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PubMed1990

Partial characterization of a C5a-inhibitor in peritoneal fluid.

We have recently described a 40-kDa protein in peritoneal fluid that neutralized the chemotactic activity of the C fraction C5a. It was deficient in peritoneal fluids of patients suffering from familial Mediterranean fever. Further characterization of the inhibitor with the use of 125I-rC5a binding to dibutyryl cAMP-induced U937 cells revealed dependence on the peritoneal fluid concentration, on the time of incubation and on temperature and pH. Fractionation of 125I-C5a on Sephadex G-50 column, before and after incubation with peritoneal fluid, revealed similar fractionation patterns despite loss of biologic activity of the treated C5a (but not its binding to polyclonal anti-C5a antibody). Analysis of rC5a by SDS-PAGE before and after treatment with partially purified C5a inhibitor, revealed slight modification of the inhibitor-treated C5a. Using various protease inhibitors (i.e., PMSF) suggested that the C5a inhibitor is a serine protease. It neutralized C5a by means of limited proteolysis which did not change C5a immunologic properties and changed only slightly its m.w. but abolished its receptor binding and chemotactic functions. It is suggested that the C5a inhibitor plays a role in the regulation of inflammation in serosal tissues and that its deficiency in familial Mediterranean fever may explain the attacks of sterile inflammation characteristic of this disease.

Ascitic Fluid↗

PubMed1995

Stimulation of glycogen phosphorylase in rat hepatocytes via prostanoid release from Kupffer cells by recombinant rat anaphylatoxin C5a but not by native human C5a in hepatocyte/Kupffer cell co-cultures.

Human anaphylatoxin C3a had previously been shown to increase glycogenolysis in perfused rat liver and prostanoid formation in rat liver macrophages. Surprisingly, human C5a, which in other systems elicited stronger responses than C3a, did not increase glycogenolysis in perfused rat liver. Species incompatibilities within the experimental system had been supposed to be the reason. The current study supports this hypothesis: (1) In rat liver macrophages that had been maintained in primary culture for 72 h recombinant rat anaphylatoxin C5a in concentrations between 0.1 and 10 micrograms/ml increased the formation of thromboxane A2, prostaglandin D2, E2 and F2 alpha 6- to 12-fold over basal within 10 min. In contrast, human anaphylatoxin C5a did not increase prostanoid formation in rat Kupffer cells. (2) The increase in prostanoid formation by recombinant rat C5a was specific. It was inhibited by a neutralizing monoclonal antibody. (3) In co-cultures of rat hepatocytes and rat Kupffer cells but not in hepatocyte mono-cultures recombinant rat C5a increased glycogen phosphorylase activity 3-fold over basal. This effect was inhibited by incubation of the co-cultures with 500 microM acetylsalicyclic acid. Thus, C5a generated either locally in the liver or systemically e.g. in the course of sepsis, may increase hepatic glycogenolysis by a prostanoid-mediated intercellular communication between Kupffer cells and hepatocytes.

Acetylglucosaminidase↗

PubMed1988

The use of high field NMR to determine homogeneity in a preparation of human C5a produced by Escherichia coli.

The polypeptide backbone of human C5a was prepared by recombinant DNA techniques. Standard biochemical analysis guided the protein separation to give a sample of C5a which was deemed homogeneous. However, nuclear magnetic resonance (NMR) studies showed the material to be significantly heterogeneous. Reanalysis by high performance liquid chromatography (HPLC) corroborated the NMR results. Further separation by HPLC and analysis by NMR spectroscopy guided the isolation of rC5a to greater than 92% purity. NMR analysis, immunochemical and biological evaluation of the impurities showed them to be C5a structural variants. These results indicate that conventional methods of protein chemistry can fail to reveal heterogeneity in recombinant proteins, and in some circumstances NMR spectroscopy can aid in their purification.

Chromatography, High Pressure Liquid↗

PubMed2004

Protective effects of a potent C5a receptor antagonist on experimental acute limb ischemia-reperfusion in rats.

