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PubMed1995

Nerve growth factor from the venom of the Chinese cobra Naja naja atra: purification and description of non-neuronal activities.

Nerve growth factor (NGF) was separated from crude Naja naja atra venom by using weak cation-exchange chromatography, followed by reversed-phase liquid chromatography. The yield of the purification was 0.2-0.5% (w/w). The mol. wt was determined to be 13,600 and the protein still induced the typical fibre outgrowth of cultured PC-12 cells in a concentration range of 5-10 ng/ml. Beside this neuronal effect we demonstrated non-neuronal effects of cobra venom NGF, such as induction of plasma extravasation and histamine release from whole blood cells. With human leucocyte preparations, including enriched basophils, there was an increase in C5a-induced histamine release, whereas NGF alone was inactive. Cobra NGF was one-tenth as potent as human recombinant NGF, with a half-maximal stimulation occurring at 10 ng/ml. Cobra NGF and human recombinant NGF showed a modulatory effect on histamine release comparable to the haematopoietic growth factor IL-3. Thus, the non-neuronal effects of cobra NGF may account for immunomodulatory activities during inflammatory events.

Animals↗
PubMed1997

Agonist-induced adherence of equine eosinophils to fibronectin.

Eosinophils are believed to play an important part in the pathogenesis of equine diseases such as helminth infestation and the allergic skin disease, sweet itch. It has been shown that adherence of human eosinophils to the connective tissue matrix protein fibronectin enhances cell activation and survival time. If adherence causes similar changes in the properties of equine eosinophils, cell-induced tissue damage at a site of parasitic infestation or allergic response would be exacerbated. However, investigation of this hypothesis requires identification of mediators that cause equine eosinophil adherence. Since the equivalent recombinant equine proteins were not available, the present study reports the effects of recombinant human (rh) C5a and IL-5 on the adherence of equine peripheral blood eosinophils (EPBEs) to fibronectin in vitro. The effects of LTB4 and PAF on EPBE adherence to fibronectin were also examined and phorbol myristate acetate (PMA) was used as a positive control. PMA caused a dose-related increase in EPBE adherence to fibronectin-coated plastic. In comparison, rh C5a produced a much smaller response which was only evident at the highest dose tested. On the other hand, rhIL-5 induced a small, but significant dose-related increase in EPBE adherence. Moreover, this response was in part dependent on the beta 1 integrin Very Late Antigen-4 (VLA4). Since adherence to serum-coated plastic was also increased by IL-5, beta 2 integrins may be activated and/or up-regulated on EPBEs by the cytokine. Neither LTB4 nor PAF caused EPBE adherence to fibronectin but prior incubation with these mediators increased the response of cells to IL-5. There were no differences between the responses of EPBEs isolated from horses with clinical signs of sweet itch and normal animals. Thus, whilst up-regulation of IL-5-induced adherence may occur locally in tissues in vivo, it does not appear to take place in the circulation. Finally, C5a, PAF and LTB4, but not IL-5, caused equine neutrophil adherence to fibronectin demonstrating the different responses of granulocytes to these mediators. The results obtained in the present study have shown that mediators which may be released at sites of inflammatory or allergic reactions can induce or enhance eosinophil adherence to tissue matrix protein. Thus, these mediators can now be used in future studies to determine if cell adherence may alter eosinophil activation or survival time.

Animals↗
PubMed2001

Psoriasis scales contain C5a as the predominant chemotaxin for monocyte-derived dendritic cells.

Dendritic cells seem to be of major importance for the pathogenesis of psoriasis. They are increased in number in lesional psoriatic skin which is thought to be due to an increased influx from the peripheral blood regulated by chemotaxins. Using a biological/biochemical approach we have addressed the question whether psoriasis scale extracts contain proteinaceous chemotaxins for dendritic cells. Human monocytes differentiated into dendritic cells by culture with GM-CSF and IL-4 (MoDC) served as responder cells. Chemotactic activity for MoDC was purified by several HPLC-steps. The results of our study show that C5a/C5adesarg is the major chemotactic peptide for MoDC in psoriasis scale extracts. In comparison to other stimuli such as fMLP or monocyte chemotactic peptide 1 (MCP-1) C5a proved to be a most potent and efficient chemotaxin for MoDC. C5a co-eluted with MRP14/calgranulin B which is present in large amounts in psoriasis scale extracts as identified by amino acid sequencing. However, MRP14/calgranulin B did not possess any chemotactic activity for MoDC. Our results provide evidence that C5a/C5adesarg although not specific for dendritic cells seems to be the major chemoattractant for these cells in lesional psoriasis skin.

