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PubMed1997

Complement partially mediates acid aspiration-induced remote organ injury in the rat.

BACKGROUND: Acid aspiration into one lung is known to cause both a local as well as remote organ injury characterized by neutrophil sequestration and subsequent edema. This study investigated investigated the role of the complement cascade in the development of acid aspiration-induced local lung and remote organ injuries using K-76 COONa (K76), an anticomplement agent that inhibits the complement pathway at the C5 step, and its usefulness as a treatment drug. METHODS: Anesthetized rats underwent tracheostomy and insertion of a cannula. K76 was intraperitoneally administrated prior to or immediately after the instillation of 0.1 ml of HCl (0.1N) or phosphate buffer solution (PBS) into the left lung. Inflammatory responses were evaluated by tumor necrosis factor alpha (TNF alpha) in the plasma and the bronchoalveolar lavage fluid (BALF) (n = 4), tissue myeloperoxidase (MPO), wet-to-dry weight ratio (W/D ratio) (n = 6), and protein concentration in the BALF (n = 6). RESULTS: Acid instillation caused an increase in the plasma TNF alpha, which was significantly attenuated by the administration of K76 prior to or after the acid instillation. Acid instillation to the left lung resulted in an increase of MPO and W/D ratios of the left lung, the right lung, and the small intestine. The administration of K76 inhibited the increase of MPO in these organs. K76 inhibited the increase of W/D ratios of the right non-instillated lung and the small intestine. Acid instillation led to increased protein concentration in the BALF of the left lung, which was not inhibited by K76. K76 administrated after the acid instillation had the same effects. TNF alpha in the BALF was not detected in all groups. CONCLUSION: These results suggest that localized acid aspiration induces, through the C5a step of the complement system-dependent mechanisms, TNF alpha formation and neutrophil sequestration, which caused the increase of endothelial permeability of the systemic organs. K76 is effective as a treatment drug in modulating some of the injuries caused by the acid instillation, but further investigations is warranted as to its potential as a therapeutic agent.

Animals↗
PubMed2004

Synergy between proinflammatory ligands of G protein-coupled receptors in neutrophil activation and migration.

The chemokine dose and the time period during which the chemotactic gradient is established determine the number of leukocytes that infiltrate inflamed tissues. At suboptimal chemokine concentrations, neutrophils may require a priming agent or a second stimulus for full activation. An interesting mode of cooperative action to reach maximal migration is synergy between chemokines. This was first observed between the plasma CC chemokine regakine-1 and the tissue CXC chemokine ligand interleukin-8 (IL-8/CXCL8) in neutrophil chemotaxis. Addition of antibodies against IL-8 or regakine-1 in the Boyden microchamber assay abrogated this synergy. Other CC chemokines, such as CC chemokine ligand-2 monocyte chemotactic protein-1 (MCP-1/CCL2), MCP-2 (CCL8), and MCP-3 (CCL7) as well as the CXC chemokine receptor-4 (CXCR4) agonist stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12), also dose-dependently enhanced neutrophil chemotaxis toward a suboptimal concentration of IL-8. These chemokines synergized equally well with the anaphylatoxin C5a in neutrophil chemotaxis. Alternatively, IL-8 and C5a did not synergize with an inactive precursor form of CXCL7, connective tissue-activating peptide-III/CXCL7, or the chemoattractant neutrophil-activating peptide-2/CXCL7. In the chemotaxis assay under agarose, MCP-3 dose-dependently increased the migration distance of neutrophils toward IL-8. In addition, the combination of IL-8 and MCP-3 resulted in enhanced neutrophil shape change. AMD3100, a specific CXCR4 inhibitor, reduced the synergistic effect between SDF-1alpha and IL-8 significantly. SDF-1alpha, but not MCP-1, synergized with IL-8 in chemotaxis with CXCR1-transfected, CXCR4-positive Jurkat cells. Thus, proinflammatory chemokines (IL-8, MCP-1), coinduced during infection in the tissue, synergize with each other or with constitutive chemokines (regakine-1, SDF-1alpha) to enhance the inflammatory response.

Calcium↗
PubMed1998

A CD18/ICAM-1-dependent pathway mediates eosinophil adhesion to human bronchial epithelial cells.