OBJECTIVES: The capacity of a potent C5a receptor antagonist to inhibit various parameters of local and remote organ injury following lower limb ischemia-reperfusion (I/R) in rats was investigated. METHODS: Rats were subjected to 2 h bilateral hindlimb ischemia and 4 h reperfusion. Drug-treated rats received AcF-[OPdChaWR] (1 mg/kg) iv either 10 min before ischemia or 10 min prior to reperfusion, or orally (10 mg/kg) 30 min prior to ischemia. Levels of circulating creatine kinase (CK), lactate dehydrogenase (LDH), alanine and aspartate aminotransferase (ALT/AST), creatinine, blood urea nitrogen (BUN), polymorphonuclear leukocytes (PMNs), and calcium (Ca(++)) and potassium (K(+)) ions were determined. Other parameters measured included urinary protein levels, muscle edema, and myeloperoxidase (MPO) concentrations in the lung, liver, and muscle along with liver homogenate tumor necrosis factor-alpha (TNF-alpha) concentrations.L RESULTS: imb I/R injury was characterized by significant elevations of CK, LDH, ALT, AST, creatinine, BUN, proteinuria, PMNs, serum K(+), muscle edema, organ MPO, and liver homogenate TNF-alpha concentrations, but a significant reduction in serum Ca(2+) concentrations. When rats were treated with AcF-[OPdChaWR], there were significant improvements in all these parameters. CONCLUSIONS: These results indicate a pivotal role for C5a in inducing local and remote organ injury and suggest a possible new drug therapeutic category for preventing anticipated tissue injury associated with I/R.

Acute Disease↗

PubMed2004

Bacterial chaperone protein, Skp, induces leukocyte chemotaxis via C5a receptor.

C5a receptor has been identified as a leukocyte chemotactic receptor to two intrinsic chemical mediators, C5a and the S19 ribosomal protein dimer, so far. We found an Escherichia coli protein that also induced the chemotactic responses of monocytes and polymorphonuclear leukocytes via the C5a receptor. We identified the E. coli-derived chemoattractant to be Skp by the molecular size and the N-terminal amino acid sequence. Skp is a periplasmic chaperone protein widely present in gram-negative bacterial species. Immunoabsorption experiments indicated that Skp was the major leukocyte chemotactic factor in the E. coli extract. Receptor-antagonizing experiments with analogue peptides of S19 ribosomal protein and of C5a suggested that Skp induced the receptor activation by the two-step binding mechanism as in the cases of the intrinsic mediators, sharing the ligand-binding site of the receptor among them at each binding step. The C5a receptor would play a role in the host defense directly recognizing the bacteria-derived protein, besides identifying the signals of the intrinsic chemical mediators.

Animals↗

PubMed2002

Inhibition by prostaglandin E(2) of anaphylatoxin C5a- but not zymosan-induced prostanoid release from rat Kupffer cells.

The proinflammatory anaphylatoxin C5a induces the release of prostanoids, ie, prostaglandins (PG) and thromboxane (TX), from the resident liver macrophages (Kupffer cells [KC]). Because KC themselves express prostanoid receptors, prostanoids--besides having paracrine functions--might regulate their own release in an autocrine loop. So far, such a possible feedback regulation has not been investigated systematically, probably because of methodological difficulties to measure newly synthesized prostanoids in the presence of added prostanoids. Here, after prelabeling of phospholipids with [(14)C]arachidonate, cellularly formed [(14)C]prostanoids were determined in the presence of added unlabelled prostanoids by thin layer chromatography. In cultured KC, recombinant rat C5a (rrC5a) rapidly increased PGD(2), PGE(2), and TXA(2) release, which was strongly reduced by PGE(2), but neither by PGD(2) nor by the TXA(2) analog U46619. The inhibitory effect of PGE(2) was mimicked by cAMP, indicating that the G(s)-coupled PGE(2) receptors type 2 or 4 were involved. Zymosan also enhanced prostanoid release from KC, but with slightly slower kinetics; this action was neither inhibited by PGE(2) nor by cAMP. Also in perfused rat livers, rrC5a enhanced prostanoid release from KC as shown by prostanoid overflow and thereby indirectly increased glucose output from hepatocytes. Again, PGE(2), but not PGD(2), inhibited rrC5a-elicited prostanoid overflow. This resulted in a complete inhibition of rrC5a-induced, prostanoid-mediated glucose output. Thus, PGE(2) can inhibit specifically the C5a-induced prostanoid release from KC via a feedback mechanism and thereby limit prostanoid-mediated hepatocellular defense reactions, eg, glucose release.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMedDate not supplied

Intraoperative blood salvage in excisional burn surgery: an analysis of yield, bacteriology, and inflammatory mediators.

