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PubMed2002

L-selectin is required for fMLP- but not C5a-induced margination of neutrophils in pulmonary circulation.

To study the role of L-selectin in neutrophil (PMN) margination and sequestration in the pulmonary microcirculation, maximally active concentrations of C5a (900 pmol/g) and N-formylmethionyl-leucyl-phenylalanine (fMLP; 0.34 pmol/g) were injected into the jugular vein of wild-type or L-selectin-deficient C57BL/6 mice. In wild-type mice administered C5a or fMLP, 92 +/- 1% and 34 +/- 9%, respectively, of peripheral blood PMN were trapped mostly in the pulmonary circulation as determined by immunohistochemistry and myeloperoxidase activity. In wild-type mice treated with F(ab')(2) fragments of the L-selectin monoclonal antibody MEL-14 or in L-selectin-deficient mice, C5a-induced neutropenia was not significantly reduced, but the decrease in peripheral PMN in response to fMLP was completely abolished, indicating that L-selectin is necessary for fMLP- but not C5a-induced pulmonary margination. Immunostained lung sections of fMLP- or C5a-treated mice showed sequestered neutrophils in alveolar capillaries with no evidence of neutrophil aggregates. We conclude that chemoattractant-induced PMN margination in the pulmonary circulation can occur by two separate mechanisms, one of which requires L-selectin.

Animals↗

PubMed2001

Role of C5a in multiorgan failure during sepsis.

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O(2), rising partial pressure of CO(2)). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.

Acidosis↗

PubMed1999

Effect of permeability on indices of haemodialysis membrane biocompatibility.

BACKGROUND: Increases in plasma anaphylatoxins frequently are used as an index of haemodialysis membrane biocompatibility; however, their plasma levels may be influenced by the loss of anaphylatoxins into the dialysate compartment. METHODS: We compared the generation and compartmental distribution of anaphylatoxins, C3a and C5a, in a high flux and a low flux polysulfone membrane dialyser when whole human blood was recirculated through an in vitro haemodialysis circuit. RESULTS: Plasma C3a levels in high flux polysulfone (2.31 +/- 0.81 microg/ml) and low flux polysulfone (3.02 +/- 0.98 microg/ml) dialysers were comparable after 120 min (P = NS). In contrast, dialysate C3a in high flux polysulfone (0.65 +/- 0.31 microg/ml) accounted for 37.5 +/- 7.0% of the total detected (plasma + dialysate) C3a mass in the dialysers, while dialysate C3a in low flux polysulfone dialysers (0.01 +/- 0.01 microg/ml) accounted for only 0.3 +/- 0.3% of the total mass (P < 0.05; high flux vs low flux). Anaphylatoxin C5a was undetectable in the dialysate compartment of either dialyser examined. CONCLUSIONS: Our results indicate that anaphylatoxins readily traverse certain high flux dialysis membranes; consequently, plasma C3a levels may not accurately reflect the C3-activating potential of these membranes.

Biocompatible Materials↗

PubMed1985

Basophil function in patients with chronic myelogenous leukaemia.

We have compared the release of histamine from basophils isolated from six patients with chronic myelogenous leukaemia (CML) and seven normal controls. No differences were noted in the release induced by rabbit anti-human IgE. However, basophils from CML patients released less than 20% of their intracellular histamine when challenged with the anaphylatoxin C5a at a concentration that caused greater than 50% release from normal basophils. Furthermore, basophils from CML donors showed a significantly lower release in the presence of the calcium ionophore A23187. These results suggest that basophils from CML patients have an inherent defect.

Basophils↗

PubMed1991

Inhibition of neutrophil chemotaxis by elastase-generated IgG fragments.

