Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.     

Production of C5a by ASP, a serine protease released from Aeromonas sobria

Aeromonas sobria causes pus and edema at sites of infection. However, the mechanisms underlying these effects have not been elucidated. C5a, the amino-terminal fragment of the complement 5th component (C5), mimics these events. To investigate the involvement of C5a in the pathophysiology of A. sobria infection, we examined release of C5a from human C5 by a serine protease (ASP), a putative virulence factor secreted by this bacterium. C5 incubated with enzymatically active ASP induced neutrophil migration in a dose-dependent manner from an ASP concentration of 3 nM and in an incubation time-dependent manner in as little as 7 min, with neutrophil accumulation in guinea pigs at intradermal injection sites and neutrophil superoxide release. These effects on neutrophils were inhibited by a C5a-receptor antagonist. The ASP incubation mixture with C5 but not C3 elicited vascular leakage in a dose- and incubation time-dependent manner, which was inhibited by a histamine H(1)-receptor antagonist. Together with these C5a-like activities, ASP cleaved C5 to release only one C5a Ag, the m.w. of which was similar to that of C5a. Immunoblotting using an anti-C5a Ab revealed generation of a C5a-like fragment from human plasma incubated with ASP. These results suggest that ASP-elicited neutrophil migration and vascular leakage via C5a production from C5 could occur in vivo, which was supported by that ASP did not affect functions of C5a and neutrophil C5a receptor. Through C5a generation, ASP could be associated with the induction of pus and edema caused by infection with this bacterium.     

Expression and function of C5a receptor in mouse microvascular endothelial cells

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.     

Endothelial changes and microvascular leakage due to hyperthermia in chick embryos

The effects of hyperthermia on the developing 2- and 3-day chick embryo were studied by vital microscopy, in vivo microangiography and electron microscopy of post-capillary venules of the pellucid area of the yolk sac. Hyperthermia of 3 degrees C and 4 degrees C produced significant microvascular changes and perivascular oedema. The microvascular defects were characterized by interruption of the endothelial lining and the presence of blood cells breaking through the vessel walls. In addition, there were numerous inter-endothelial gaps with wide subendothelial spaces. Microangiography showed leakage from the vessel walls. It is concluded that hyperthermia produces vessel wall injury and induces the formation of gaps between endothelial cells resulting in extravasation of plasma and blood cells. These gaps are similar to those produced by biochemical mediators of inflammation. It is suggested that these microvascular changes with pathological leakage may play important roles in abnormal vascular and embryonic development.     

Complete primary structure of human C4a anaphylatoxin

C4a anaphylatoxin is derived from the fourth component (C4) of the blood complement system. The C4 alpha-chain is selectively cleaved between positions 77 and 78 by the protease C1s, a subcomponent of C1, generating the fragments C4a and C4b. Human C4a was isolated directly from fresh serum after C1 of the classical pathway of complement was activated by heat-aggregated gamma-globulin. The C4a anaphylatoxin is a cationic polypeptide of Mr = 9000 composed of 77 residues and devoid of histidine, tryptophan, and carbohydrate. The primary structure of human C4a was deduced from sequence analysis of two cyanogen bromide fragments and of peptides obtained after chymotryptic digestion of the COOH-terminal cyanogen bromide fragment. The proposed sequence is: (formula, see text) Manual alignment of the linear structures of human C3a, C4a, and C5a, based primarily on the location of two Cys-Cys sequences in each indicate a 30% homology between C3a and C4a and a 36% homology between C5a and C4a. It was concluded from the sequence comparison that C3a, C4a, and C5a are a family of bioactive factors derived from precursor molecules that share a common genetic origin. Although the human anaphylatoxins share a partial structural identity and express similar biological activities, these factors ae immunologically distinct molecules having no antigenic determinants in common as judged by radioimmunoassay.     

Helical conformation at the carboxy-terminal portion of human C3a is required for full activity