The diminution of intraoperative hemorrhage remains a fundamental goal of the burn surgeon. We hypothesized that intraoperative blood salvage during burn excisions would be feasible if predicated on yield, bacteriology, and concentration of inflammatory mediators in the washed product. Reinfusion of culture-positive blood has a clear precedent in the trauma literature. Eight operations with immediate and complete collection of shed blood into a cell-saver device were prospectively studied. A median salvage rate of 43% of total shed red blood cells was estimated to have been recovered. Actual volumetric measurement of intraoperative blood loss was achieved. Bacterial contamination was consonant with the abdominal trauma experience. The levels of C3a, C5a, TNF alpha, and IL-1 beta in the final cell-saver product were all found to be at clinically insignificant levels.

Adolescent↗

PubMed1998

Platelet adhesion, contact phase coagulation activation, and C5a generation of polyethylene glycol acid-grafted high flux cellulosic membrane with varieties of grafting amounts.

Grafting of polyethylene glycol chains onto cellulosic membrane can be expected to reduce the interaction between blood (plasma protein and cells) and the membrane surface. Alkylether carboxylic acid (PEG acid) grafted high flux cellulosic membranes for hemodialysis, in which the polyethylene glycol chain bears an alkyl group at one side and a carboxyl group at the other side, have been developed and evaluated. PEG acid-grafted high flux cellulosic membranes with various grafting amounts have been compared with respect to platelet adhesion, the contact phase of blood coagulation, and complement activation in vitro. A new method of quantitating platelet adhesion on hollow-fiber membrane surfaces has been developed, which is based on the determination of lactate dehydrogenase (LDH) activity after lysis of the adhered platelets. PEG acid-grafted high flux cellulosic membranes showed reduced platelet adhesion and complement activation effects in grafting amounts of 200 ppm or higher without detecting adverse effects up to grafting amounts of 850 ppm. The platelet adhesion of a PEG acid-grafted cellulosic membrane depends on both the flux and grafting amounts of the membrane. It is concluded that the grafting of PEG acid onto a cellulosic membrane improves its biocompatibility as evaluated in terms of platelet adhesion, complement activation, and thrombogenicity.

Animals↗

PubMed1998

Anti-C5a monoclonal antibody reduces cardiopulmonary bypass and cardioplegia-induced coronary endothelial dysfunction.

OBJECTIVE: Because C5a induces tissue injury by activating polymorphonuclear leukocytes, the hypothesis was that inhibition of C5a activity would reduce cardioplegia-related injury. METHODS: Pigs were placed on cardiopulmonary bypass. The hearts were arrested for 1 hour with hyperkalemic cardioplegia. Pigs were then separated from bypass, and the hearts were reperfused for 2 hours. Anti-porcine C5a monoclonal antibody (1.6 mg/kg, intravenously; n = 6) was administered 20 minutes before the onset of cardiopulmonary bypass. Six pigs received saline solution vehicle. Reactivity of coronary arterioles was studied in vitro with videomicroscopy. Microvessels from uninstrumented pigs served as controls for vascular studies. RESULTS: Endothelium-dependent relaxation to adenosine diphosphate (percent relaxation of precontraction) was reduced after cardioplegic reperfusion (63% +/- 14% vs 77% +/- 10% in control at 10 micromol/L; P =.01). This impairment in endothelium-dependent relaxation was improved with anti-porcine C5a monoclonal antibody (80% +/- 22%; P =.01 vs saline solution), as was the impaired endothelium-dependent relaxation to clonidine (64% +/- 12% control; 26% +/- 17% saline solution; 55% +/- 24% anti-porcine C5a monoclonal antibody at 10 micromol/L; P =.01 saline solution vs control or anti-porcine C5a monoclonal antibody). Myeloperoxidase activity was significantly decreased (0.2 +/- 0.2 units/g protein; P =.04) in the anti-porcine C5a monoclonal antibody group compared with 5.2 +/- 2.7 in the saline solution group. CH50 2 hours after bypass was not statistically different (0.57 +/- 0.41 unit and 0.65 +/- 0.41 unit, respectively) between the anti-porcine C5a monoclonal antibody and saline solution groups. Despite less myocardial polymorphonuclear leukocyte infiltration after C5a inhibition, maximum rate of rise of left ventricular pressure, percent segmental shortening, and blood flow through the left anterior descending coronary artery were similar in the anti-porcine C5a monoclonal antibody and saline solution groups. CONCLUSIONS: Inhibition of C5a limits neutrophil-mediated impairment of endothelium-dependent relaxation after cardiopulmonary bypass and cardioplegic reperfusion, but it has no effect on short-term myocardial functional preservation.

