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PubMed1985

Escherichia coli and human neutrophils. Effect of bacterial supernatant with hemolysin activity upon chemotaxin receptors.

A greater proportion of Escherichia coli strains isolated from clinical extraintestinal infections produce alpha-hemolysin than do strains isolated from normal fecal flora. Proposed mechanisms to explain this observation have stressed the fact that hemolysis liberates hemoglobin, which may provide a nutritional boost for E coli growth. Alternatively, a cytolytic effect of hemolysin upon host neutrophils has been postulated. Our previous studies have suggested a third possibility: Human neutrophils incubated in the supernatant of an alpha-hemolysin-producing E coli strain produced a selective inhibition of chemotaxis toward C5a. Bacteria-free supernatants from 14 clinical strains of E coli were therefore evaluated for an ability to lyse sheep erythrocytes, alter human polymorphonuclear neutrophil (PMN) chemotaxin receptors, and affect release of PMN enzymes. Supernatants possessing hemolytic activity decreased the C5a receptor activity of human PMNs and increased the number of peptide receptors. A stimulation of secondary granule release, as evidenced by the release of PMN lysozyme, may account for the increased expression of peptide receptors. Perturbation of host defenses through a loss of neutrophil migratory function and secondary granule contents may allow for enhanced survival of E coli, which produce alpha-hemolysin.

Bacterial Proteins↗
PubMed1997

Studies on the role of plasminogen activator in systemic infection by virulent Yersinia pestis strain C092.

Plasminogen activator is an outer membrane protease of Yersinia pestis encoded by the pla gene on plasmid pPst. Pla of the KIM-10 strain of Y. pestis appears to be required for the virulence from a subcutaneous (sc) but not an intraperitoneal (ip) or intravenous (iv) route of infection in mice. However, other strains of Y. pestis are highly virulent by the sc route yet lack pPst and pla. In this study, the pPst- Pestoides F strain was lethal to mice inoculated sc, with an LD50 (3 cfu), equal to that of C092, a virulent pPst+ strain. To analyse further the role of Pla in invasive infection, isogenic derivatives of C092, including one harboring pla with a frameshift mutation and another cured of pPst, were made. Although the ip LD50 of pPst- C092 and of the pla mutant were nearly identical to that of the wild type, the subcutaneous LD50 of the cured and mutant strains were 4 to 6 logs greater than that of wild type. Thus, pPst appears to be required for development of a lethal infection by some strains after sc inoculation but not after direct ip inoculation. Pla-associated virulence did not appear to be mediated by interference with the phagocyte chemoattractant C5a, as shown by the lack of correlation of C5a production with susceptibility to Y. pestis in C5a+ and C5a- congenic mice. In a footpad model of the early host response to subcutaneous infection, pPst- C092 proliferated at the subcutaneous injection site to a similar extent as did the wild type parent strain, and elicited a similarly large, local inflammatory response. However, the wild type was present at higher concentrations at more distant sites such as the popliteal lymph node and spleen.

Animals↗
PubMed1985

Stimulus interactions in release of superoxide anion (O2-) from human neutrophils. Further evidence for multiple pathways of activation.

A wide variety of agents stimulate superoxide anion (O2-) release from human neutrophils. To determine whether the same or different cellular pathways are utilized, neutrophils were stimulated to release O2- with combinations of f-Met-Leu-Phe (FMLP) (10(-7)M), C5a (25 nM), Con A (100 micrograms/ml), arachidonic acid (100 microM), and PMA (1 microgram/ml). These concentrations produced maximal O2- production when used alone. A synergistic response was observed when Con A was used in combination with FMLP or C5a. This response was twice the expected release in cytochalasin B-treated cells and three to five times the expected release in untreated cells. Additional studies showed that synergism was dependent upon the simultaneous presence of both agents. Additive O2- responses were observed when either FMLP, Con A, or C5a was tested in combination with arachidonic acid and when FMLP and C5a were tested together. When PMA was tested with C5a, FMLP, or Con A, a nonadditive O2- response resulted, whereas mixtures of PMA and arachidonic acid resulted in a less than additive response. These contrasting results using different soluble stimuli in combination suggest that multiple pathways exist for the stimulation of neutrophil O2- release, with some stimuli being totally independent of each other (possibly activating separate pools of oxidase), while other stimuli show cooperative effects on oxidase activation.

Adult↗
PubMed1994

Effects of low-density lipoproteins on polymorphonuclear leukocyte functions in vitro.