Human C3a, a 77-residue fragment released during complement activation, is a potent spasmogen that contracts smooth muscle, enhances vascular permeability, and suppresses humoral immune responses. Studies with synthetic peptides have shown that the active site of this anaphylatoxin resides in the COOH-terminal portion of C3a; the minimal peptide structure capable of expressing activity contains residues 73-77, Leu-Gly-Leu-Ala-Arg (C3a-73-77). Longer synthetic C3a analogue peptides, e.g., C3a-57-77 containing the 21 COOH-terminal amino acids, exhibit activity nearly equivalent to that of intact C3a. Circular dichroism spectra of peptide C3a-57-77 in aqueous buffer containing 25% (v/v) trifluoroethanol indicated helical structure (41% helix), and analysis of the sequence suggested an amphipathic surface. We have synthesized several 21-residue peptide analogues of the natural C3a sequence containing residues 57-77 that were designed to enhance helix and to accentuate amphipathy. Syntheses were designed to include strategic placement of the helix-promoting residues 2-aminobutyric acid (beta-methylalanine) and 2-aminoisobutyric acid (alpha-methylalanine). Two 21-residue C3a analogue peptides that were designed to enhance helical content were shown to exhibit greater biological activity than either the native factor C3a or C3a-57-77. Moreover, activity was abrogated by the appropriate placement of helix-breaking residues, e.g., proline, suggesting that a conformational requirement for activity is genuine. These observations suggest that a helical conformation is requisite for optimal C3a activity and that in intact C3a the NH2-terminal portion (residues 1-21) and the disulfide-linked core (residues 22-57) function primarily to stabilize ordered conformation at the COOH-terminal region of the molecule.     

Circular dichroism of C3a anaphylatoxin. Effects of pH, heat, guanidinium chloride, and mercaptoethanol on conformation and function.

Circular dichroism spectra for C3a anaphylatoxins (protein fragments generated enzymatically in serum during activation of the third component of complement (C3)) from human and porcine sources were compared in the region of 190 to 250 nm. The spectra were indistinguishable in this region, although an estimated difference of approximately 20% exists between the primary structures of human and porcine C3a. Calculations indicated a total of 40 to 45% alpha helical content based on either the 208 or 222 nm extremum of the CD spectra. Addition of either mercaptoethanol or 6 M guanidinium chloride to human C3a produced a marked decrease in the mean residue ellipticity centered at 222 nm without irreversibly affecting the biological activity. Simultaneous addition of mercaptoethanol and guanidinium chloride virtually eliminated the CD contribution at 222 nm and resulted in more than 90% inactivation of the anaphylatoxin. Removal of the denaturant and reducing agent restored both the biological activity and the CD spectrum of C3a. Heat treatment or reduction and alkylation of human C3a produced biologically inactive and conformationally modified anaphylatoxin. In contrast, C3a inactivated by enzymatic removal of the COOH-terminal arginyl residue was structurally unchanged as judged by CD measurements. Consequently, it is proposed that in addition to the essential COOH-terminal arginyl residue, a highly ordered conformation is required for biological functionality of the C3a molecule.     

The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74).

The substantial variations in the responses of cells to the anaphylatoxin C5a and its desarginated form, C5adR(74), suggest that more than one type of cell surface receptor for these ligands might exist. However, only a single receptor for C5a and C5adR(74), CD88, has been characterized to date. Here we report that the orphan receptor C5L2/gpr77, which shares 35% amino acid identity with CD88, binds C5a with high affinity but has a 10-fold higher affinity for C5adR(74) than CD88. C5L2 also has a moderate affinity for anaphylatoxin C3a, but cross-competition studies suggest that C3a binds to a distinct site from C5a. C4a was able to displace C3a, suggesting that C5L2, like the C3a receptor, may have a low binding affinity for this anaphylatoxin. Unlike CD88 and C3a receptor, C5L2 transfected into RBL-2H3 cells does not support degranulation or increases in intracellular [Ca(2+)] and is not rapidly internalized in response to ligand binding. However, ligation of C5L2 by anaphylatoxin did potentiate the degranulation response to cross-linkage of the high affinity IgE receptor by a pertussis toxin-sensitive mechanism. These results suggest that C5L2 is an anaphylatoxin-binding protein with unique ligand binding and signaling properties.     

Characterization of synthetic C3a analog peptides on human eosinophils in comparison to the native complement component C3a

The C3a anaphylatoxin is a potent proinflammatory mediator derived from the complement system inducing biologic effects of human eosinophils like Ca2+ transients and the activation of the respiratory burst. These findings support an important role for C3a in diseases typically associated with a peripheral blood or tissue eosinophilia. Synthetic human C3a analogue peptides with variations at the C-terminal effector domain have been evaluated with respect to their binding affinity and signaling potency on human eosinophils. Flow cytometrical analysis and RT-PCR revealed that the C3a receptor is constitutively expressed on human eosinophils. Peptides bearing an N-terminal 9-fluorenylmethoxycarbonyl and the 6-aminohexanoyl motif were the most powerful peptides tested. Amino acid replacements in the conserved C-terminal pentapeptide decreased binding affinity and functional potency substantially. In addition, synthetic C3a analogue peptides induced C3aR internalization, led to transient changes of intracellular Ca2+ concentration, and did release reactive oxygen species in human eosinophils indicating the in vivo relevance of C3a-related sequences. The tripeptide LAR was found to be essential for C3a receptor binding on human eosinophils. Moreover, the putative binding motif of C3a anaphylatoxin is also crucial for the induction of biologic effects in the human system such as changes of intracellular Ca2+ concentration and the release of reactive oxygen species. This study demonstrates that the carboxyl terminus is important for the interaction with the C3aR and the biologic potency of C3a anaphylatoxin in the human system and plays a key role in the activation process of human eosinophils.     