IgG1 is cleaved in vitro by granulocyte elastase into Fc and Fab fragments. The elastase-specific Fc fragment has been previously detected in vivo. Biological activity of the fragments has been described in modulating neutrophil oxidative metabolism and enzyme release. To investigate further effects granulocyte chemotaxis (CT) was tested. The CT was assayed in Boyden chambers and the chemotactic index (CI) was calculated which represents the mean distance travelled by the activated cells. Stimulation of leucocyte CT by casein, activated serum and FMLP gives maximal values of delta CI = 46.7, 26.4 and 7.2 microns, respectively. Native IgG1 and the elastase-produced IgG fragments do not stimulate leucocyte CT. FMLP-stimulated CT is specifically inhibited by the elastase-produced Fc fragments. Addition of 7 nmol Fc to stimulus concentrations of 16 to 125 nM FMLP results in total inhibition of chemotaxis demonstrated by CI values which are lower than those for unstimulated cells. The inhibition of CT is concentration dependent in the range of 2 to 7 nmol Fc/10(6) PMN. Number and affinity of FMLP receptors are not influenced by Fc fragments, so Fc binds neither to FMLP nor the FMLP receptor. CT stimulated by casein shows a large portion of chemokinesis. Only at suboptimal casein concentrations do Fc and IgG have an inhibitory effect on CT (0.63 mg casein/ml, 10 nmol peptide/10(6) PMN). C5a-stimulated CT is not influenced by IgG or IgG fragments which indicates that the samples are not cytotoxic. So the FMLP and casein-stimulated CT is specifically inhibited by the elastase-produced Fc fragments in a low concentration range.

Binding Sites↗

PubMed1997

Role of resident mast cells and macrophages in the neutrophil migration induced by LTB4, fMLP and C5a des arg.

In the present study, we have investigated the participation of resident peritoneal cells (macrophages and mast cells) in the neutrophil migration induced in rats by the intraperitoneal administration of LTB4, fMLP or C5a des arg. The intraperitoneal injection of LTB4 (10 nmol), fMLP (10 nmol) and C5a des arg (zymosan-activated plasma, 1 ml) caused an intense neutrophil migration compared to the saline control (1,000, 1,500 and 2,000%, respectively). An 83% depletion in the number of resident cells following peritoneal lavage reduced the LTB4-stimulated neutrophil migration by 73.6% without affecting that caused by fMLP and C5a des arg. Increasing the peritoneal macrophage population (236%) by pretreating the cavities with thioglycollate enhanced the neutrophil migration induced by LTB4 (129%), but did not alter that induced by fMLP and C5a des arg. Similarly, reducing the population of peritoneal mast cells containing toluidine-blue-staining granules by subchronically pretreating the cavities with compound 48/80 diminished the LTB4-induced NM by 69% but had no effect on the responses to fMLP and C5a des arg. Pretreating the animals with dexamethasone strongly inhibited (70%) the neutrophil migration induced by the intraperitoneal injection of LTB4, fMLP and C5a des arg. Indomethacin, BW A4C and NDGA had no such effect. The incubating medium from peritoneal macrophages and mast cells stimulated with LTB4 induced neutrophil migration when injected into the peritoneal cavity of rats. This migration was strongly reduced (70%) by treating the cells with dexamethasone. In contrast, stimulating the cells with fMLP or C5a des arg did not result in the release of any promigratory activity into the incubating fluid. Our results suggest that LTB4 induces neutrophil migration via a mechanism dependent on resident mast cells and macrophages while that induced by C5a des arg and fMLP seems to be independent of such cellular involvement. The neutrophil migration induced by LTB4 is apparently mediated by factor(s) whose release is blocked by dexamethasone. fMLP and C5a appear to cause in vivo migration by the formation of a concentration gradient and by a glucocorticoid-sensitive mechanism different from that stimulated by LTB4.

Animals↗

PubMed2005

C5a-mediated leukotriene B4-amplified neutrophil chemotaxis is essential in tumor immunotherapy facilitated by anti-tumor monoclonal antibody and beta-glucan.

