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PubMed1992

C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)trisphosphate formation, but not phosphatidylinositol (4,5) bisphosphate hydrolysis and superoxide production, in human neutrophils.

We investigated phospholipid signal transduction, calcium flux, O2- anion production and actin polymerization after stimulation with the C fragment and chemoattractant, C5a, and then determined how C5a pretreatment affected subsequent responses to formyl peptide in human neutrophils. We have previously demonstrated that the novel lipids, phosphatidylinositol trisphosphate (PIP3) and phosphatidylinositol(3,4)P2 (PI(3,4)P2), rise transiently in neutrophils after activation with formyl peptide. Furthermore, the rise in PIP3 parallels actin polymerization. In this study, neutrophils activated with C5a exhibited two distinct G protein-dependent signal pathways involving different phosphoinositides: 1) [32P]PI(4,5)P2 hydrolysis and [32P]PA production, and 2) the transient formation of D-3-phosphorylated phosphoinositides, [32P]PIP3 and [32P]PI(3,4)P2. When neutrophils were preincubated with C5a for 5 min before stimulation with formyl peptide, [32P]PI(4,5)P2 hydrolysis was unchanged, and [32P]PA production and O2- formation were slightly enhanced compared with controls stimulated with formyl peptide in the absence of C5a. In contrast, [32P]PIP3 production, right angle light scatter, and actin polymerization were all reduced 35 to 40%. Therefore, these data support the hypothesis that PIP3 plays a role in chemotaxis but not superoxide formation.

Actins↗
PubMed1995

C3a is a chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation.

Inflammatory action of the potent chemotaxin C5a has been well characterized on a variety of human cell types, including neutrophils, monocytes, basophils, and eosinophils. The cellular effects of C3a are less well defined. Contradictory reports have been published for C3a activation of neutrophils. Recent reports that C3a activates both basophils and eosinophils prompted us to reinvestigate the effects of C3a stimulation on eosinophils. We hypothesized that C3a activation of eosinophils, cells that are present in most neutrophil preparations, might lead to neutrophil activation. Using neutrophils of 98% purity, we observed no evidence of cellular activation after stimulation with either C3a, recombinant human C3a (rhC3a), or the synthetic C3a analogue C3a 57-77, Y57. Eosinophils purified to > 98% purity displayed concentration-dependent polarization, chemotaxis, and enzyme release by stimulation with C3a, rhC3a, and the synthetic C3a analogue. An inactive form of C3a, C3adesArg, failed to stimulate either eosinophils or neutrophils. Using neutrophil preparations containing 5-9% eosinophils, up to 20% of neutrophils became polarized after exposure to C3a. Likewise, we demonstrated that supernatant from C3a-stimulated eosinophils promotes neutrophil chemotaxis. Eosinophil polarization experiments were repeated in the presence of antibody to the C5a receptor (C5aR) to show that C3a and C5a interact with different receptors. C3a activates eosinophils in the presence of anti-C5aR antibody at concentrations that fully block C5a activation. We conclude that eosinophils are directly activated by either C3a or C5a, whereas C3a failed to activate neutrophils. C3a acts on eosinophils via a receptor that is distinct from C5aR. Since neutrophils are indirectly stimulated by C3a, eosinophils contaminating neutrophil preparations may explain earlier reports that C3a activates human neutrophils.

Amino Acid Sequence↗
PubMed1990

Anaphylatoxin and terminal complement complexes in red cell salvage.

Thirteen patients undergoing elective orthopedic surgery were studied regarding anaphylatoxin (C3a and C5a) and terminal complement complex (TCC) formation in association with red cell salvage. The auto-transfusion equipment gave a centrifuged and washed erythrocyte fraction. The concentrations of C5a and TCC were not increased but elevated C3a levels were found in the suspension. After infusion of the erythrocyte fraction to the patient, no signs of systemic complement activation were observed. Thus, plasma levels of C3a, C5a and TCC were within the normal range in all the patients before and after autotransfusion. This study indicates that the complement system is activated in the cellsaver equipment. The washing procedure, however, seems to eliminate most of the anaphylatoxins and terminal complement complexes. No extensive systemic activation of complement seems to occur in association with autotransfusion to patients undergoing elective surgery.

Aged↗
PubMed2000

Demonstration that platelet-activating factor is capable of activating mast cells and inducing a chemotactic response.