In the circulation, human polymorphonuclear leukocytes (PMN) are exposed to various factors, such as lipoproteins, which could alter their metabolic and functional characteristics. In this work, the effects of low-density lipoproteins (LDL) on PMN oxidative metabolism and migration were studied in vitro. LDL stimulated PMN superoxide generation. This effect lasted for 15-20 min and was concentration-dependent. Staurosporine, a potent inhibitor of protein kinase C, did not suppress this stimulating effect. The chemotactic response of PMN to formyl-methionyl-leucyl-phenylalanine and C5a was inhibited by LDL and this effect was conserved after trypsination of LDL. LDL from normolipidemic subjects were more potent than LDL from hypertriglyceridemic subjects for both effects. LDL had no effect on superoxide generation by opsonized zymosan-stimulated PMN. These data showed that PMN responses may be modified by environmental conditions such as the presence of lipoproteins.

Alkaloids↗
PubMed1986

Production of C5a antagonist by synovial and peritoneal tissue fibroblasts.

Fibroblasts grown from synovial and peritoneal tissues release into the medium an inhibitor of neutrophil chemotaxis. The inhibitor resembles the antagonist previously described in synovial and peritoneal fluids. It is a heat stable (56 degrees C) protein of MW approximately 40 kDa that counteracts the chemotactic activity of zymosan-activated serum or purified C5a but not the peptide chemoattractant F-met-leu-phe. No chemotactic inhibitor was detected in media from skin fibroblast cultures or in formal human sera. It is suggested that the inhibitor is produced locally by synovial and peritoneal fibroblasts and that it might play a role in the regulation of inflammation at sites lined with mesothelium.

Chemotactic Factors↗
PubMed1987

Effect of blood cooling on cuprophan-induced anaphylatoxin generation.

We investigated whether cooling of the extracorporeal blood during hemodialysis could prevent anaphylatoxin generation and leukopenia caused by blood-cuprophan contact. After preliminary in vitro studies confirming the temperature dependence of C5a generation, we carried out hypothermic dialysis on nine patients by manipulating blood and dialysate temperature in such a way that blood temperature within the dialyzer averaged 25 degrees C. In comparison with the control procedure (blood temperature within the dialyzer 35 degrees C) hypothermic dialysis reduced peak C5a generation from 41.7 +/- 17 ng/ml to 9.7 +/- 3.4 ng/ml (P less than 0.01), and white blood cell fall from 72 +/- 15 to 25 +/- 20% (P less than 0.01). Arterial PO2 decreased less in hypothermic dialysis (-19 +/- 9% of pre-HD value) than in the control procedure (-30 +/- 11%) (P less than 0.05). We conclude that blood cooling attenuates cuprophan-induced anaphylatoxin generation and leukopenia.

Adult↗
PubMedDate not supplied

Comparison of histamine release from human blood monocytes, lymphocytes, adenoidal and skin mast cells.

Monocytes and lymphocytes from human blood contain 0.043 +/- 0.007 and 0.053 +/- 0.014 pg histamine/cell, respectively, which can be released by a number of stimulants (A 23187, C5a, substance P, specific allergen). The release process takes 60-120 min to reach its end point, in contrast to tissue mast cells which complete the release within 1-3 min. Both, ketotifen (10(-7) - 10(-5) M) and disodium cromoglycate (10(-5) - 10(-3) M) inhibited histamine release dose dependently up to 40-45%, which might be particularly relevant during the later stages of acute allergic or pseudoallergic reactions.

Adenoids↗
PubMed1988

Identification of a histamine release inhibitory factor produced by human mononuclear cells in vitro.

Human mononuclear cells (MNC) secrete histamine-releasing factor(s) (HRF) when cultured in vitro. HRF induces the release of histamine and other mediators from basophils and mast cells. We have shown that MNC also produced a histamine release inhibitory factor (HRIF), and that the synthesis is augmented by culture with physiologic concentrations of histamine (10(-10) to 10(-6) M) and by the mitogen concanavalin A (Con A). HRIF does not affect release initiated by other secretagogues such as allergen, anti-IgE, C5a, Con A, and phorbol myristate acetate. HRIF requires a preincubation with the cells for 5-10 min for maximal inhibition, and this effect is not abolished by washing the cells after the preincubation. The biological activity of HRIF is protease-sensitive, neuraminidase-resistant, and relatively heat-stable. HRIF can be distinguished from HRF by a lower apparent molecular mass (8,000-10,000 D).