Animals↗

PubMed2003

Regulation by C5a of neutrophil activation during sepsis.

In sepsis, there is evidence that excessive C5a generation leads to compromised innate immune functions, being associated with poor outcome. We now report that in vitro exposure of neutrophils to C5a causes increased levels of IkappaBalpha, decreased NF-kappaB-dependent gene transcription of TNFalpha, and decreased lipopolysaccharide (LPS)-induced TNFalpha production. Similar findings were obtained with neutrophils from cecal ligation/puncture (CLP)-induced septic rats. Such changes were reversed by antibody-induced in vivo blockade of C5a. In contrast, in vitro exposure of alveolar macrophages to C5a and LPS resulted in enhanced production of TNFalpha and no increase in IkappaBalpha. These data suggest that CLP-induced sepsis causes a C5a-dependent dysfunction of neutrophils, which is characterized by altered signaling associated with NF-kappaB activation.

Animals↗

PubMed1986

Kinetics of the generation and action of chemical mediators in zymosan-induced inflammation of the rabbit peritoneal cavity.

Acute inflammation was induced by intraperitoneal injection of zymosan (yeast cell walls) in the rabbit. Peritoneal inflammation was monitored by the local accumulation of intravenously-injected Evans blue dye (which binds to plasma albumin) and of polymorphonuclear leukocytes (PMNLs). The zymosan-induced exudate fluid contained a microvascular permeability-increasing factor or factors which, unlike histamine and bradykinin, had a long duration of action when tested in rabbit skin and was dependent on circulating PMNLs. Using radioimmunoassay, high levels of rabbit C5a, or C5a des Arg, were detected in the exudate fluid and accounted for much of the permeability-increasing activity, as judged by skin bioassay after separation on Sephadex G-100. The vasodilator prostaglandin, prostaglandin I2 (PGI2), was generated in the inflammatory reaction, as judged by the presence of high levels of 6-oxo-PGF1 alpha detected in the exudate by radioimmunoassay. However, in contrast to observations in rabbit skin, inhibition of prostaglandin generation had a relatively small effect on peritoneal oedema formation. C5a and C5a des Arg increase microvascular permeability by a PMNL-dependent mechanism in the rabbit. However, in response to zymosan, protein leakage was detected considerably earlier than PMNL accumulation. A hypothesis to account for this difference is proposed.

Animals↗

PubMed1988

Synthesis of porcine C5a anaphylatoxin by the solution procedure and confirmation of the reported structure.

Porcine C5a anaphylatoxin, the primary structure of which was first determined by Gerard and Hugli in 1980 as a 74-amino acid peptide having three intramolecular disulfide bonds, was synthesized by the solution procedure applying our maximum protection strategy. The fully deprotected peptide was subjected to air oxidation in an acetate buffer at pH 7.5, and the product was isolated as a single entity by HPLC. Amino acid analysis and biological activities of the synthetic peptide agreed well with the reported values. However, the retention time of the synthetic C5a was different from that of the natural product, supplied by Dr. Hugli, on both reversed phase (RP) and ion-exchange (IEX) HPLC systems. The tryptic peptide mapping on HPLC revealed that Gln which was incorporated into the peptide at position 65 was replaced by Glu in the natural product. The elution pattern of tryptic peptides containing three disulfide bonds was identical with natural and synthetic C5a. It was also identical with that of a peptide which was synthesized following the estimated secondary structure proposed by Zimmermann and Vogt in 1984.

Amino Acid Sequence↗

PubMed1995

C5a- and tumor necrosis factor-alpha-induced leukocytosis occurs independently of beta 2 integrins and L-selectin: differential effects on neutrophil adhesion molecule expression in vivo.