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.     

Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin-shock

The complement anaphylatoxin C3a, on binding the C3aR, mediates numerous proinflammatory activities. In addition, recent in vitro studies with C3a have implicated C3aR as a possible anti-inflammatory receptor. Because of its possible dual role in modulating the inflammatory response, it is uncertain whether C3aR contributes to the pathogenesis of endotoxin shock. Here, the targeted-disruption of the C3aR in mice is reported. These mice exhibit an enhanced lethality to endotoxin shock with a pronounced gene dosage effect. In addition, the plasma concentration of IL-1beta was significantly elevated in the C3aR(-/-) mice compared with their littermates following LPS challenge. These findings demonstrate an important protective role for the C3aR in endotoxin shock and indicate that, in addition to its traditionally accepted functions in mediating inflammation, the C3aR also acts in vivo as an anti-inflammatory receptor by attenuating LPS-induced proinflammatory cytokine production.     

Postischemic anti-inflammatory effects of bradykinin preconditioning

We sought to determine the mechanisms whereby brief administration of bradykinin (bradykinin preconditioning, BK-PC) before prolonged ischemia followed by reperfusion (I/R) prevents postischemic microvascular dysfunction. Intravital videomicroscopic approaches were used to quantify I/R-induced leukocyte/endothelial cell adhesive interactions and microvascular barrier disruption in single postcapillary venules of the rat mesentery. I/R increased the number of rolling, adherent, and emigrated leukocytes and enhanced venular albumin leakage, effects that were prevented by BK-PC. The anti-inflammatory effects of BK-PC were largely prevented by concomitant administration of a B(2)-receptor antagonist but not by coincident B(1) receptor blockade, nitric oxide (NO) synthase inhibition, or cyclooxygenase blockade. However, NO synthase blockade during reperfusion after prolonged ischemia was effective in attenuating the anti-inflammatory effects of BK-PC. Pan protein kinase C (PKC) inhibition antagonized the beneficial effects of BK-PC but only when administered during prolonged ischemia. In contrast, specific inhibition of the conventional PKC isotypes failed to alter the effectiveness of BK-PC. These results indicate that bradykinin can be used to pharmacologically precondition single mesenteric postcapillary venules to resist I/R-induced leukocyte recruitment and microvascular barrier dysfunction by a mechanism that involves B(2) receptor-dependent activation of nonconventional PKC isotypes and subsequent formation of NO.     

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.     

人过敏毒素C5a反义cDNA的克隆、表达和拮抗作用

采用引物二聚体配对搭桥法成功扩增了人过敏毒素C5a全长的反义cNDA ,将扩增的反义cNDA克隆到原核表达载体pPROEXTM HTc上 ,与 6×His头形成融合基因 ,转化E .coliDH5α ,30℃下经IPTG诱导 ,该融合蛋白在大肠杆菌中以可溶性形式表达 ,表达量达 10 % ;利用金属螯合亲和层析一步法纯化 ,得到纯度 80 %的融合蛋白 ;烟草蚀刻病毒蛋白酶酶切超滤浓缩后 ,得到高浓度高纯度人过敏毒素C5a反义肽 ,经N端蛋白测序鉴定 ,其氨基酸序列正确 .髓过氧化物酶 (myeloperoxi dase ,MPO)为单核细胞PMN所特有 ,其活性可以间接反映C5a趋化白细胞的能力 ,C5a刺激血管内皮细胞表达胞间粘附分子 (ICAM 1)、血管间粘附分子 (PCAM 1)等 ,后者能增加对PMN的粘附 ,检测MPO活性可间接反映出C5a的功能 .还观察了纯化的C5a反义肽对C5a刺激内皮细胞胞浆[Ca2 + ]浓度变化的影响 .高浓度高纯度的C5a反义肽对C5a过敏毒素存在拮抗作用 ,说明反义肽在补体系统C5a分子中是存在的 .这为深入研究C5a反义肽的结构与功能关系提供了物质保证     