Biomarkers, Tumor↗
PubMed2000

C5a-stimulated human neutrophils use a subset of beta2 integrins to support the adhesion-dependent phase of superoxide production.

Isolated human polymorphonuclear neutrophils (PMN) responded to human C5a with an immediate, transient release of superoxide lasting from 0.5 to 5 min. This was followed by a second release of superoxide, which began at 10 min after addition of C5a, was sustained for more than 30 min, and required ICAM-1 immobilized in the wells. F(ab')2 monoclonal antibody (mAb) preparations were used to dissect the role of individual beta2 integrins and to avoid the confounding effects of ligating Fc receptors. Anti-CD18 mAb treatment of the PMN had no effect on the immediate first phase but completely inhibited the second, adhesion-dependent phase of superoxide production. Anti-CR3 mAb only inhibited the adhesion phase of superoxide production partially, implying that other beta2 integrins were involved. A mixture of anti-CD11a, anti-CD11b, and anti-CD11c was not able to block superoxide production completely, suggesting a role for alphad/beta2. Surprisingly, blocking anti-LFA-1 mAb had no effect on superoxide production. Consistent with this observation, immobilized, purified ICAM-2, a specific counter-receptor for LFA-1, did not support the adhesion-dependent phase of-superoxide production. Thus, PMN treated with C5a used signals via CR3, P150/95, and alphad/beta2, but not LFA-1, to support superoxide production. LFA-1 has been shown by others to mediate most of the adhesion necessary for transendothelial migration in vivo. The inability of LFA-1 ligation to stimulate superoxide production may be an important means of preventing blood-vessel damage when PMN migrate across the endothelium.

Antibodies, Monoclonal↗
PubMed1994

Regulation of neutrophil activation by oleic acid.

Physiological concentrations of oleic acid inhibited C5a-induced myeloperoxidase release from neutrophils. The inhibition occurred promptly following the addition of oleic acid, was dose-dependent and saturable, and was greater at low concentrations of C5a. Kinetic analysis of the curve for 1/myeloperoxidase release against 1/[C5a] in the presence of oleic acid, was compatible with a cooperative pattern of interaction. The inhibitory effect persisted after repeated washings of cells preincubated with oleic acid. The effect of oleic acid was not specific for C5a-induced neutrophil activation; oleic acid also inhibited myeloperoxidase release induced by the Ca2+ ionophore A-23187, as well as zymosan activated serum-induced chemotaxis. In experiments designed to localize the site of action of oleic acid on neutrophil activation, an intracellular oleic acid-binding protein M(r) = 13,000) was isolated. The results suggest that acute changes in plasma fatty acid levels may have significant effects on neutrophil function under physiological conditions.

Complement C5a↗
PubMed1994

Disparate role of the beta 2-integrin CD18 in the local accumulation of neutrophils in pulmonary and cutaneous inflammation in the rabbit.

The leukocyte adhesion glycoprotein complex CD11/CD18 has been shown to be important in mediating neutrophil accumulation at sites of inflammation in many experimental models. The exception is the lung, where neutrophil accumulation into the airspaces can be CD18-dependent and -independent, according to the stimulus used to induce pulmonary inflammation. By using the anti-CD18 mAb 60.3, this study examined the role of CD18 on neutrophil accumulation in the lungs of rabbits induced by a local intrabronchial instillation of C5a or interleukin-1 alpha (IL-1 alpha) into the upper lung lobes. For comparison, cutaneous inflammation was induced in the same animals by intradermal injection of the same mediators. Pretreating rabbits with 60.3 abolished accumulation of 111In-labeled neutrophils in skin induced by both C5a and IL-1 alpha. In contrast, in the same animals, C5a-induced accumulation of neutrophils in the lung was not significantly affected by 60.3, while neutrophil accumulation in response to IL-1 alpha showed a significant, but not absolute, dependency on CD18. External gamma scintigraphy of 111In-labeled neutrophils demonstrated that the kinetics of cell retention in the lung was similar for both C5a and IL-1 alpha. In summary, accumulation of neutrophils to sites of inflammation in cutaneous inflammation shows an absolute dependency on CD18, while migration of these cells to sites of inflammation in the lung can be largely independent of this adhesion molecule. These data indicate that the mechanisms responsible for accumulation of neutrophils in cutaneous and pulmonary inflammation are different.

