I-labeled C5a, bind C5a in solid-phase enzyme immunoassay, and block 125I-labeled C5a binding to polymorphonuclear leukocytes. Four of these MAbs had affinity constants for C5a in the 1 X 10(9) to 3 X 10(9) M-1 range. These MAbs blocked C5a-induced neutrophil polarization and chemiluminescence. They blocked the ability of passively administered C5a to cause neutropenia in rabbits. These anti-C5a neutralizing MAbs may have potential therapeutic use in states of complement activation.

Antibodies, Monoclonal↗
PubMed2001

Systemic complement depletion diminishes perihematomal brain edema in rats.

BACKGROUND AND PURPOSE: The complement cascade is activated after experimental intracerebral hemorrhage (ICH). It remains unclear, however, whether depleting the complement system will improve injury resulting from ICH. This study investigated the effects of systemic complement depletion on brain edema formation after ICH. METHODS: Fifty-six pentobarbital-anesthetized Sprague-Dawley rats were used. Treatment animals were complement-depleted with cobra venom factor (CVF) (intraperitoneally). Control rats received an equal volume of saline injection (intraperitoneally). In both treatment and control rats, autologous blood (100 microL) was infused stereotaxically into the right basal ganglia. Rats were killed 2, 24, or 72 hours later for brain water, ion, and tumor necrosis factor-alpha (TNF-alpha) measurements, for Western blot analysis, and for immunohistochemical studies. Brain edema was quantitated by wet/dry weight. TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Western blot analysis was applied for C9 semiquantification. Immunohistochemistry was used to detect complement C3d, C5a, C9, and myeloperoxidase. RESULTS: Perihematomal brain edema was reduced by systemic complement depletion at 24 hours (78.8+/-0.6% versus 81.5+/-0.8% in control, P:<0.01) and 72 hours (81.5+/-1.5% versus 83.6+/-0.9% in control, P:<0.05), while cerebellar water content was unaffected (78.2+/-0.3% versus 78.0+/-0. 1%). Complement depletion reduced TNF-alpha production 2 hours after ICH. Immunocytochemistry showed that complement depletion significantly reduced perihematomal C9 deposition, C3d production, and the number of C5a- and myeloperoxidase-positive cells. CONCLUSIONS: Complement depletion by CVF attenuates brain edema in ICH, indicating that complement activation plays an important role in ICH-induced brain edema. Preventing complement activation may be effective in the treatment of ICH.

Animals↗
PubMed1992

Contractile actions of C5a on isolated porcine myocardium.

Although intracoronary administration of the complement component, C5a, produces deleterious effects on regional coronary blood flow and segmental ventricular function, it is unclear whether a direct myocardial action contributes to the dysfunction induced by the anaphylatoxin. We therefore evaluated the effects of purified porcine C5a on contractile tension of isolated supported ventricular trabeculae from pig hearts. Muscles were studied in a myograph bath at 30 degrees C, electrically stimulated 12 times per minute, and stretched to produce maximal isometric developed tension. C5a concentrations of 30, 100, and 300 ng/ml increased tension (P less than 0.05) 9.8, 5.5, and 20.9%, respectively. In seven of nine muscles exposed to 300 ng/ml C5a, tension initially decreased 10.5% (P less than 0.05) before the positive inotropic effect. Tachyphylaxis was demonstrated by lack of contractile response to a second administration of C5a greater than 70 min after the initial exposure to the complement fragment. Blockade of histamine H1 receptors with diphenhydramine (10(-6) M) markedly attenuated both the positive and negative contractile responses to C5a. beta-Adrenoceptor blockade with propranolol (5.6 x 10(-7) M) did not alter the response to C5a. Levels of thromboxane (Tx)B2, the stable metabolite of TxA2, were augmented in the bath after exposure to C5a (59 +/- 27.3 to 104 +/- 28.2 pg/ml, P less than 0.05). Although the TxA2 agonist, U-46619 (50 ng/ml), significantly increased tension, TxA2 receptor blockade with SQ 29548 (50 ng/ml) did not alter the response to C5a.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1994

The degradation product of the C5a anaphylatoxin C5adesarg retains basophil-activating properties.