Eosinophil adhesion to airway epithelium is believed to facilitate eosinophil accumulation and retention in asthmatic airways. Monoclonal antibodies (mAb) against intercellular adhesion molecule-1 (ICAM-1) and its CD18 leukocyte integrin ligands have been shown to inhibit airway eosinophilia in animal models of asthma, although the role of this pathway in eosinophil-epithelial adhesion is not fully understood. To investigate the role in vitro of CD18 and ICAM-1, we measured adhesion of fluorescently labeled human eosinophils to normal human bronchial epithelial cell (NHBEC) monolayers pretreated for 24 h with culture medium (low constitutive ICAM-1) or tumor necrosis factor-alpha (TNF-alpha; 1 ng/ml) and interferon-gamma (IFN-gamma) (10 ng/ml; increased ICAM-1). Stimulation of eosinophils with C5a (10(-7) M) increased adhesion measured at 30 min to unactivated NHBEC from 11.4 +/- 0.7 to 15.5 +/- 0.4% (n = 4), and this increase was CD18/ICAM-1-independent, whereas phorbolmyristate acetate (PMA) (10(-8) M)-induced adhesion (20.7 +/- 1.7%) was abolished by anti-CD18 and reduced by anti-ICAM-1. In contrast, C5a- and PMA-induced adhesion to TNF-alpha/IFN-gamma-activated NHBEC (increased from 11.1 +/- 1.3% to 21.9 +/- 1.0% and 27.6 +/- 1.9%, respectively) was CD18- and ICAM-1-dependent. Eotaxin, but not regulated on activation normal T cells expressed and secreted, macrophage inflammatory protein-1, formyl methionyl leucyl phenylalanine, leukotriene B4 or platelet-activating factor, also induced CD18/ICAM-1-dependent adhesion to activated NHBEC. In the absence of added chemoattractants, eosinophil adhesion to NHBEC increased with time and, at 120 min, was significantly greater (P < 0.01) to activated NHBEC (37.3 +/- 2.4%, n = 5) than to unactivated monolayers (24.3 +/- 1.9%); mAb against CD18 or ICAM-1 abolished increased, but not basal, adhesion. These results suggest that CD18/ICAM-1 mediated eosinophil adhesion to activated NHBEC but that adhesion to resting NHBEC was largely independent of this pathway.

Antibodies, Monoclonal↗
PubMed1991

Regulation of mediator release by human basophils: importance of the sequence and time of addition in the combined action of different agonists.

Biologically active molecules affecting basophil function can be divided into 4 groups according to their capacity to induce basophil degranulation and/or leukotriene generation: (1) full agonists such as anti-IgE or fMLP, which induce both histamine and leukotriene release; (2) partial agonists such as C5a, which induces degranulation only; (3) incomplete agonists such as neutrophil-activating peptide-1, platelet-activating factor or C3a, which induce mediator release only after cytokine preincubation, and (4) basophil response modifiers, such as interleukin-3, interleukin-5 and granulocyte/macrophage- colony-stimulating factor, which (a) enhance the releasability to all basophil agonists, (b) change the mediator profile, (c) enhance the rate of mediator release, (d) render basophils responsive to lower agonist concentrations and (e) render basophils responsive to incomplete agonists. We demonstrated that histamine release and de novo synthesis of lipid mediators are clearly separately regulated, and that combined actions of different molecules are of importance. In particular, the type(s), the time interval and the sequence of action of basophil-activating molecules are crucial for the final outcome of the basophil release reaction.

Basophils↗
PubMed2001

Differential inhibition of inflammatory effector functions by petasin, isopetasin and neopetasin in human eosinophils.