Animals↗
PubMed1992

Binding of fluorescein-labeled anaphylatoxin C5a to human peripheral blood, spleen, and bone marrow leukocytes.

The expression of C5a receptors (C5aR) on human leukocytes was evaluated by flow cytometry using fluorescein-labeled human C5a (C5a-F). Granulocytes and CD14+ mononuclear cells (MNL) but not CD3+, CD20+, CD16+, CD56+, or CD11b+ lymphocytes in peripheral blood and spleen bound C5a-F. C5a-F binding was saturable and inhibitable by anti-C5a monoclonal antibody (MoAb) C17/5 or unlabeled C5a. During hemodialysis, which led to the generation of C5a, only granulocytes and monocytes increased their expression of the adhesion molecule CD11b (CR3). In vitro, C5a induced an increase of CR3 and p 150/95 (CD11c/CR4) only on myeloid cells. However, treatment of leukocytes with phorbol 12-myristate 13 acetate increased CR3 and CR4 expression on both myeloid cells and a lymphocyte subpopulation. Stimulation of MNL in mixed lymphocyte cultures or by treatment with conditioned medium or with IFN-gamma did not induce binding sites for C5aR on lymphocytes and reduced the binding of C5a-F to monocytes. The expression of C5aR on low-density bone marrow cells was analyzed by setting appropriate gates during flow cytometry. Cells that bound C5a-F were found in all populations that contained granulocyte and monocyte precursors, but not in lymphocyte precursor populations. All C5aR+ bone marrow cells were CD34 and expressed high levels of CR3, which suggests a late appearance of C5aR during myeloid cell maturation. Our results indicate that C5aR is exclusively expressed on myeloid cells within the hematopoetic cell population.

Antibodies, Monoclonal↗
PubMed1997

Requirement for C-X-C chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) in IgG immune complex-induced lung injury.

The C-X-C chemokines of the IL-8 family possess potent chemotactic activity for neutrophils, but their in vivo role in inflammatory responses is not well understood. In the IgG immune complex-induced model of acute lung inflammatory injury in the rat we have evaluated the roles of two rat chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC). Both mRNA and protein for MIP-2 and CINC appeared in a time-dependent manner after initiation of IgG immune complex deposition in lung. There exists a 69% homology between the amino acid sequences for these proteins, and we found cross-reactivity between polyclonal Abs raised to these chemokines. By purifying the blocking Abs using double affinity methods (with Ag-immobilized beads), this cross-reactivity was removed. Individually, anti-MIP-2 and anti-CINC Ab significantly reduced lung injury (as measured by 125I-labeled albumin leakage from the pulmonary vasculature) and reduced neutrophil accumulation in the lung (as determined by myeloperoxidase (MPO content) and neutrophil counts in bronchoalveolar lavage (BAL) fluids); however, no change in TNF-alpha levels in BAL fluids was found. Chemotactic activity in BAL fluids collected 2 h after injury from animals undergoing immune complex deposition could be shown to be chiefly due to the combined contributions of MIP-2 (39%), CINC (28%), and C5a (21%). When either MIP-2 or CINC was blocked in vivo, up-regulation of Mac-1 expression on neutrophils obtained from BAL fluids was significantly reduced. These data suggest that, in the model studied, both MIP-2 and CINC contribute significantly to the influx of neutrophils and their activation.

Animals↗
PubMed1981

Induction of ocular inflammation by synthetic mediators.

Chemotactic mediators, N-formylmethionyl-leucyl-phenylalanine (FMLP) and the complement component C5a, were injected into the rabbit cornea, vitreous, and skin to induce a reaction resembling the "Arthus phenomenon." Injection of these mediators induced edema and granulocytic infiltration in the cornea, conjunctiva, and skin. These histologic changes resembled the inflammation produced by antigen (ovalbumin [OVA]) in specifically immunized rabbits. Keratitis began after two hours and subsided six hours after the injection. Conversely, the vitreous response started six hours after injection of FMLP and C5a and peaked between 24 and 48 hours. All the inflammatory reactions induced by FMLP, C5a, and rechallenge with antigen could be inhibited in varying degrees by subconjunctival injection of 0.1 mL of 10(-5)M dexamethasone, quinacrine, 5,8,11,14-eicosatetraynoic acid (ETYA), or indomethacin, agents that suppress different sites of chemotaxis of polymorphonuclear leukocytes. However, only the inflammation induced by FMLP could be inhibited by carbobenzoxy-phe-met, a competitive inhibitor of FMLP.