Platelet-activating factor (PAF) is generated in a variety of inflammatory conditions in which mast cells accumulate. However, little is known about the ability of PAF to influence mast cell function directly. In this study we examine the ability of PAF to activate mast cells and regulate mast cell chemotaxis. PAF was found to induce intracellular calcium mobilization and chemotactic responses in both murine and human mast cells. PAF induced transient increases in intracellular Ca2+ concentrations with a 50% effective dose of 1 nM and induced significant migratory responses at PAF concentrations of 1 nM to 1 microM in the human leukaemia mast cell line (HMC-1). Using signal transduction inhibitors, both PAF-induced calcium mobilization and migration of mast cells were shown to require activation of pertussis toxin-sensitive G proteins. PAF-induced calcium and chemotactic responses were cross-desensitized by C5a. Together, these data demonstrate that PAF is capable of activating distinct signalling pathways in mast cells associated with calcium mobilization and cell migration; and that PAF may thus contribute to the regulation of mast cell responses and hyperplasia at sites of inflammation.

Animals↗
PubMed1992

Neutrophil chemotactic factors promote leukocytosis. A common mechanism for cellular recruitment from bone marrow.

We investigated cellular responses in a rabbit to i.v. administration of five established chemotactic factors (leukotriene B4 (LTB4), platelet-activating factor (PAF), C5a, N-Formyl-Met-Leu-Phe (F-MLF), and IL-8), and each exerted a characteristic effect on circulating white blood cell levels. All five factors induced a rapid and transient leukopenia. The blood was nearly devoid of circulating neutrophils 5 min after administration of each chemotactic factor. Other leukocytes were also variably depleted during the leukopenic phase, including eosinophils, basophils, monocytes, and lymphocytes. The lymphocyte numbers remained significantly depressed (approximately 30%) for as long as 3 h after administration of PAF or f-MLF. Each chemotactic factor produced a marked neutrophilia (i.e., 250-400% of baseline levels) after the initial leukopenia. Eosinophil numbers were elevated along with the neutrophil response in the C5a- and LTB4-treated animals. Basophil levels were significantly elevated only in LTB4-treated animals. The cellular response to PAF, f-MLF, and IL-8 appeared to be specific for the neutrophils. The kinetic profiles of the neutrophilia induced by PAF (10 micrograms/kg) or f-MLF (2.5 micrograms/kg) were similar, with maximal responses occurring 3 to 4 h after administration. In contrast, LTB4 (10 micrograms/kg), IL-8 (2.5 micrograms/kg), and C5a (5 micrograms/kg) induced a more rapid neutrophilia, with peak responses occurring 1 to 1.5 h after injection, and remaining elevated for 3 to 4 h. In all animals the neutrophilia was accompanied by a relative increase in the number of nonsegmented neutrophils (bands), suggesting that a major component of leukocytosis is caused by the release of bone marrow reserves. Phenidone (10 mg/kg), a dual cyclooxygenase/5-lipoxygenase inhibitor, affected neither the neutropenia nor the neutrophilia induced by C5a, f-MLF, or PAF. The protein synthesis inhibitor actinomycin D also failed to suppress neutrophil responses induced by either C5a or PAF. These results suggest that leukocytosis is a common response induced by all neutrophil chemotactic factors. Leukocytosis appears to be a direct result of the dynamic adaptive response of neutrophils to chemotactic factor stimulation without involvement of a secondary mediator system.

Animals↗
PubMed1995

OK-432 induces production of neutrophil chemotactic factors in malignant pleural effusion.

We investigated the changes in cellular components and neutrophil chemotactic factors in pleural fluid from 19 lung cancer patients who received intrapleural injection of OK-432 to treat malignant pleurisy. Not only neutrophil chemotactic activity (NCA) but also neutrophil count and percentage were increased significantly at 6 hours after OK-432 injection. The neutrophil count was significantly correlated with NCA level. The levels of C5a and IL-8 in pleural fluid were increased significantly after OK-432 injection. The increased IL-8 level was associated with a increase of both NCA and neutrophil count. OK-432 treatment also induced a marked increase of IL-1 beta and IL-6 in pleural fluid. Thus, intrapleural injection of OK-432 induced production of neutrophil chemotactic factors (IL-8 and C5a) and cytokines (IL-1 beta and IL-6), which eventually attracted neutrophils into the pleural space. These observations suggest that neutrophil migration mediated by these factors and cytokines may contribute to the sclerosing effects of OK-432 treatment.

Adult↗
PubMed1994

Dual pathways for GTP-dependent regulation of chemoattractant-activated K+ conductance in murine J774 monocytes.