Amino Acid Sequence↗
PubMed2003

Constitutively active Galpha16 stimulates STAT3 via a c-Src/JAK- and ERK-dependent mechanism.

The hematopoietic-specific Galpha16 protein has recently been shown to mediate receptor-induced activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we have delineated the mechanism by which Galpha16 stimulates STAT3 in human embryonic kidney 293 cells. A constitutively active Galpha16 mutant, Galpha16QL, stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyr705 and Ser727. Galpha16QL-induced STAT3 activation was enhanced by overexpression of extracellular signal-regulated kinase 1 (ERK1), but was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Inhibition of phospholipase Cbeta, protein kinase C, and calmodulin-dependent kinase II by their respective inhibitors also suppressed Galpha16QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Galpha16QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants. In contrast, c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42, phosphatidylinositol 3-kinase, and the epidermal growth factor receptor did not appear to be required. Similar observations were obtained with human erythroleukemia cells, where STAT3 phosphorylation was stimulated by C5a in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Galpha16. Demonstration of the involvement of different kinases in Galpha16QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins.

Blotting, Western↗
PubMed1995

Polyamines found in gingival fluid inhibit chemotaxis by human polymorphonuclear leukocytes in vitro.

Putrescine and spermidine occur at concentrations approaching 1 mM in gingival fluid at diseased periodontal sites. Previous work demonstrates that these polyamines potentiate Ca2+ signaling in polymorphonuclear leukocytes (PMNs), resulting in enhanced degranulation and superoxide generation. The present study extends this work by characterizing the effects of polyamines on PMN chemotaxis and phagocytosis, in which Ca2+ signaling plays a less defined regulatory role. Putrescine (1 mM) and spermidine (0.1 to 0.5 mM) significantly inhibited chemotaxis to fMet-Leu-Phe and C5a (P < 0.05). This inhibition was not strongly related to any effect polyamines have on PMN adhesion, actin polymerization, or formyl peptide receptor expression. Neither putrescine nor spermidine had a significant impact on phagocytosis of opsonized bacteria by PMNs. Thus, at concentrations similar to those found in gingival fluid, polyamines could potentially inhibit recruitment of PMNs to diseased pockets without impairing their ability to engulf invading bacteria.

Analysis of Variance↗
PubMed1999

Role of 5-lipoxygenase products in the local accumulation of neutrophils in dermal inflammation in the rabbit.

Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.

Animals↗
PubMed1983

Platelet-activating factor-mediated contraction of rabbit lung strips: pharmacologic modulation.

Synthetic platelet-activating factor (PAF) (1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) has been shown to induce a slowly developing contraction of rabbit lung parenchymal strips in an isolated organ bath. The spasmogenic effect of AGEPC appeared to be mediated by specific receptors distinct from H1, H2, cholinergic and C5a anaphylatoxin receptors. Prior exposure to AGEPC induced specific desensitization of lung parenchymal strips. Experiments with several pharmacological agents indicated that AGEPC-induced contraction was independent from cyclooxygenase, but was blocked when phospholipase A2 and lipoxygenase were inhibited and when the Ca++ channels were antagonized. Corticosteroids exhibited an inhibitory effect specific for AGEPC. Intracellular levels of cyclic AMP or cyclic GMP seemed to have a modulatory role in AGEPC-induced contraction of rabbit lung parenchymal strips.

Acetylcholine↗
PubMed1987

The phenomenon of leukergy: induction and detection of leukocyte aggregation in whole human blood.