Intravenous and orally administered beta-glucans promote tumor regression and survival by priming granulocyte and macrophage C receptor 3 (CR3, iC3bR and CD11b/CD18) to trigger the cytotoxicity of tumor cells opsonized with iC3b via anti-tumor Abs. Despite evidence for priming of macrophage CR3 by oral beta-glucan in vivo, the current study in C57BL/6 and BALB/c mice showed that granulocytes were the essential killer cells in mAb- and oral beta-glucan-mediated tumor regression, because responses were absent in granulocyte-depleted mice. Among granulocytes, neutrophils were the major effector cells, because tumor regression did not occur when C5a-dependent chemotaxis was blocked with a C5aR antagonist, whereas tumor regression was normal in C3aR(-/-) mice. Neutrophil recruitment by C5a in vivo required amplification via leukotriene B(4), because both C5a-mediated leukocyte recruitment into the peritoneal cavity and tumor regression were suppressed in leukotriene B(4)R-deficient (BLT-1(-/-)) mice.

Administration, Oral↗

PubMed1986

C5a receptor modulation on neutrophils and monocytes from chronic hemodialysis and peritoneal dialysis patients.

Chronic renal failure patients have an increased risk for infection which may partially be due to altered chemotactic ability of their white blood cells. This study was designed to evaluate chemotactic factor and Fc receptor expression on neutrophils (PMN) and monocytes from chronic renal failure patients on hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Analysis of these receptors was performed using flow cytometry and fluorescent chemotactic factors (C5a, f-Met-Leu-Phe-Lys [fMLPL] and casein) and heat-aggregated human IgG. Peripheral blood PMN and monocytes obtained from 14 HD patients (in the predialysis period) and 14 CAPD patients were analyzed for their ability to bind each of the fluoresceinated ligands. PMN and monocytes from both patient groups had a significant reduction in their ability to bind C5a. The average percentage (+/- s.e.m.) of PMN that bound C5a was 93.9 +/- 1.1 for the controls, 72.9 +/- 3.8 for HD patients, and 79.3 +/- 4.0 for CAPD patients. Similar results were obtained with monocytes with 69.7 +/- 1.9% for controls, 54.6 +/- 4.5% for HD patients, and 31.0 +/- 4.5% for CAPD patients. These differences in C5a binding were also reflected in the average intensity of fluorescence. There was no significant difference in the percentage or fluorescence intensity of PMN or monocytes that bound casein or aggregated IgG when either group of dialysis patients was compared to the control values. Binding of fMLPL by PMN and monocytes from the HD patients and PMN from the CAPD patients were similar to control values but the binding of fMLPL by monocytes from CAPD patients was significantly suppressed (p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Complement C3↗

PubMed1986

Degranulation inhibition. A potential mechanism for control of neutrophil superoxide production in sepsis.

Previous studies with neutrophils from patients with intra-abdominal sepsis have provided convincing evidence of in vivo exposure to C5a. However, in contradistinction to normal cells pretreated with C5a, patient cells showed depressed superoxide response to N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) and enhanced FMLP receptor affinity. To identify possible mechanisms responsible for these findings, we examined the effects of lysosomal alkalinization with the weak base clindamycin on normal neutrophils with and without C5a. Our results showed a specific suppression of FMLP-induced superoxide production and a loss of low-affinity FMLP receptors. These results occurred in the presence of clindamycin levels that did not interfere with other cellular processes. These findings suggest that regulation of neutrophil function during the course of intra-abdominal sepsis may be due to effectors active both at the cell surface (C5a) and within the lysosome. The clinical significance of our findings relates to a possible mechanism for specific pharmacologic suppression of oxide-radical production by neutrophils. Such oxide radicals are believed to be important in the capillary injury accompanying severe sepsis.

Blood Proteins↗

PubMed1992

Signal transduction for interleukin-3-dependent leukotriene synthesis in normal human basophils: opposing role of tyrosine kinase and protein kinase.