We previously demonstrated in rabbits that various neutrophil chemotactic factors share an ability to induce recruitment of polymorphonuclear leukocytes (PMN) from bone marrow when administered intravenously (Jagels and Hugli, J Immunol 148:1119, 1992). In the study reported here, we investigated the effects of chemotactic factors on the expression of beta 2 integrins and L-selectin in vivo and the roles of these adhesionmolecules in the recruitment process. Leukocytosis was induced by infusion of either C5a (5 micrograms/kg), N-formyl-methionyl-leucyl-phenylalanine (f-MLP; 2.5 micrograms/kg), or tumor necrosis factor alpha (TNF-alpha; 100 ng/kg). C5a increased the expression of CD18 (the common subunit of beta 2 integrins) on PMN by nearly twofold and decreased levels of L-selectin by 50% within 15 minutes after administration. Levels of beta 2 integrins returned to baseline 2 to 3 hours after induction of leukocytosis. L-selectin remained depressed for more than 5 hours, demonstrating that shedding was induced in the recruited bone marrow leukocytes as well as in circulating PMN. In contrast to the response to C5a, TNF-alpha did not cause upregulation of CD18 or shedding of L-selectin. Levels of L-selectin were consistently increased 60 minutes after administration of TNF-alpha, coinciding with a rapid rise in the number of band-form PMN in the circulation. Intact IgG and F(ab)2 forms of the anti-CD18 monoclonal antibody IB4 or the anti-L-selectin antibody DREG-200 were administered intravenously 15 minutes before induction of leukocytosis by the chemotactic factors. Neither IB4 nor its F(ab)2 fragments blocked leukocytosis induced by C5a, f-MLP, or TNF-alpha. DREG-200 also did not block leukocytosis induced by f-MLP, C5a, or TNF-alpha. These results suggest that leukocyte emigration from the bone marrow into the circulation proceeds through interactions distinct from those involved in neutrophil chemotaxis and diapedesis. Shedding of L-selectin from C5a-recruited bone marrow leukocytes demonstrates activation of these cells in the recruitment process and may reflect a potential mechanism for their release. The dissimilar effects of C5a and TNF-alpha on expression of adhesion molecules may result from distinct stimulatory pathways and suggests differential activation states for cellular recruitment by these inflammatory factors.

Animals↗

PubMed1990

Responses of isolated pulmonary arteries to the C5a anaphylatoxin.

The anaphylatoxin C5a possesses spasmogenic activity in smooth muscle tissues. In this study, we examined the effect of C5a on isolated rabbit pulmonary artery (PA) ring segments under a variety of experimental conditions. C5a (1-100 nM) caused transient constriction of PA at resting tension. C5a-induced PA constriction was not affected by the histamine H1-receptor antagonist pyrilamine (1 microM) but was significantly decreased by the cyclooxygenase inhibitor indomethacin (1 microM). Disruption of the endothelial layer of the vessel had no effect on C5a activity in resting PA. PA, which had been preconstricted with norepinephrine (5 microM), exhibited a different response to C5a than PA at resting tension, however. C5a (1-10 nM) induced a biphasic response in preconstricted PA, initially causing further constriction followed by a greater degree of relaxation resulting in an overall decrease in PA tension. All responses of preconstricted PA to C5a were completely eliminated by indomethacin and significantly depressed by endothelial disruption. These data indicate that C5a interacts directly with isolated rabbit PA, but the nature of the PA response to this peptide depends on several variables including the presence or absence of active PA tension and cyclooxygenase metabolites, and the presence of intact endothelium.

Anaphylatoxins↗

PubMed1988

Identification of the C5a des Arg cochemotaxin. Homology with vitamin D-binding protein (group-specific component globulin).

The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. The cochemotaxin attaches to sialic acid residues within the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the nature of the cochemotaxin and found that vitamin D-binding protein is the putative cochemotaxin. Vitamin D-binding protein enhanced the chemotactic activity of native C5a des Arg, but had no effect on the chemotactic activity of either native C5a or FMLP. Sialic acid prevented both enhancement by vitamin D-binding protein of the chemotactic activity of native C5a des Arg and formation of C5a des Arg-vitamin D-binding protein complexes, detected by molecular sieve chromatography. Furthermore, vitamin D-binding protein and cochemotaxin exhibited identical molecular weights, isoelectric points, antigenic reactivity, and amino acid composition.

Amino Acids↗

PubMed1991

CD45 is not involved in the processing of spatial information required for chemotaxis.