Leukocyte aggregation is involved in the generation of vascular damage during various inflammatory conditions. So far, in vitro leukocyte aggregation has been studied by mixing patients' plasma or serum with leukocytes of a normal donor in a platelet aggregometer. We evaluated the leukergy phenomenon, that is, the occurrence of aggregated leukocytes in peripheral blood of patients with inflammatory disorders, as an alternative tool to the former method. Leukocyte aggregation could be induced by zymosan-activated plasma, phorbol myristate acetate, and formyl-methionyl-leucyl-phenylalanine, as well as by the calcium ionophore A23187 in vitro in whole human blood. This aggregation was blocked by various lipoxygenase and cyclooxygenase pathway inhibitors. Leukergy was diagnosed in peripheral blood of patients with inflammatory disorders and was generally not associated with elevated concentrations of immune complexes, lactoferrin, C3a des Arg, or C5a des Arg. To the best of our knowledge, this is the first report on leukocyte aggregation studies performed in whole blood. Such study permits evaluation of the aggregation phenomena of leukocytes in their natural milieu. Furthermore, it is possible with this method to obtain direct visualization of leukocytic aggregates in the peripheral blood of patients. The significance of our results and their possible implications are discussed in light of the pertinent literature.

Adult↗
PubMed1989

The effect of adherence on the generation of reactive oxygen species by human neutrophilic granulocytes.

Human neutrophilic granulocytes (PMN) suspended in protein containing salt solution or adherent on protein coated nylon fibers were tested for the production of H2O2 and O2- in response to various PMN stimulants. Upon stimulation with the chemotactic factors formyl-methionyl-leucyl-phenylalanine, C5a and platelet activating factor, the non-chemotactic ionophore A23187, and the chemotaxis inhibitors tumor necrosis factor (TNF alpha) and lymphotoxin (TNF beta) adherent PMN produced considerably more reactive oxygen metabolites than suspended cells. The relative amounts of the two metabolites varied with the stimulus and its concentration, TNF alpha and TNF beta favoring H2O2 production, C5a eliciting more O2- than H2O2 and the other active stimulants being in between. Leukotriene B4 and a novel monocyte-derived chemotaxin were inactive in releasing either oxygen derivative from adherent or suspended PMN. The data indicate that attachment of PMN to endothelial cells or to connective tissue substances can strongly enhance its ability to respond to a given stimulus with the production of reactive oxygen metabolites. The findings may in part explain the "priming" phenomenon since many PMN-priming mediators increase the cells' adherence.

Calcimycin↗
PubMed1987

Oscillatory motion in human neutrophils responding to chemotactic stimuli.

Human neutrophils treated with the secretion inhibitor 17-hydroxywortmannin were stimulated with fMLP, C5a, PAF or LTB4, and the ensuing shape change was studied. The cells rapidly extended lamellipodia and showed regular oscillatory behaviour. The oscillations were observed in both light transmission and 90 degrees light scattering, had the same frequency in each case, and disappeared within 30-50 seconds. Light scattering theory suggests that they reflect rhythmic changes in the shape and/or size of the chemotactically stimulated cells, possibly related to crawling or swimming movements associated with migration.

Androstadienes↗
PubMedDate not supplied

Inflammatory response after implantation of a left ventricular assist device: comparison between the axial flow MicroMed DeBakey VAD and the pulsatile Novacor device.