The complement cleavage product C5a is a potent agonist of different leukocyte types and also has anaphylatoxic properties through the release of mediators by basophils and tissue mast cells. C5a is very rapidly degraded by serum carboxypeptidase N which cleaves the functionally important carboxy-terminal arginine, generating C5desarg, a chemotactic agonist with little mast cell-activating ability. Here we show that natural human C5adesarg is still a trigger for basophil mediator release superior to other endogenous IgE-independent agonists such as monocyte chemotactic protein (MCP)-1, interleukin (IL)-8, C3a and platelet-activating factor. On a molar basis C5adesarg is only one order of magnitude less potent and about half as efficacious as C5a at inducing basophil degranulation. Priming of basophils with either IL-3, IL-5, granulocyte-macrophage-colony-stimulating factor (GM-CSF) or nerve growth factor (NGF) (with comparable efficacies, but different potencies: IL-3 > NGF > IL-5 > GM-CSF) enhanced histamine release and conditioned the cells to produce large amounts of leukotriene C4 (LTC4), which is not generated by basophils exposed to C5adesarg alone. The efficacy of C5a and C5adesarg at inducing histamine and LTC4 release by primed basophils was similar. Thus, C5adesarg is a stable inducer of release of inflammatory mediators by human basophils, particularly in primed cells, and complement may, therefore, play a role in immediate-type hypersensitivity diseases in allergic late-phase reactions.

Basophils↗
PubMed1997

Contractile actions of C5a on isolated porcine coronary resistance and conductance arteries.

The comparative effects of the complement component C5a on coronary resistance and conductance arteries have not been evaluated. To clarify the coronary contractile actions of this anaphylatoxin, we studied the effects of C5a on development of isometric tension in isolated porcine coronary conductance and resistance arteries. Internal diameters of conductance and resistance vessels were 367 +/- 21 and 88 +/- 4 microns, respectively. Vessel ring segments were suspended in a microvessel myograph, stretched to the peaks of their length-tension curves, and precontracted with 30 mM K+ physiological salt solution. Dose-response curves to C5a (2, 10, and 50 nM) were obtained. At 50 nM, the C5a-induced increase in tension in resistance arteries (4.1 +/- 0.9 to 5.7 +/- 1.4 mN, 35.8 +/- 3.4%) was significantly greater (P < 0.05) than in conductance arteries (10.7 +/- 2.2 to 12.4 +/- 2.6 mN, 15.6 +/- 3.0%). A specific thromboxane A2 receptor antagonist, SQ-29548, virtually eliminated C5a-induced increases in tension. C5a did not impair endothelium-dependent relaxation in either conductance or resistance vessels, as indicated by the half-maximal effective dose (ED50) calculated from bradykinin dose-response curves before and after exposure of the vessels to 50 nM C5a (resistance: pre-C5a ED50 = 2 nM, post-C5a ED50 +/- 3 nM; conductance: pre-C5a ED50 +/- 13 nM, post-C5a ED50 +/- 14 nM). These results indicate that 1) C5a has a greater vasoconstrictive effect on isolated porcine resistance than conductance coronary arteries; 2) C5a-induced coronary constriction is mediated by thromboxane A2; and 3) C5a does not impair endothelium-dependent relaxation of isolated porcine coronary arteries.

Animals↗
PubMed1989

Characterization of a receptor for C5a anaphylatoxin on human eosinophils.

The complement anaphylatoxin peptide C5a is well known to activate human polymorphonuclear leukocytes through receptor-mediated processes. C5a has also been reported to activate eosinophils for both chemotaxis and hexose uptake. We characterized the receptor molecule for human C5a on human eosinophils and compared it with the receptor on human neutrophils. At 4 degrees C, uptake of 1 nM 125I-C5a reaches equilibrium within 10 min on both cell types. Binding of 125I-C5a occurs over a concentration range comparable to that which stimulates lysosomal enzyme release and hexose uptake in both cell types. Scatchard analyses of the data indicate the presence of two receptor populations on eosinophils; a high affinity receptor with 15,000-20,000 sites/cell and a Kd of 3.1 +/- 0.6 x 10(-11) M, and a low affinity receptor with approximately 375,000 sites/cell and a Kd of 1 x 10(-7) M. Parallel experiments with neutrophils indicate the presence of a single receptor population with approximately 90,000 sites/cell and a Kd of 4.8 +/- 0.1 x 10(-10)M. The eosinophil receptor molecule was further characterized by covalently cross-linking 125I-C5a to cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material. Autoradiography indicates the presence of a dominant C5a-eosinophil receptor complex with an apparent mass of 60-65 kDa. The corresponding neutrophil-C5a receptor complex has an apparent mass of 50-52 kDa as observed by others. When the cross-linked 125I-C5a-receptor complex was treated with cyanogen bromide, different patterns were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for neutrophils and eosinophils. Thus, human eosinophils have a receptor for C5a anaphylatoxin which appears to be distinct from the C5a receptor present on human neutrophils.