To examine the possible role of CD45 (a cell surface tyrosine phosphatase) in processing spatial information leading to the orientation of neutrophils in a gradient of chemoattractant, we used antibodies to cap CD45. If the phosphatase activity of CD45 was processing spatial information coming from occupied receptors and leading to the directional cell response, sequestering CD45 in one region of the cell surface should lead to a decreased ability of the cells to orient in a gradient of chemoattractant. CD45 was capped by sequential addition of KC56, a mAb against CD45, and a rabbit anti-mouse antiserum. Cells with capped CD45 were able to orient toward N-formylnorleucylleucylphenylalanine and C5a at levels similar to those achieved by cells processed simultaneously but without capping CD45. This indicates that the spatial arrangement of CD45 is not critical for the ability of cells to orient in a gradient of chemoattractant.

Antigens, CD↗

PubMed1984

Structure and function of the human C3b receptor.

The human C3b receptor (C3bR) is a glycoprotein that exists in two allotypic forms having Mr values of approximately 250,000 (F) and 260,000 (S). The number of receptors present on erythrocytes varies by eightfold among normal individuals and is genetically regulated by two codominant alleles that are distinct from the alleles determining the structural polymorphism. C5a and C5ades Arg induce rapid increases in the number of receptors expressed by neutrophils in vitro, and probably account for the increased receptor expression on neutrophils in patients undergoing hemodialysis. Cytoskeletal association of the C3bR on monocytes and neutrophils is suggested by experiments demonstrating receptor-mediated phagocytosis and adsorptive endocytosis through coated pits, and by the reciprocal coredistribution of cross-linked C3b and Fc receptors and the detergent insolubility of cross-linked C3bR. The factor H-like cofactor activity of the C3bR promotes the cleavage of bound C3b to iC3b, C3c, and C3d,g, which may enhance the clearance of circulating immune complexes and the generation of ligands for CR2 and CR3. The inherited partial deficiency of the erythrocyte C3bR in patients with systemic lupus erythematosus and the absence of glomerular C3bR in these patients with proliferative glomerulonephritis may contribute to systemic and organ-specific abnormalities in the clearance of immune complexes in this disease.

Complement C4↗

PubMed1995

CD11/CD18-independent transendothelial migration of human polymorphonuclear leukocytes and monocytes: involvement of distinct and unique mechanisms.

Monocytes and polymorphonuclear leukocytes (PMNLs) migrate across cytokine (interleukin-1, tumor necrosis factor) activated endothelium or unstimulated endothelium in response to chemotactic factors in vitro and in vivo utilizing the CD11/CD18 (i.e., beta 2 integrin) adhesion molecule complex. However, in vivo studies have suggested that under some conditions and/or in certain tissues, leukocyte migration can also proceed via CD11/CD18-independent mechanisms. Here we compared adhesion mechanisms involved in the migration of 51Cr-labeled blood monocytes and PMNLs across human umbilical vein endothelium (HUVE) monolayers. We observed that monocyte transendothelial migration was not inhibited by monoclonal antibody (mAb) to CD18, when the HUVE was activated with IL-1 and the chemotactic factor C5a induced the migration. This CD18-independent monocyte migration was blocked by treatment of the monocyte with mAb to beta 1 or alpha 4 integrin, suggesting that very late activation antigen 4 (VLA-4) on the monocyte served as the alternative migration mechanism. In contrast to monocytes, mAb to CD18 inhibited PMNL migration to C5a across IL-1-activated HUVE, but only by 66%, significantly less than with C5a alone (84%) or IL-1-activated HUVE alone (95%). The migration of anti-CD18 mAb-treated PMNLs was not inhibited by function-blocking mAbs to sialyl Lewisx, L-selectin, beta 1 or alpha 4 integrin, the beta 3-related leukocyte response integrin, IL-8, or platelet-activating factor (PAF) antagonists, alone or in combination. Antibody-blocking studies of the ligands on HUVE indicated that E-selectin may be partially involved in this CD18-independent PMNL migration but that ICAM-1, VCAM-1, PECAM-1, and P-selectin are not involved. Of several chemotactic factors tested, C5a and C5adesArg in activated plasma were the most active in inducing CD18-independent migration of PMNLs across IL-1-activated HUVE. These results demonstrate that (1) monocytes can utilize VLA-4 for optimal transendothelial migration and (2) PMNLs may have a novel CD18-independent migration mechanism that is activated by C5a in conjunction with one or more ligands on cytokine-activated endothelium. This may involve, in part, E-selectin interacting with a yet to be identified counterreceptor on PMNLs.

Antibodies, Monoclonal↗