The implantation of a ventricular assist device (VAD) is associated with a stimulation of the inflammatory system. We compared changes in the inflammatory response after implantation of a pulsatile Novacor left (L) VAD and the axial flow MicroMed DeBakey VAD. Six consecutive patients after implantation of a Novacor LVAD (NC) and six patients after implantation of a MicroMed DeBakey VAD (MD) were included in the investigation. Patients received LVADs for medically non treatable end-stage heart failure. Tumor necrosis factor alpha (TNF), C3a, C5a, interleukin 6 (IL-6), and neutrophil elastase were measured twice a week over a period of 3 months after implantation of the device. All tests were performed with an enzyme-linked immunosorbent assay. There was no significant difference in the clinical course of the two groups. All inflammatory parameters were elevated in both groups during the entire period of the investigation. There was no difference in TNF, polynuclear leukocyte elastase, or C3a levels between the two groups; however, IL-6 (NC: 23.6+/-37.6 pg/ml vs. MD: 63+/-114 pg/ml, p < 0.001) and C5a (NC: 708+/-352 microg/L vs. MD: 1,745+/-1,305 microg/L, p < 0.001) were increased significantly more in patients following implantation of the axial flow MicroMed DeBakey VAD. Compared with the pulsatile Novacor device, the implantation of the axial flow MicroMed DeBakey LVAD seems to be associated with an increased stimulation of one part of the inflammatory system. Further investigations are necessary for evaluation of the pathophysiologic mechanism and clinical implications of these findings.

Adult↗
PubMed1998

Moderate inhibitory effect of interleukin-10 on human neutrophil and monocyte chemotaxis in vitro.

Neutrophils and monocytes are the major classes of phagocytes that migrate from the blood stream and accumulate in inflamed tissues in response to various chemoattractants. Because IL-10 is a potent anti-inflammatory cytokine, we analyzed its in vitro effect on chemotaxis using an under agarose method. We found that, as compared to prednisolone, IL-10 alone was a modest inhibitor of C5a, fMLP and IL-8-induced neutrophil chemotaxis, and C5a-induced monocyte chemotaxis. However, GM-CSF pretreatment of the cells potentiated this inhibitory effect. Similarly, the IL-10 induced modulation of the beta2 integrin CD11b/CD18 adhesion molecule expression was only observed on GM-CSF-preactivated neutrophils and monocytes. Taken together, these results suggest that the migration and accumulation of phagocytes at infection sites would not be significantly affected by IL-10 given as an immunomodulatory therapy.

Antigens, CD↗
PubMed1997

Differential effects of a mitogen-activated protein kinase kinase inhibitor on human neutrophil responses to chemotactic factors.

Chemotactic factors, i.e., an N-formyl peptide, C5a, interleukin-8, and leukotriene B4, induced neutrophils to activate mitogen-activated protein (MAP) kinases, as defined by the tyrosine phosphorylation and decrease in electrophoretic mobility of immunodetected 44-, 42-, and 40-kDa proteins. PD 98059, an inhibitor of MAP kinase kinase activation, blocked these changes. The drug likewise blocked neutrophil chemotaxis but did not alter superoxide anion production and paradoxically enhanced degranulation responses to the stimuli. The MAP kinase pathway appears to have a highly selective role in mediating motility but not other cellular responses.

Chemotactic Factors↗
PubMed1992

Interleukin-8 induces changes in human neutrophil actin conformation and distribution: relationship to inhibition of adhesion to cytokine-activated endothelium.

Interleukin-8 (IL-8) induces diverse biological responses in neutrophils, including inhibition of adhesion to cytokine-activated endothelium, which we have termed the leukocyte adhesion inhibition (LAI) effect. Pretreatment of neutrophils with cytochalasin B abolished the LAI effect of IL-8, suggesting a microfilament-dependent mechanism. Interleukin-8 induced a rapid increase (less than or equal to 15 s) in the polymerization of actin filaments in human neutrophils that was blocked by pretreatment with cytochalasin B. F-actin depolymerization occurred gradually at a rate inversely proportional to IL-8 concentration. This temporal pattern of actin polymerization-depolymerization was similar to that induced by other chemotactic factors such as C5a and N-formylmethionyl-leucyl-phenylalanine, which also exhibit a marked LAI effect, but the lipid mediators, leukotriene B4 and platelet-activating factor, lack any significant LAI effect. Scanning confocal microscopy demonstrated that neutrophil actin microfilaments undergo a dramatic rearrangement prior to detachment of the neutrophil from a surface. We suggest that the ability of IL-8 and certain other leukocyte agonists to regulate the actin polymer network of neutrophils may play an important role in adhesive interactions with the vascular endothelium.

Actins↗
PubMed1992

A rapid microtiter plate method for the detection of lysozyme release from human neutrophils.