BACKGROUND: Priming of eosinophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with platelet-activating factor (PAF) or the anaphylatoxin C5a is associated with a rapid production of leukotrienes (LTs) and release of eosinophil cationic protein (ECP). OBJECTIVE: This study was designed to determine the effects of the sesquiterpene esters petasin, isopetasin and neopetasin on LT generation and ECP release in eosinophils in vitro. METHODS: The model of eosinophil activation described above was used to induce LT production and ECP release. Cells were incubated with petasins and control inhibitors prior to priming and stimulation. To analyse intracellular steps of eosinophil activation and determine potential drug targets, some key signalling events were studied. Activity of cytosolic phospholipase A2 (cPLA(2)) was measured by analysing the generation of arachidonic acid (AA). Translocation of 5-lipoxygenase (5-LO) was observed using immunofluorescence microscopy. Intracellular calcium concentrations [Ca2+]i were measured by a bulk spectrofluorometric assay. RESULTS: Whereas all three compounds inhibited LT synthesis, ECP release from eosinophils was blocked by petasin only, but not isopetasin or neopetasin. Similarly, PAF- or C5a-induced increases in [Ca2+]i were completely abrogated by petasin only, whereas isopetasin and neopetasin had significant lower blocking efficacy. Moreover, only petasin, but not isopetasin or neopetasin, prevented increases in cPLA(2) activity and 5-LO translocation from the cytosolic compartment to the nucleus envelope in calcium ionophore-stimulated eosinophils. CONCLUSION: These data suggest that different petasins may at least partially block different intracellular signalling molecules. To reduce LT synthesis, isopetasin and neopetasin may act at the level of or distal to 5-LO. In contrast, petasin may inhibit inflammatory effector functions in human eosinophils by disrupting signalling events at the level of or proximal to phospholipase Cbeta (PLCbeta), besides its potential inhibitory activity within mitogen-activated protein kinase (MAPK) and LT pathways.

Active Transport, Cell Nucleus↗
PubMed1987

Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface.

Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min. Human alveolar and murine peritoneal macrophages, cells that had previously diapedised, could not be induced to upregulate Mac-1 or p150,95. Detergent permeabilization studies in monocytes indicated that these proteins were stored in internal latent pools, which were reduced upon stimulation. Electron microscopy utilizing rabbit antiserum against p150,95 revealed these proteins on the plasma membrane, and in intracellular vesicles and peroxidase negative granules. Together with other functional studies, these findings suggest that the mobilization of Mac-1 and p150,95 from an intracellular compartment to the plasma membrane regulates the monocyte's ability to adhere and diapedese.

Animals↗
PubMed2000

Effects of human lung fibroblasts on eosinophil degranulation.

BACKGROUND: Although eosinophils and fibroblasts are thought to contribute to the mechanisms of chronic asthmatic inflammation, the interaction between eosinophils and fibroblasts has not been thoroughly clarified. We examined eosinophil cationic protein (ECP) release from human eosinophils cultured in the presence of human lung fibroblast HFL-1. METHODS: Eosinophils from healthy donors were cultured with or without C5a for 16 h in the presence of human fetal lung fibroblasts which had previously been incubated with or without TNF for 4 h. ECP in supernatants was measured by RIA. RESULTS: ECP release was potentiated only when both eosinophils and fibroblasts were activated by C5a and TNF, respectively, while it was not significantly potentiated when either eosinophils or fibroblasts were activated. Paraformaldehyde fixation of fibroblasts had some suppressive effect on ECP enhancement, and mAb against GM-CSF partly inhibited ECP enhancement. Coculture of eosinophils and fibroblasts with stimulus treatment resulted in the enhancement of both eosinophil adhesion and ECP release. The potentiation of ECP release was partially inhibited by anti-ICAM-1 mAb, anti-CD18 mAb, and anti-CD29 mAb, which caused partial and comparable inhibition of the enhancement of eosinophil adhesion. CONCLUSIONS: This study suggests that the activation of fibroblasts may have some role in the potentiation of ECP release from cocultured eosinophils, and that adhesion of eosinophils to fibroblasts may partly be involved in the mechanism of ECP potentiation.

Antibodies, Monoclonal↗
PubMed1993

Clinical study of high-flux cuprammonium rayon hemodialysis membranes.

The aim of this crossover clinical study was to gain basic information on the hemocompatibility and effectiveness of recently developed high-flux membranes made of cuprammonium rayon with ultrafiltration coefficients of 10, 17, and 19 ml/mm Hg/h (S12W, SU12W, and SS12W dialyzers, respectively), and to identify any possible differences from a conventional membrane made of the same material with an ultrafiltration coefficient of 6 ml/mm Hg/h (C12W dialyzer). All the tested membranes led to an abrupt drop in leukocyte count in the initial phase of hemodialysis. In high-flux membranes, C5a anaphylatoxin would pass into the dialysate, but mean C5a anaphylatoxin concentrations in the dialysate were lower by orders of magnitude than its plasma concentrations, which behaved, in high- and low-flux membranes alike, typically of those made of nonsubstituted cellulose with no intermembrane differences. As judged by the concentrations of the thrombin-antithrombin III complex, the coagulation system was activated--again, without differences between membranes. The reduction rates for urea, creatinine, and phosphates were comparable for all the tested membranes. Compared with baseline, the post-dialysis serum concentrations of beta 2-microglobulin in high-flux membranes, unlike the low-flux membrane, were significantly lower. We conclude that there are no significant differences between the tested high- and low-flux membranes made of cuprammonium rayon in the monitored hemocompatibility parameters, and that high-flux membranes are capable of reducing serum beta 2-microglobulin concentrations.

