Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Study of chemotactic activity developed by neutrophils from rheumatoid arthritis patients

Neutrophils are the predominant cells accumulated in the synovial fluid (SF) of rheumatoid arthritis (RA) patients. Accumulation of neutrophils may be regarded as a possible way by which neutrophils exert cytotoxic functions. The aim of the present study was to analyze the chemotactic response of neutrophils (PMNs) isolated from the peripheral blood or SF of patients with RA by performing the chemotaxis assay, in which N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as chemotactic agent. Our results showed that FMLP induced response of peripheral blood neutrophils from 12 patients with RA was similar with the response of 15 healthy controls. A decreased chemotactic response to FMLP was, however, observed in PMNs isolated from the SF of RA patients as comlipared with peripheral blood cells. Therefore, this defective chemotactic ability of neutrophil, was inversely correlated with the number of infiltrating cells in SF. These results indicate that chemotactic ability of neutrophils may be reduced after migration to the SF. Because PMNs chemotaxis in vivo has likely occurred in the presence of serum or SF, we tried to simulate the same conditions in vitro. Therefore, we analyzed the effect of serum or SF on the RA-PMNs chemotaxis. Heat-inactivated serum produced a marked reduction of chemotactic activity developed by PMNs isolated from patients with RA. Notably, a significant increase of chemotactic activity was observed when FMLP and serum stimuli were used together, as compared with the same stimuli used alone. The results suggested that complement activation might interfere with neutrophils chemotaxis. SF amplifies the chemotactic activity of PMNs isolated from peripheral blood of RA patients, but does not affect the chemotaxis developed by PMNs isolated from SF. The data might suggest that several components of SF (IL-8, leukotrien B4, thrombin, platelet-activating factor, etc.) could serve as a potent stimulus for recruitment of neutrophils from periphery into the RA joint. In conclusion, serum or SF components seem to contribute to chemotaxis of neutrophils and play a role in differential killing of PMNs and incidence of infection.     

Mechanisms of pulmonary vasoconstriction induced by chemotactic peptide FMLP in isolated rabbit lungs.

The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) has been shown to constrict both bronchial and coronary vascular smooth muscle through the action of cyclooxygenase or lipoxygenase products. We observed that intravenous FMLP increased pulmonary vascular resistance (PVR) in isolated buffer-perfused rabbit lungs. FMLP increased the PVR (primarily in the middle segment of the pulmonary vascular bed) at concentrations greater than or equal to 10(-7) M. Maximum vasoconstriction occurred at 5 min and then slowly declined to a level that remained above baseline at 30 min. Tachyphylaxis was observed in response to FMLP. When polymorphonuclear leukocytes (PMNs) were added to the perfusate, FMLP caused a greater increase in PVR. PMN depletion with dimethylmyleran significantly reduced the PVR response to FMLP. Pretreatment with two dissimilar cyclooxygenase inhibitors, meclofenamate and ibuprofen, and the leukotriene synthesis blocker MK 886 had no effect on the FMLP-induced vasoconstriction. However, the reactive oxygen species scavenger catalase significantly reduced the vasoconstriction. These results suggest that FMLP induces vasoconstriction that is dependent on PMNs and mediated by reactive oxygen species with no involvement of cyclooxygenase or lipoxygenase products.     

Deactivation of guinea pig pulmonary alveolar macrophage responses to N-formyl-methionyl-leucyl-phenylalanine: chemotaxis, superoxide generation, and binding

Incubation of pulmonary alveolar macrophages (PAM) with the synthetic chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in deactivation of PAM chemotaxis. The chemotactic response to 10(-8) M FMLP was inhibited 85% after 30 min of preincubation with 10(-6) M FMLP and 48% by 10(-8) M FMLP. Only the higher dose of FMLP (10(-6) M) caused deactivation of the chemotactic response to C5a (20%). Preincubation with partially purified C5a at a concentration of 100 microliter/ml produced a 32% inhibition of the PAM response to 10(-8) M FMLP. In contrast, preincubation with FMLP had no significant effect on superoxide generation, either at baseline or after stimulation. Levels of intracellular cyclic adenosine-3',5'-monophosphate (cAMP) increased in response to PGE1 in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, but FMLP failed to induce a change in cAMP levels. Studies of 3H-FMLP binding were consistent with two populations of membrane receptors with different affinities. Preincubation of PAM with FMLP did not result in a reduction of maximal binding. We conclude that FMLP induces deactivation of PAM chemotaxis, but cross-deactivation occurs only after high dose treatment. Unlike the PMN, macrophage chemotactic activation is not accompanied by an elevation in cAMP levels. These observations suggest that PAM chemotaxis is influenced by prior exposure to chemotactic stimuli, but other aspects of the PAM response diverge from that of PMN. The mechanism of deactivation of PAM does not appear to result from a shift in the dose-response curve or decreased availability of membrane receptors, but may involve uncoupling of post-receptor cellular responses.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Formyl peptide-induced contraction of human airways in vitro