An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng/ml. Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t-Boc-L-methionyl-L-leucyl-L-phenylalanine (N-t-Boc), LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC50 of 2 microM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC50 of 2 microM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Lack of correlation between calcium mobilization and respiratory burst activation induced by chemotactic factors in rabbit polymorphonuclear leukocytes.

Low concentrations of FMLP, partially purified rabbit C5a, leukotriene B4 and platelet activating factor induced a rapid rise of intracellular free Ca2+ in rabbit polymorphonuclear leukocytes. However, the four factors differed markedly in their ability to activate the respiratory burst. The peptides FMLP and C5a induced a single, strong chemiluminescence response whereas the lipids leukotriene B4 and platelet activating factor induced a markedly less intense response with a two-peak profile. Respiratory burst activation by the peptides was dependent on extracellular Ca2+ whereas the lipids required both Mg2+ and Ca2+. The results indicate that mobilization of intracellular Ca2+ is insufficient by itself to induce respiratory burst activation and that the intracellular pathways leading to activation differ depending on the nature of the stimulus.

Animals↗
PubMed1994

A high potency nonformylated peptide agonist for the phagocyte N-formylpeptide chemotactic receptor.

Analysis of synthetic tri- and tetrapeptides has previously indicated that N-formylation is required for high biological activity when they react with the phagocyte N-formylpeptide receptor, suggesting that the natural ligand for the receptor is from bacterial and/or mitochondrial sources. To explore this requirement further, we synthesized the pentapeptide methionyl-norleucyl-leucyl-phenylalanyl-phenylalanine (MNleLFF) and studied the effects of different NH2-terminal modifications on its activity. N-formyl-MNleLFF induced transient alterations of [Ca2+]i and superoxide production in human neutrophils with 10- and 100-fold greater potency, respectively, than the proto-type N-formylpeptide, N-formylmethionyl-leucyl-phenylalanine (fMLF). Surprisingly, N-acetyl-MNleLFF was a potent as N-formyl-MNleLFF. Moreover, the unacylated counterpart H-MNleLFF was also highly active, having an EC50 for calcium mobilization of 10 nM, and for respiratory burst activation of 100 nM. All three pentapeptides could completely desensitize calcium transients elicited by stimulation of neutrophils with fMLF, whereas the neutrophil chemoattractants C5a and interleukin 8 only weakly affected fMLF-induced transients, suggesting that they activate neutrophils via the same receptor as fMLF. Finally, all three pentapeptides activated the recombinant human N-formylpeptide receptor expressed in frog oocytes, but did not effectively activate related phagocyte receptors. These data broaden the potential sources of natural ligands for the N-formyl-peptide receptor from N-formylated bacterial and mitochondrial products to other nonformylated endogenous peptides.

Amino Acid Sequence↗
PubMed1985

Effect of captopril on the vascular permeability changes induced by C5a, histamine and compound 48/80.

The modulating effect of captopril, an inhibitor of angiotensin-converting enzyme, on the vascular permeability changes induced by intradermal injections of C5a, histamine and compound 48/80, was evaluated in guinea-pigs. Cutaneous vascular permeability changes were measured by the extravasation of intravenously injected 125I-bovine serum albumin. Intraperitoneal injection of 12.5, 25, or 50 mg/kg of captopril 30 min prior to the injection of C5a (10(-11) mol) significantly enhanced the increase in vascular permeability induced by this agent (P less than 0.02). This may be explained by the known property of captopril as an inhibitor of carboxypeptidase. No effect was observed when captopril was injected either 2 or 4 h before the injection of C5a. In contrast, the same doses of captopril, when injected intraperitoneally 2 h before the injection of histamine (10(-8) mol) or compound 48/80 (10 micrograms), significantly suppressed the increase in vascular permeability induced by these agents (P less than 0.02). This suppressive effect occurred in a captopril dose-dependent manner. The ability of captopril to modulate the vascular permeability response induced by vasoactive agents indicates that it is a potentially useful tool to dissect the relative roles of mediators involved in inflammatory processes.

Animals↗