Adult↗
PubMed2006

Bovine prion (PrP) and Doppel (Dpl) proteins expression after in vitro leukocyte activation or Dpl/PrP blocking.

It has been postulated that Doppel (Dpl) and Prion (PrP) proteins have yet undetermined interactions, since Dpl is overexpressed in transgenic PrP-deficient mice. In this study we investigated the expression levels of Dpl and PrP on lymphocytes, monocytes and neutrophils (PMNs) isolated from bovine blood and incubated (2 and 18 h) with TNFalpha, IL-1, IL-2, IL-8, C5a, IFNgamma, anti-PrP, and anti-Dpl antibodies by flow cytometry. The isolation procedures yielded cell populations with high purity, viability and recovery rates. After 2 h incubation, expression of PrP or Dpl was altered only in PMNs. These cells overexpressed PrP when incubated with TNFalpha and IFNgamma, and both PrP and Dpl when incubated with C5a; incubation with TNFalpha, IL-8 and IFNgamma led to down-regulation of Dpl. Lymphocytes incubated for 18 h with IL-2 and with IFNgamma overexpressed Dpl. Incubation with the anti-PrP antibody induced down-regulation of Dpl in PMNs after 2 h and overexpression of Dpl in lymphocytes after 18 h. The differences recorded after 2 h were likely due to redistribution of pre-existing PrP or Dpl molecules, while those seen at 18 h were most probably due to increased protein synthesis. The variations seen using the different activators depend on different receptors and/or signaling pathways. These results demonstrate that is possible to alter the expression of Dpl and PrP in blood cells in vitro by incubation with either cytokines or anti-PrP antibodies. This opens an interesting opportunity to study the biology of these proteins using in vitro systems.

Animals↗
PubMed1994

Regulation of TNF-alpha receptor expression on human neutrophils by various cell activating factors.

Since tumor necrosis factor-alpha (TNF-alpha) has been implicated in a wide range of inflammatory states, we sought to examine the regulation of TNF-alpha receptors on human neutrophils (PMNs). We were particularly interested in the relationship of TNF-alpha binding characteristics to various known neutrophil-priming stimuli such as temperature, N-formylmethionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), C5ades Arg, and human recombinant C5a (HrC5a). Competition binding studies indicated that there was no significant change in receptor number or affinity due to the temperature used for neutrophil isolation. PMNs isolated at 4 or 25 degrees C had a single class of receptors with a Kd of 83.1 +/- 3.3 vs 90.0 +/- 8.3 pmole/liter and a similar number of receptors, 1575 +/- 99 vs 1876 +/- 279 receptors/cell. However, when either group of isolated PMNs was further incubated in buffer at 37 degrees C for 15 min, a significant increase in Kd (4-37 degrees C, 147.9 +/- 23.7 pmole/liter; 25-37 degrees C, 165.2 +/- 29.2 pmol/liter) and a decrease in receptor number (4-37 degrees C, 1116 +/- 86 receptors/cell; 25-37 degrees C, 957 +/- 71 receptors/cell) were seen. To determine the effect of other activating substances such as fMLP, PMA, C5ades Arg, HrC5a, 4 degrees C isolated PMNs were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement C5a↗
PubMed2001

Role of contact system activation in hemodialyzer-induced thrombogenicity.