The intracellular signaling pathways regulating the synthesis of leukotrienes by myeloid cells are largely unknown. In addition, the signal transduction mechanisms utilized by the cytokine receptor family are still poorly understood. The fact that in mature human basophils the synthesis of leukotriene C4 (LTC4) induced by C5a is strictly dependent on a short preincubation with the cytokine interleukin-3 (IL-3), allowed us to investigate the metabolic requirements for LTC4 synthesis, and also to provide some information on early signal transduction mechanisms of IL-3 in these differentiated, non-dividing blood leukocytes. IL-3 itself does not alter intracellular free calcium concentration ([Ca2+]i) in basophils, whereas C5a induces a transient rise independent of IL-3 pretreatment, indicating that the priming effect of IL-3 cannot be explained by alterations in [Ca2+]i changes. The protein kinase C inhibitor staurosporine did not inhibit C5a-induced histamine release nor IL-3-dependent LTC4 formation in contrast to the IgE receptor-dependent basophil response. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) induced histamine release without leukotriene formation. PMA-treated basophils did not produce LTC4 in response to C5a. Rather, PMA blocked the IL-3 effect on C5a-induced LTC4 synthesis. Only the C5a signal but not the IL-3 effect was pertussis toxin sensitive. Two unrelated tyrosine kinase inhibitors, tyrphostin RG-50864 and herbimycin A, were both very efficient blockers of IL-3-dependent lipid mediator formation whereas C5a-induced histamine release was preserved. Thus LTC4 formation does not require activation of a staurosporine-sensitive serine/threonine kinase. To the contrary, IL-3-dependent LTC4 formation appears to be regulated by serine/threonine and tyrosine phosphorylation in an antagonistic manner.

Alkaloids↗

PubMed2000

Metabolism and function of 3-D-phosphorylated phosphoinositides in C5a-stimulated eosinophils.

Eosinophils play a central role in the pathogenesis of parasitic infections, atopic diseases, and bullous dermatoses. To understand the regulative function of phosphatidylinositol 3-kinases in cell responses of eosinophils, phospholipid metabolism and production of reactive oxygen metabolites were followed after stimulation with C5a. Measurements of phosphatidylinositol lipids and analysis of deacylated products of separated lipid extracts showed fast and transient formation of phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). Cell studies in the presence of the tyrosine kinase blocker genistein indicated that C5a-stimulated PIP(3) formation occurred independently of tyrosine kinase activity. To analyze the function of PI4,5P(2)-3-kinase in eosinophils, the influence of wortmannin and LY294002 on production of reactive oxygen metabolites was studied. Both compounds inhibited with similar concentration dependency C5a-induced formation of PIP(3) and production of reactive oxygen metabolites. In summary, these data showed for the first time the involvement of PI4,5P(2)-3-kinase in the production of reactive oxygen metabolites in eosinophils.

Androstadienes↗

PubMed2006

Parkinson disease IgG and C5a-induced synergistic dopaminergic neurotoxicity: role of microglia.

Increasing evidence suggests the involvement of immune/inflammatory system in Parkinson's disease (PD). Many immune/inflammatory factors may synergistically participate in PD. In this study, we demonstrated that immunoglobulin G from the serum of 4/11 PD patients (PDIgG, 60microg/ml) and recombinant human C5a (0.1nM) synergistically induced selective dopaminergic neurodegeneration in rat mesencephalic neuron-glia cultures, while that PDIgG alone or C5a alone was minimally toxic or nontoxic. IgG from 17 disease controls and from 7 normal controls did not significantly induce dopaminergic neurotoxicity in the cultures even in the presence of C5a. Using mesencephalic neuron-enriched cultures, we found that the synergistic dopaminergic neurotoxicity was mediated by glia. The results from microglia-supplemented neuronal cultures, astroglia-supplemented neuronal cultures and neuron-astroglia cocultures indicated that microglia, not astroglia, played a pivotal role in the neurotoxicity. Through immunocytochemistry analysis and assay of proinflammatory factors, we observed that each of the four PDIgGs (60microg/ml) and C5a (0.1nM) synergistically induced microglia activation and production of superoxide and nitric oxide (NO) in neuron-glia cultures. Further investigations indicated that superoxide and NO were both responsible for the synergistic neurotoxicity. Finally, using F(ab')(2) fragments of PDIgG, we demonstrated that microglial Fc receptors may play an important role in the neurotoxicity. Our work provides new evidence for the involvement of the immune/inflammatory system in PD and helpful clues for studying the combined effect of antibody and complement on microglia.

Animals↗

PubMed2002

The unique property of the CC chemokine regakine-1 to synergize with other plasma-derived inflammatory mediators in neutrophil chemotaxis does not reside in its NH2-terminal structure.