Cold Temperature↗
PubMed1993

Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC).

We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.

Animals↗
PubMed1975

Release of histamine from rat mast cells by the complement peptides C3a and C5a.

Suspensions of rat mast cells were used to study the histamine-releasing actions of anaphylatoxins C3A and C5a in vitro. The peptides, derived from human or porcine complement proteins C3 and C5, were less potent than 48/80 but more potent than bradykinin in stimulating release of histamine from mast cells. The pattern of release resembled that of the anaphylactic release action, e.g. release was limited to less than 30 per cent of the cell histamine, the reaction was calcium-dependent and was potentiated by phosphatidyl serine. When C3a and C5a were added together to mast cell suspensions, the amount of histamine released was additive. Similarly, release by either peptide combined with bradykinin was additive. Histamine-releasing activity (as well as smooth muscle-stimulating activity) was abolished when the peptides were treated with pancreatic carboxy-peptidase B. Active or inactive peptides were bound by mast cells and addition of active C3a in combination with the inactive, des-arginine derivative, C3ai, resulted in partial inhibition of histamine release.

Anaphylaxis↗
PubMed2000

[A novel monocyte chemotactic factor that connects innate immunity and acquired immunity].

A monocyte chemotactic factor was separated from rheumatoid arthritis synovium, and identified as a homo-dimer of S19 ribosomal protein. When S19 protein was treated with the plasma transglutaminase, an inter-molecular isopeptide bond was formed between Lys122 and Gln137, and the chemotactic activity appeared. The S19 protein dimer caused chemotaxis via the receptor on monocytes to C5a, the complement C5-derived chemotactic factor. This dimer antagonized the C5a receptor on neutrophils. This dimer was released from apoptotic cells, and functioned in the phagocytic clearance of these cells by recruiting circulating monocytes. After engulfment, the macrophages moved to regional lymph nodes, and presented apoptotic cell-derived antigens to T cells. T cells proliferated and activated B cells, and eventually the IgM antibody response was observed. Cooperation between the innate immune response and the acquired response would induce an effective host defense primarily against viral infection.

Animals↗
PubMed1994

Primary structure and functional characterization of rat C5a: an anaphylatoxin with unusually high potency.

The anaphylatoxin C5a is a pro-inflammatory factor generated from C5 during complement activation. C5a derived from rat C5 exhibits significantly greater potency compared to C5a from other species. Rat C5a was 25-fold more potent than human C5a for eliciting spasmogenic contraction of guinea pig ileum. Proteolytic removal of the C-terminal arginine of C5a (C5adesArg) reduced spasmogenic potency of rat C5a by only 4-fold compared to a 3,000-fold reduction for human C5adesArg. In addition, rat C5adesArg was 50-fold more potent than human C5adesArg in a guinea pig vascular permeability (in vivo) assay and as a chemotactic factor for human neutrophils. C5a and C5adesArg were purified from zymosan-activated rat serum. Rat C5a, like human C5a, is glycosylated but contains 77 amino acid residues instead of the 74 residues of human C5a. Comparison of the primary structures of rat and human C5a indicated differences at 30 positions including an insert of 3 residues (LLH) in the rat molecule between residue positions 3 and 4 in human C5a. Insertion of residues LLH between Gln-3 and Lys-4 in a recombinant human C5a molecule using site-directed mutagenesis failed to enhance potency. Synthetic C-terminal analogues of rat C5a proved to be measurably more potent than the corresponding human C5a analogues (Ember JA et al., 1993, Protein Sci 2(Suppl 1):159 [Abstr]). We conclude that multiple sequence differences in the C-terminal effector portion and/or elsewhere in rat C5a, but not the LLH insert, account for the significant enhancement in potency of rat C5a over C5a from other species.

Amino Acid Sequence↗