Animals↗
PubMed1990

Agonist-sensitive binding of a photoreactive GTP analog to a G-protein alpha-subunit in membranes of HL-60 cells.

Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [alpha-32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 alpha-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3-50 microM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, Gi2, requires Mg2+ for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.

Affinity Labels↗
PubMed1989

Platelet-activating factor production in human neutrophils by sequential stimulation with granulocyte-macrophage colony-stimulating factor and the chemotactic factors C5A or formyl-methionyl-leucyl-phenylalanine.

Besides its function as a growth factor, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) "primes" polymorphonuclear leukocytes (PMN) for enhanced biologic responses to a number of secondary stimuli. We examined the effect of priming PMN with GM-CSF on the production of [3H] platelet-activating factor (PAF) from [3H]acetate upon stimulation with the chemotactic factors FMLP and C5a. In PMN stimulated with the individual peptide mediators alone [3H]PAF levels were close to controls, whereas considerable amounts of [3H]PAF are formed after stimulation of PMN which have been preexposed to GM-CSF. The priming effect was concentration and time dependent. It was optimal after a preincubation period of 2 h. A maximum of [3H]PAF accumulation is reached within 2.5 min (C5a) and 5.0 min (FMLP) after activation of GM-CSF-primed PMN. In addition, we show that PAF isolated from PMN preincubated with GM-CSF and triggered with chemotactic factors is able to enhance the respiratory burst in PMN. PAF formed by sequentially activated PMN could contribute to the enhanced oxygen radical production and cytotoxicity in effector cells and play a role in modulating and amplifying inflammatory reactions.

Adult↗
PubMed1995

Polymorphonuclear leucocyte migration through human dermal fibroblast monolayers is dependent on both beta 2-integrin (CD11/CD18) and beta 1-integrin (CD29) mechanisms.

Accumulation of leucocytes in inflammation involves their migration through vascular endothelium and then in the connective tissue. We investigated human polymorphonuclear leucocyte (PMNL) migration through a biological barrier of human dermal fibroblasts grown on microporous filters, as a model of PMNL migration in the connective tissue. PMNL did not migrate through a fibroblast monolayer unless a chemotactic factor, e.g. C5a, interleukin-8 (IL-8) or zymosan-activated plasma (ZAP; C5adesArg), was added. This migration was partially inhibited (35-70%, depending on the stimulus) by treatment of PMNL with monoclonal antibody (mAb) to CD18 (beta 2-integrins). Most of the CD18-independent migration was inhibited by mAb to beta 1-integrins (CD29). Inhibition by mAb to beta 1 was observed when the PMNL, but not the fibroblasts, were treated with mAb. The role of beta 1-integrins in PMNL transfibroblast migration was detectable only when the function of the CD11-CD18 complex was blocked, because mAb to beta 1-integrin alone had no significant effect on PMNL migration. Migration induced by C5a was more CD18-independent compared to IL-8 or C5adesArg. The CD18-independent migration was also inhibited by mAb to the beta 1-integrin subunits alpha 5 (of very late antigens-5; VLA-5) and alpha 6 (of VLA-6). Treatment of the fibroblasts (4 hr) with tumour necrosis factor-alpha (TNF-alpha) or IL-1 alpha enhanced C5a-induced PMNL transfibroblast migration and increased the proportion of migration utilizing the CD11-CD18 mechanism. However, TNF-alpha treatment had no effect on the degree of beta 1-integrin-dependent migration. These findings suggest that in response to the chemotactic factors C5a, IL-8 and C5adesArg, PMNL migration in the connective tissue is mediated by both CD11-CD18 (beta 2) and beta 1-integrins on the PMNL. The VLA-5 and VLA-6 members of beta 1-integrins are involved in this process. This is in contrast to PMNL migration across endothelium in this system, which is virtually all CD18 dependent with no significant role for beta 1-integrins.

CD18 Antigens↗
PubMed1995

[Chemico-enzymatic synthesis, cloning and expression of a gene for an analog of human anaphylatoxin C5a].

Chemical-enzymatic synthesis and cloning of a gene for a human anaphylatoxin C5a analog were carried out. Recombinant plasmid pRC5a providing the expression of the synthetic gene in the Escherichia coli cells was obtained. The biological activity of the expression product was demonstrated by the chemotaxis activity test and by the release of myeloperoxidases from rat peritoneal cells that was induced by bacterial cell lysates containing the recombinant protein.

Amino Acid Sequence↗