Whole-cell patch clamp recording and digital imaging microscopy were used to investigate the electrical and calcium signaling responses of murine J774 monocytes to chemoattractants and other calcium-mobilizing agonists. A latent outwardly rectifying K+ conductance, GkOR, was elicited within seconds by each of the following agonists: C5a, ATP, ADP, interleukin-8, and the adenosine analog 5'-(N-ethylcarboxamido)-adenosine. In terms of its pharmacologic profile and current-voltage (I-V) relation, GkOR was very similar to a P2 purinoceptor-activated K+ conductance previously described in rat mast cells and to a K+ conductance elicited in J774 cells by the GTP analog guanosine 5'-O-(3-thiotriphosphate). Agonist-induced elevation of calcium, primarily due to intracellular release, and the induction of GkOR both required a GTP-binding protein of the Gi family, as both events were blocked by pertussis toxin; intracellular dialysis with guanosine 5'-O-(2-thiodiphosphate) also prevented the induction of GkOR, further implicating mediation by a G protein. Induction of GkOR did not depend upon influx of Ca2+, as it occurred equally well when the concentration of external Ca2+ was 100 nM or 2 mM. We attempted to uncouple agonist-induced calcium release from induction of GkOR by dialyzing the cell cytoplasm with Ca(2+)-EGTA or Ca(2+)-1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffers before agonist application. When the concentration of free Ca2+ ([Ca2+]i) was set to approximately 15 nM with 1.1 or 11 mM EGTA, a "slow"Ca2+ buffer, 10 nM C5a induced a large GkOR (11 nS at 1.1 mM EGTA versus 13.4 nS at 11 mM EGTA). Surprisingly, when [Ca2+]i was buffered at 15 nM with 10 mM BAPTA, a "rapid" Ca2+ buffer, C5a elicited a much smaller although significant K+ conductance (approximately 3 nS). Systematic increase in cytosolic [Ca2+]i upon dialysis with a series of 10 mM BAPTA-Ca2+ buffers (15-2400 nM [Ca2+]i) revealed activation of a very large K+ conductance (maximum 17.4 nS), even in the presumed absence of receptor stimulation. This conductance had a similar I-V relationship to GkOR, and activation occurred within the range of [Ca2+]i observed in intact cells following stimulation with C5a or ADP (EC50 approximately 475 nM [Ca2+]i). Activation of Gk thus may proceed in part via the release of intracellular calcium from a source in close proximity to the channel or other calcium-binding regulatory protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1983

Neutrophil activation as a mechanism of tissue injury.

C5A is capable of causing granulocyte/granulocyte interactions that lead to clumping, vasoocclusion, and the extension of infarctive damage in patients with coronary artery disease. Employing a new rheologic procedure, laser transillumination of thin vascular beds, one directly observes in vivo granulocyte aggregation in the mesentery of the rat. Nonsteroidal antiinflammatory drugs (NSAIDs) were shown to stop granulocyte agglomeration and to limit the extension of experimental myocardial infarcts in the cat. There may be application for NSAIDs for myocardial infarction in humans. NSAIDs block granulocyte aggregation to both a traditional activated complement and a complement activated by cryoglobulins. Patients with severe cryoglobulinemic cutaneous vasculitis who failed on steroids responded to NSAID administration.

Animals↗
PubMed1991

The sequential generation of neutrophil chemoattractant proteins in acute inflammation in the rabbit in vivo. Relationship between C5a and proteins with the characteristics of IL-8/neutrophil-activating protein 1.

An in vivo experimental peritonitis model was investigated in the rabbit using zymosan as the inflammatory stimulus. After an i.p. injection of zymosan, exudate was removed at intervals and tested in the back skin of assay rabbits. Assay rabbits received i.v. injections of 125I-albumin and 111In-neutrophils, and the local accumulation of each label was measured in response to intradermal injections of exudate samples mixed with a potentiating dose of PGE2. When peritoneal exudate samples were tested in the presence of a specific anti-C5a antibody, virtually all the edema-inducing and neutrophil chemoattractant activity was abolished in samples taken up to 2 h after the zymosan injection. Later samples, however, contained increasing levels of a non-C5a component. In C5a-depleted 6-h exudate two peaks of inflammatory activity were separated using cation exchange HPLC. Evidence is presented that C5a itself is unable to stimulate the production of these activities. Both peaks of activity appear related to IL-8/NAP-1 as they inhibited the binding of 125I-IL-8/NAP-1 to human neutrophils.