The recently discovered CC chemokine, regakine-1, is constitutively present in bovine serum and synergizes with the CXC chemokine interleukin-8 (IL-8) to chemoattract neutrophils. Here we show that regakine-1 cooperates with the CXC chemokine receptor 2 ligand neutrophil activating protein-2 (NAP-2) and the anaphylatoxin C5a, two other mediators of inflammation present in the circulation. Neutrophil chemotaxis was 3-fold enhanced when regakine-1 (100 ng/ml) and C5a (30 ng/ml) were combined at concentrations present in bovine or human plasma, respectively. This synergy was also observed when neutrophils were preincubated with regakine-1. Plasma chemokines such as NAP-2, beta-thromboglobulin, and hemofiltrate CC-chemokine-1 did not affect C5a chemotactic activity. The capability of regakine-1 to synergize with C5a, NAP-2, or N-formyl-methionyl-leucyl-phenylalanine (fMLP) was not observed for monocyte chemotactic protein-3 (MCP-3), another CC chemokine that weakly chemoattracts neutrophils. Regakine-1 also failed to cooperate with MCP-3 and macrophage inflammatory protein-1alpha in neutrophil chemotaxis. The receptor of regakine-1 is not known yet. Competition with labeled fMLP or C5a for binding to neutrophils or receptor transfected cell lines demonstrated that regakine-1 did not alter receptor recognition. The protein kinase inhibitors 2'-amino-3'-methoxyflavone (PD98059), wortmannin and staurosporin had no effect on the synergy between C5a and regakine-1. Although NH2-terminal truncation affects the chemotactic potency of most chemokines, it did not affect the synergistic capacity of regakine-1 with C5a on neutrophils. These findings indicate that the constitutive plasma chemokine regakine-1 is a stable enhancer of the inflammatory response and that its blockade might be beneficial in acute and systemic inflammatory disorders.

Chemokine CCL4↗

PubMed2000

Selective suppression of IL-12 production by chemoattractants.

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.

Cells, Cultured↗

PubMed1994

C3a activates the respiratory burst in human polymorphonuclear neutrophilic leukocytes via pertussis toxin-sensitive G-proteins.

In contrast to C5a, which represents a well-established potent activator of the respiratory burst in polymorphonuclear neutrophilic granulocytes (PMN), the functional role of C3a in the activation of PMN is, so far, poorly understood. Herein, the potential role of human C3a in the activation of the respiratory burst in human PMN was investigated. The release of reactive oxygen species (ROS) of PMN from healthy donors was measured by lucigenin-dependent chemiluminescence. C3a dose-dependently induced the production of ROS in human PMN in the range between 10 ng/mL and 1,000 ng/mL, whereas C3a-desArg was inactive. Flow cytometric measurement of H2O2 by dihydrorhodamine-123 labeling of anti-CD16-stained PMN showed that predominantly neutrophilic PMN are responsible for the C3a-induced activation of the respiratory burst. To exclude that C3a stimulation was caused by contamination with C5a, the specificity of C3a-induced activation of PMN was shown using monoclonal antibodies (MoAbs). Accordingly, the effect of C3a was completely abolished in the presence of Fab fragments of a blocking anti-C3a MoAb. In addition, blockade of the C5a receptor by the anti-C5a receptor (anti-C5aR) MoAb, S5/1, totally inhibited the C5a-induced production of ROS, whereas the C3a response in the presence of the anti-C5aR MoAb was unaffected. The specificity of the response was further confirmed by homologous desensitization after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. The C3a-induced ROS production by PMN was inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi proteins) in the signal transduction process initiated by C3a. In addition, stimulation of PMN by C3a resulted in a transient increase in the cytosolic free calcium concentration ([Ca2+]i) in a dose-dependent manner. In contrast to C3a-induced ROS production, C3a did not induce a chemotactic response in PMN, indicating functional qualitative differences as compared with C5a. In summary, these results show that C3a is a potent activator of the respiratory burst in human PMN. Therefore, these findings point to a novel role of C3a in the pathogenesis of inflammatory diseases associated with increased C3a levels and PMN activation.