BACKGROUND: The contact system is generally believed to be the main trigger of the coagulation cascade during extracorporeal circulation. However, the extent of contact activation, its role for intradialytic thrombin generation as well as the influence of different dialyzer membranes have not been well established. METHODS: In a novel full-scale ex vivo recirculation dialysis model, we investigated the thrombogenicity of three widely used hemodialyzers (Cuprophan Renak RA15-U, Polysulfone F6HPS and AN69XT Nephral 200). The activation of the contact system was evaluated using a newly developed ELISA for factor XIIa-C1-inhibitor complexes. Additionally, we determined free FXIIa (ELISA), thrombin-antithrombin (TAT) complexes, platelet factor 4 (PF4), complement activation (C5a), granulocyte elastase and blood cell counts. The findings in blood from normal volunteers were compared with factor XII-deficient blood. RESULTS: With normal blood AN69 exhibited the highest thrombogenicity in comparison to Cuprophan and Polysulfone, as assessed by TAT generation and platelet consumption. AN69 caused a rapid increase of the FXIIa-C1-inhibitor complexes and of free FXIIa. Despite significant TAT generation with Cuprophan and Polysulfone free FXIIa remained unchanged and the FXIIa-C1-inhibitor complexes stayed below the detection limit. With factor XII-deficient blood Polysulfone exhibited the same TAT generation, whereas the thrombogenicity of AN69 was greatly reduced. CONCLUSIONS: Our data challenge the common assumption that activation of the contact system with generation of FXIIa is the main trigger for coagulation and thrombus formation in hemodialysis. Only the negatively charged AN69 membrane with enhanced thrombogenicity strongly induced contact activation.

Adult↗
PubMed1985

The role of C5 in septic lung injury.

One proposed mechanism for the pathogenesis of lung injury in septic animals is the stimulation by C5a of granulocytes to produce and release toxic oxygen radicals that damage cellular membranes in pulmonary capillaries. The authors have investigated the possible role of C5 in septic lung injury, utilizing C5-sufficient and C5-deficient twin mice strains. In this lethal sepsis model, mean survival time is increased in C5-deficient mice in comparison to the survival of their C5-sufficient twins. Morphometric results demonstrate a significant increase in intracapillary granulocrit and air-blood barrier thickness 24 hours after cecal ligation and puncture in C5-sufficient septic mice. Similarly, mean arterial pO2 is significantly decreased in the C5-sufficient animals. Intracapillary granulocrit, air-blood barrier thickness, and arterial pO2 are normal in the septic C5-deficient twins of these animals. These data support the hypothesis that C5 is involved in the pathogenesis of septic lung injury.

Animals↗
PubMed1993

Quantification of chemotactic peptides (C5a anaphylatoxin and IL-8) in psoriatic lesional skin.

BACKGROUND AND DESIGN: Psoriatic scale extracts contain a unique chemotactic peptide fraction that is likely to be involved in the induction of rhythmic transepidermal leukocyte chemotaxis. Recent studies have identified the presence of two unrelated chemotactic peptides in this fraction, ie, C5a/C5a des Arg and interleukin 8 (IL-8), and its related cytokines. To investigate their relative contribution to the transepidermal leukocyte migration as well as their interrelationship in psoriatic lesions, we have quantified concentrations of immunoreactive C5a/C5a des Arg and IL-8 in psoriatic lesional scale extracts and those from related sterile pustular dermatoses such as subcorneal pustular dermatosis and pustulosis palmaris et plantaris. RESULTS: The concentrations of C5a/C5a des Arg and IL-8 were more significantly increased in the horny-tissue extracts from lesional skin than in those from noninflammatory orthokeratotic skin (P < .01). The increase of C5a/C5a des Arg concentration was specific to the lesional scale extracts, but showed a rather wide range of variation. By contrast, IL-8 concentration, although consistently increased in the lesional scale extracts, was also moderately increased even in noninflammatory scale extracts prepared from ichthyosis vulgaris. The elevation of IL-8 levels in psoriatic lesions was also confirmed by measuring their levels in cutaneous tissue fluid samples collected from suction blisters. However, unexpectedly, some control samples obtained from normal skin also showed a moderate increase in the IL-8 level. Neutrophil chemotactic activity correlated significantly only with the levels of C5a/C5a des Arg in the scales (P < .05). No such significant correlation was found between chemotactic activity and IL-8 or between C5a/C5a des Arg and IL-8. CONCLUSION: Based on these results, we speculate that, although IL-8 may exert a synergistic effect with C5a/C5a des Arg in the induction of transepidermal leukocyte chemotaxis, it constitutes a proinflammatory cytokine that is involved in the production of the persistent inflammatory changes characterized by a T-lymphocyte infiltration. In contrast, C5a/C5a des Arg seems to be generated only in the inflammatory lesional skin under specific circumstances that preferentially favor complement activation and also seems to play a major role in the induction of cyclic transepidermal leukocyte chemotaxis from "squirting papillae."