Acute-Phase Reaction↗
PubMed1997

Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils.

At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.

Actins↗
PubMed1996

Adhesion mechanisms involved in C5a-induced eosinophil homotypic aggregation.

During the process of migration into tissues, leukocytes interact primarily with vascular endothelial cells but they have also been shown to interact with each other. In this study we investigated the adhesion mechanisms involved in guinea pig eosinophil homotypic aggregation as assessed by changes in light transmission. The anti-CD18 monoclonal antibody (mAb) 6.5E, at concentrations in excess of those previously shown to abrogate CD18-dependent eosinophil adherence to serum-coated plastic, inhibited C5a-induced eosinophil aggregation to a maximum of 49-68%. In contrast, the anti-intercellular adhesion molecule-1 (ICAM-1) mAb RR1/1, which binds to guinea pig eosinophils and has been shown to block guinea pig ICAM-1 function, had no effect on C5a-induced responses. Similarly, two functionally active anti-very late antigen-4 (VLA-4) mAbs has no effect on eosinophil aggregation and did not affect the CD18-independent component of the aggregation response. The role of L-selectin in eosinophil aggregation was investigated by using heparin, the selectin-binding polysaccharide fucoidin, and the anti-L-selectin mAb MEL-14. Heparin concentration dependently inhibited C5a- and platelet- activating factor- (PAF) induced aggregation but C5a-induced responses were inhibited more potently. Fucoidin, but not the carbohydrate dermatan sulphate, effectively inhibited C5a-induced eosinophil aggregation. PMA- and PAF- induced responses were also inhibited by fucoidin. Moreover, fucoidin and 6.5E were additive in their ability to inhibit C5a-induced aggregation. Similarly, MEL-14 effectively inhibited C5a-induced eosinophil aggregation. In conclusion, we have demonstrated that guinea pig eosinophil homotypic aggregation is mostly dependent on CD11/CD18 and L-selectin present on the eosinophil surface. In addition, VLA-4 plays no role in mediating this aggregation response.

Animals↗
PubMed2004

Covalently immobilized thrombomodulin inhibits coagulation and complement activation of artificial surfaces in vitro.

Thrombomodulin (TM) serves as the endothelial cell receptor for thrombin and alters its characteristics from pro- to anticoagulant. Additionally, it promotes the formation of activated protein C. We evaluated the conservation of the overall outcome of these functions in recombinant TM linked to artificial surfaces by incubation with human whole blood in vitro. TM was covalently immobilized through poly(ethylene glycol) (PEG) spacers onto thin films of poly(octadecene alt maleic anhydride) covering planar glass substrates. TM binding to the polymer films was achieved after active ester formation at the carboxylic acid terminus of the PEG spacers and thoroughly characterized by HPLC-based amino acid analysis, immunofluorescence and ellipsometry. TM-coated samples were incubated for 3h with freshly drawn whole human blood anticoagulated with heparin (5IU/ml) using in-house developed incubation systems. The substantially reduced activation of blood coagulation (TAT) for TM-coated samples correlates well with the degree of contact activation (bradykinin and FXIIa formation) while no significant effects were observed for the platelet activation (PF4). Further, complement activation (C5a levels), was strongly diminished at the TM-containing surfaces. We conclude that the suggested method for preparation of TM immobilization may serve to prepare model substrates for studies on TM interactions but similarly provides a promising coating strategy for blood contacting medical devices.

Adsorption↗
PubMed1988

Human C5a induces a substantial histamine release in human basophils but not in tissue mast cells.

The histamine-releasing effect of human C5a was compared with that of concanavalin A in blood basophils and isolated adenoidal mast cells from the same donors. In basophils, C5a (10 ng/ml) induced a significant histamine release (7.5 +/- 2.1%, corrected value). In isolated adenoidal mast cells C5a had only a marginal effect (2% histamine release), although the cells responded markedly to concanavalin A stimulation (about 13% release). The results support the view that the heterogeneity of mast cells and basophils is also reflected in the expression of anaphylatoxin receptors on their surface.

Adenoids↗
PubMed1999

Regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury.

Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-alpha and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a.

Acute Disease↗
PubMed1998

Reduction of mononuclear cytokine production in hemodialysis patients treated with steam-sterilized low-flux polysulphone membranes.