Calcium↗

PubMed1993

IL-3 alters free arachidonic acid generation in C5a-stimulated human basophils.

IL-3 is known to enhance the secretion of several mediators from human basophils activated by receptor-mediated stimuli. IL-3 can cause a qualitative change in the mediator release pattern for C5a-mediated stimulation; without IL-3, C5a causes no leukotriene release whereas in the presence of IL-3, significant leukotriene occurs. This study examines the influence of a 15-min pretreatment of basophils with IL-3 (10 ng/ml) on several signal transduction events. Basophils stimulated with C5a typically displayed only a transient cytosolic Ca2+ response [Ca2+]i, attributed to the release of intracellular calcium stores. IL-3 had sporadic, statistically nonsignificant, effects on the peak of this initial response as well as inconsistent effects on the generation of a second phase in the [Ca2+]i response (i.e., that which is caused by the influx of extracellular Ca2+). IL-3 also had no effect on resting [Ca2+]i levels. Challenge of basophils in the presence of EGTA had little effect on the amount of leukotrienes generated. This maneuver did not influence the initial transient elevation of [Ca2+]i. Although basophil leukotriene release is usually slow, after exposure to IL-3 it was found that leukotriene release occurred rapidly, during the brief window of time (0 to 45 s) in which the [Ca2+]i transient occurred. These results suggested that the generation of AA was accelerated by pretreatment with IL-3. Subsequent mass measurements of free AA by gas chromatography-mass spectroscopy showed: 1) IL-3 itself caused no free AA generation; 2) without IL-3, C5a stimulated little or no free AA generation; and 3) in the presence of IL-3, C5a generated substantial levels of free AA at an accelerated rate. A similar acceleration in the rate of free AA generation, without any apparent increase in the amount, occurred in IL-3-primed basophils stimulated with FMLP. Additional studies showed that IL-3 had no effects on whole cell ATP levels and that the kinase inhibitor, staurosporine, did not inhibit the ability of IL-3 to cause enhanced responses to C5a. We conclude that the primary effect of a short period of pretreatment with IL-3 is to couple the generation of free AA to C5a-mediated stimulation in particular and to accelerate its generation after other stimuli. Enhancements of the [Ca2+]i response, while sporadic and at best modest, may have some influence on enhanced mediator release but did not clearly explain the functional effects of IL-3.

Arachidonic Acid↗

PubMed1992

Priming of human neutrophil functions by tumor necrosis factor: enhancement of superoxide anion generation, degranulation, and chemotaxis to chemoattractants C5a and F-Met-Leu-Phe.

Recombinant human tumor necrosis factor-alpha (rTNF) stimulated increased generation of superoxide anion (O2-) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2-2200 units/ml) for 10 min enhanced the subsequent generation of O2- in response to C5a and f-Met-Leu-Phe (FMLP). Recombinant TNF did not enhance O2- generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001-50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001-0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100-10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.

Chemotaxis, Leukocyte↗

PubMed2001

Protective effects of anti-C5a peptide antibodies in experimental sepsis.

We evaluated antibodies to different peptide regions of rat C5a in the sepsis model of cecal ligation and puncture (CLP) for their protective effects in rats. Rabbit polyclonal antibodies were developed to the following peptide regions of rat C5a: amino-terminal region (A), residues 1-16; middle region (M), residues 17-36; and the carboxyl-terminal region (C), residues 58-77. With rat neutrophils, the chemotactic activity of rat C5a was significantly inhibited by antibodies with the following rank order: anti-C > anti-M >> anti-A. In vivo, antibodies to the M and C (but not A) regions of C5a were protective in experimental sepsis, as determined by survival over a 10-day period, in a dose-dependent manner. The relative protective efficacies of anti-C5a preparations (in descending order of efficacy) were anti-C > anti-M >> anti-A. In CLP rats, a delay in infusion of antibodies, which were injected at 6 or 12 h after CLP, still resulted in significant improvement in survival rates. These in vivo and in vitro data suggest that there are optimal targets on C5a for blockade during sepsis and that delayed infusion of anti-C5a antibody until after onset of clinical evidence of sepsis still provides protective effects.

Animals↗