Adolescent↗
PubMed1982

Low zone desensitization: a stimulus-specific control mechanism of cell response. Investigations on anaphylatoxin-induced platelet secretion.

The biologic activity of the anaphylatoxic peptides C5a and C3a is regulated efficiently at the target-cell level by the phenomenon of desensitization. Desensitization of platelets is stimulus specific and can be induced by low concentrations of anaphylatoxins without any preceding secretory event. In contrast to activation to secretion, desensitization is Ca++ independent but much more time consuming, especially at lower temperatures where both processes differ markedly in reaction velocity. This low zone desensitization insures that secretion from platelets only occurs when high amounts of anaphylatoxins are rapidly generated in the vicinity of the target-cell. Consequently, stimulus-specific unresponsiveness of the target cells can be induced by slowly increasing the concentration of the respective stimuli in their vicinity. Cellular control seems to act as a first-line mechanism of regulation, whereas the role of fluid-phase control is considered as preventing longer persistence and systemic accumulation of active anaphylatoxins.

Anaphylatoxins↗
PubMed1997

Eicosanoid production from porcine neutrophils and platelets: differential production with various agonists.

Polymorphonuclear neutrophils (PMN) and platelets interact to produce both inflammatory and anti-inflammatory lipid mediators during human disease. Because swine models of human disease are used, it is important to understand the mechanisms involved in the formation of lipid mediators from porcine PMN-platelet interactions. In the present study, we investigated the mechanism of thromboxane (Tx) A2 and lipoxin A4 (LXA4) formation from porcine PMN and platelets, respectively. PMN (10(7)/ml) and platelet (30 x 10(7)/ml) suspensions stimulated with porcine C5a (pC5a), but not recombinant human C5a (rhC5a), significantly enhanced TxB2 formation. After cytochalasin B treatment, pC5a or rhC5a significantly and equally enhanced TxB2 formation from PMN-platelet suspensions. A-23187-induced TxB2 formation from platelets was not significantly augmented by the presence of PMN in these suspensions. A-23187 induced significant LXA4 production from porcine PMN that was not augmented by addition of platelets. Flow cytometric analysis of PMN-platelet suspensions revealed activated platelets adherent to PMN following pC5a stimulation. CV-6209, a platelet-activating factor (PAF) receptor antagonist, dose dependently prevented pC5a-induced platelet adherence to PMN and TxB2 formation. These data demonstrate that 1) porcine PMN alone can biosynthesize LXA4 without the assistance of platelets, which is in sharp contrast to human PMN-platelet interactions, and 2) in the absence of cytochalasin B, pC5a stimulates PAF biosynthesis from porcine PMN, resulting in TxB2 formation from platelets.

Animals↗
PubMed1974

Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors.

The interaction of human neutrophils adherent to plastic petri dishes with the purified chemotactic factors C5a and kallikrein increased their rate of aerobic glycolysis 25-120% and the activity of their hexose monophosphate shunt (HMPS) 100-600%, reaching a plateau after 2 hr at 37 degrees C. The stimulation of either pathway required a chemotactically active stimulus since neither C5 nor prekallikrein or inactivated kallikrein could enhance metabolic activity. Marked suppression of the neutrophil chemotactic response by preincubation with a chemotactic factor to achieve deactivation, 5 x 10(-7) M diisopropyl fluorophosphate, or the neutrophil immobilizing factor (NIF) did not prevent the stimulation of HMPS activity or glycolysis by chemotactic factors. The metabolic inhibitors iodoacetate and 6-aminonicotinamide at concentrations which blocked enhancement of glycolysis or HMPS activity, respectively, partially suppressed the chemotactic response of neutrophils to the chemotactic factors. The capacity of a chemotactic factor to stimulate glucose metabolism of human neutrophils is associated with a maximal chemotactic response, but this stimulation is not alone sufficient for chemotaxis.

Aerobiosis↗
PubMed2003

Expression and function of the endothelial protein C receptor in human neutrophils.

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti-endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation-blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C-based treatment of severe sepsis.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1985

Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes.

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.

Cell Fractionation↗