Homocysteine causes cerebrovascular leakage in mice

Elevated plasma homocysteine (Hcy) is associated with cerebrovascular disease and activates matrix metalloproteinases (MMPs), which lead to vascular remodeling that could disrupt the blood-brain barrier. To determine whether Hcy administration can increase brain microvascular leakage secondary to activation of MMPs, we examined pial venules by intravital video microscopy through a craniotomy in anesthetized mice. Bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was injected into a carotid artery to measure extravenular leakage. Hcy (30 microM/total blood volume) was injected 10 min after FITC-BSA injection. Four groups of mice were examined: 1) wild type (WT) given vehicle; 2) WT given Hcy (WT + Hcy); 3) MMP-9 gene knockout given Hcy (MMP-9-/- + Hcy); and 4) MMP-9-/- with topical application of histamine (10(-4) M) (MMP-9-/- + histamine). In the WT + Hcy mice, leakage of FITC-BSA from pial venules was significantly (P < 0.05) greater than in the other groups. There was no significant leakage of pial microvessels in MMP-9-/- + Hcy mice. Increased cerebrovascular leakage in the MMP-9-/- + histamine group showed that microvascular permeability could still increase by a mechanism independent of MMP-9. Treatment of cultured mouse microvascular endothelial cells with 30 microM Hcy resulted in significantly greater F-actin formation than in control cells without Hcy. Treatment with a broad-range MMP inhibitor (GM-6001; 1 microM) ameliorated Hcy-induced F-actin formation. These data suggest that Hcy increases microvascular permeability, in part, through MMP-9 activation.     

Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells

A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.     

Chimeric receptors of the human C3a receptor and C5a receptor (CD88)

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Galpha-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a.     

The alpha 4 beta 1 (very late antigen (VLA)-4, CD49d/CD29) and alpha 5 beta 1 (VLA-5, CD49e/CD29) integrins mediate beta 2 (CD11/CD18) integrin-independent neutrophil recruitment to endotoxin-induced lung inflammation

The beta(2) integrin cell adhesion molecules (CAM) mediate polymorphonuclear leukocyte (PMNL) emigration in most inflamed tissues, but, in the lung, other yet to be identified CAMs appear to be involved. In Lewis rats, the intratracheal injection of Escherichia coli-LPS induced acute (6-h) PMNL accumulation in the lung parenchyma (280 x 10(6) by myeloperoxidase assay; PBS control = 35 x 10(6)) and bronchoalveolar lavage fluid (BALF = 27 x 10(6); PBS = 0.1 x 10(6)). Parenchymal accumulation was not inhibited by a blocking Ab to beta(2) integrins and only minimally inhibited (20.5%; p < 0.05) in BALF. We examined the role of alpha(4)beta(1) and alpha(5)beta(1) integrins and of selectins in this PMNL recruitment. Treatment with mAbs to alpha(4)beta(1) or alpha(5)beta(1), even in combination, had no effect on PMNL accumulation induced by intratracheal LPS. However, anti-alpha(4) combined with anti-beta(2) mAbs inhibited PMNL recruitment to the parenchyma by 56% (p < 0.001) and to BALF by 58% (p < 0.01). The addition of anti-alpha(5) mAb to beta(2) plus alpha(4) blockade inhibited PMNL accumulation further (by 79%; p < 0.05). In contrast, blockade of L-, P-, and E-selectins in combination or together with beta(2), alpha(4), and alpha(5) integrins had no effect. LPS-induced BALF protein accumulation was not inhibited by treatment with anti-beta(2) plus alpha(4) mAbs, but was prevented when alpha(5)beta(1) was also blocked. Thus, while selectins appear to play no role, alpha(4)beta(1) and alpha(5)beta(1) function as major alternate CAMs to the beta(2) integrins in mediating PMNL migration to lung and to pulmonary vascular and epithelial permeability.     

Circular dichroism of C5a anaphylatoxin of porcine complement

The far-ultraviolet circular dichroism spectrum of C5a anaphylatoxin of porcine complement implies that it has a substantial content of helical structure. The circular dichroism spectra of C5a in the 200–250 nm region at pH 7.2 and 3.7 are nearly identical and resemble those of C3a anaphylatoxin. Treatment of C5a with 2-mercaptoethanol progressively diminishes the ellipticity at 222 nm and its anaphylatoxic activity to limiting values. Removal of the reducing agent by dialysis completely restored both the ellipticity at 222 nm and the activity. This finding indicates that the integrity of the secondary conformation of the C5a molecule is largely dependent on disulfide bonds and is essential for its full activity.