An increased cytokine production, correlated with long term complications of uremic disease, has been described during hemodialysis. To identify possible differences in the cytokine release of differently sterilized membranes, we enrolled six uremic patients on chronic hemodialysis. The patients underwent dialysis with ETO-sterilized low-flux polysulphone membranes (F6, Fresenius AG) for at least three months (A1), they were then switched to steam-sterilized polysulphone membranes (F6-HPS Fresenius AG) and further evaluations after one (B1) and two months (B2) were carried out. A final evaluation (A2) was made one month after switching back to F6 dialyzers. At each time period, samples were drawn to measure IL-1beta released by cultured mononuclear cells (MN). Moreover, dialysate samples were collected to test endotoxin levels. C3a and C5a levels were assessed at 0, 5, 15 and 60 min from starting hemodialysis. Anti-ETO IgE levels were also assayed at A1, B1 and A2. The LAL test revealed a good quality dialysate. The mean pre-dialysis IL-1beta levels were 215 pg/million cells at A1; falling to 49 at B1, and 54 at B2 (p<0.01); there was then a sharp rebound at A2: 284, p<0.01. Post-dialysis levels followed the same pattern. No correlation between the dialysate endotoxin level and cytokine release was found. Complement activation did not change and in all the phases of the study no anti-ETO IgE was detected in any of the subjects. Our data suggest that the steam sterilized polysulphone membrane induces a lower cytokine release than the ETO sterilized membrane, although the mechanism by which it does so remains to be clarified.

Adult↗
PubMed1986

Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium.

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.

Animals↗
PubMed2005

Surface CD88 functionally distinguishes the MCTC from the MCT type of human lung mast cell.

BACKGROUND: MC(T) and MC(TC) types of human mast cells (MCs) are distinguished from one another on the basis of the protease compositions of their secretory granules, but their functional and developmental relationships have been uncertain. OBJECTIVE: These studies better define the functional properties and developmental relationship of MC(T) and MC(TC) cells. METHODS: Mast cells were dispersed from human skin and lung, purified with anti-Kit antibody, and separated into CD88+ and CD88- populations by cell sorting. These cells were evaluated by immunocytochemistry with antitryptase and antichymase mAbs; for chymase and tryptase mRNA by real-time RT-PCR; for conversion of MC(T) to MC(TC) cells during cell culture with recombinant human stem cell factor and recombinant human IL-6; and for degranulation and leukotriene C 4 (LTC 4 ) secretion when stimulated with anti-FcepsilonRI, substance P, C5a, and compound 48/80. RESULTS: Mature MC(T) and MC(TC) cells were separated from one another on the basis of selective expression of CD88, the C5aR, on MC(TC) cells. Lung MC(T) cells had negligible levels of chymase mRNA and retained their MC(T) phenotype in culture. Mature MC(TC) cells from skin and lung degranulated in response to FcepsilonRI cross-linking, C5a, compound 48/80, and substance P. Lung MC(TC) cells released LTC 4 on activation, but no LTC 4 was detected when skin-derived MC(TC) cells were activated. MC(T) cells from lung degranulated and released LTC 4 in response to anti-FcepsilonRI and substance P, but not to C5a and compound 48/80. CONCLUSION: These observations functionally distinguish MC(T) from MC(TC) types of human mast cells and suggest important differences that may affect their participation in diseases such as asthma and urticaria.

Cell Degranulation↗
PubMed1994

Neutrophil dysfunction in end-stage renal failure: reduced response to priming by C5a.

We studied the effect of C5a pretreatment on phosphatidyl-inositol-4,5-bisphosphate (PIP2) hydrolysis and on the increase in peak and resting cytosolic calcium levels induced by C5a (0.1 and 10 nM) and/or N-formyl hexapeptide (FLPEP; 10 nM) in neutrophils isolated from patients with end-stage renal failure (ESRF) and those from healthy controls. We also investigated superoxide anion production under the same conditions using the fluorescent para-hydroxyphenylacetic acid assay. The hydrolysis of PIP2 induced by C5a or FLPEP alone was similar in neutrophils from patients with ESRF and in control cells. Likewise, pretreatment of patients' neutrophils with C5a prior to FLPEP did not affect hydrolysis or the increase in cytosolic calcium concentration as shown previously for control neutrophils. Resting calcium levels in both ESRF and control neutrophils, however, were significantly increased after priming with low C5a concentrations. After priming with low C5a, prior to FLPEP, there was also a significant increase in superoxide production. This increase was significantly lower in cells from uremic patients than in those from healthy controls. Our data suggest that priming-induced superoxide production in neutrophils is reduced in patients with ESRF.

Amino Acid Sequence↗