Formylmethionylleucylphenylalanine (FMLP) is a synthetic analogue of bacterial chemotactic factors. We studied the contraction of human airway tissue in vitro by FMLP. FMLP induced a concentration-dependent contraction of all bronchial spiral strips studied (n = 45). The maximum tension generated in response to FMLP was 86.6 +/- 7.0% (SE) of the maximum response to histamine. The contraction was not reduced by the histamine H1-receptor antagonist pyrilamine, the cyclooxygenase and lipoxygenase inhibitors indomethacin and BW755C, the muscarinic antagonist atropine, or capsaicin which depletes stores of substance P. The concentration-response curve was shifted to the right by the polypeptide antagonist N-t-BOC-phenylalanylleucylphenylalanylleucylphenylalanine and the leukotriene antagonist FPL 55712. When 2 successive FMLP concentration-response curves were performed the maximum response was significantly reduced from 114.8 +/- 9.1% of the histamine maximum to 39.3 +/- 6.1%. The contraction of human airways in vitro by an agent that is structurally and functionally similar to chemotactic peptides released from bacteria may have important implications in airway disease.     

The metabolism of arachidonic and eicosapentaenoic acids in human neutrophils stimulated by A23187 and FMLP

A23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5-lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187-stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP-stimulated neutrophils.     

No Role for Phospholipase A2 and Protein Kinase C in the Potentiation by α-Adrenoceptors of β-Adrenoceptor-Mediated Cyclic AMP Formation in Rat Brain

This study was undertaken to examine the role of phospholipase A2 and protein kinase C in the potentiation of beta-adrenoceptor-mediated cyclic AMP formation by alpha-adrenoceptors in rat cerebral cortical slices. Inhibition of arachidonic acid metabolism by a range of cyclooxygenase and lipoxygenase inhibitors had no effect on the potentiation of isoprenaline-stimulated cyclic AMP. Conversely, stimulation of leukotriene formation had no effect on the response to isoprenaline. The phospholipase A2 activator, melittin, stimulated cyclic AMP and potentiated the effect of isoprenaline, but these responses were not influenced by cyclooxygenase or lipoxygenase inhibitors. Indomethacin was also ineffective against the potentiation of vasoactive intestinal peptide-stimulated cyclic AMP by noradrenaline. Phorbol ester potentiated the cyclic AMP response to isoprenaline, and this potentiation was antagonized by three different putative protein kinase C inhibitors. However, the same inhibitors did not affect the alpha-adrenoceptor-stimulated enhancement of the response to isoprenaline. We have found no evidence, therefore, to support the suggestion that arachidonic acid and its metabolites and/or protein kinase C mediate the alpha-adrenoceptor modulation of beta-adrenoceptor function.     

A formyl peptide contracts guinea pig lung: role of arachidonic acid metabolites

We studied the role of cyclooxygenase and lipoxygenase products of arachidonic acid metabolism in mediating N-formyl-methionyl-leucyl-phenylalanine- (FMLP) induced contractions of guinea pig lung parenchymal strips. The cyclooxygenase inhibitors indomethacin (10(-5) M) and aspirin (3 X 10(-5) to 10(-4) M), the lipoxygenase inhibitor nordihydroguaiaretic acid (10(-5) to 3 X 10(-5) M), and the combined cyclooxygenase/lipoxygenase inhibitors 1-phenyl-3-pyrazolidinone (Phenidone) (3 X 10(-5) to 3 X 10(-4) M) and BW 755C (10(-5) to 10(-4) M) each caused a decrease in the maximum force induced by FMLP (Fmax) and an increase in the concentration of FMLP required to produce 50% of Fmax (EC50). The thromboxane synthesis inhibitor imidazole (3 X 10(-3) M) also decreased Fmax. The leukotriene D4 receptor antagonist FPL 55712 (5.7 X 10(-6) to 1.9 X 10(-5) M) increased the EC50 for FMLP, whereas desensitization of lung parenchymal strips to leukotriene B4 by pretreatment with this leukotriene (10(-7) M) had no effect on FMLP-induced contraction. After exposure to FMLP (10(-6) M), guinea pig lung produced (as determined by high-performance liquid chromatography and radioimmunoassay) leukotrienes C4 and B4, thromboxane A2 (as measured by its stable degradation product thromboxane B2), and prostaglandin F2 alpha. Lung strips not exposed to FMLP showed no evidence of leukotriene production. We conclude that thromboxane A2 and leukotriene C4 generated in response to FMLP mediate a substantial fraction of the force induced by this peptide in guinea pig lung parenchymal strips.     

The involvement of calcium and protein kinase C in modulating agonist-stimulated chemotaxis of human neutrophils

Chemotaxis of human neutrophils in response to a gradient of the chemotactic peptide, fmet-leu-phe (FMLP), was measured by the under-agarose technique. The dose-response curve for FMLP was biphasic; low concentrations were stimulatory, and the response was reduced at higher concentrations. The response to FMLP was partially inhibited (about 50%) in the absence of extracellular Ca2 (EGTA added). NiCl2 dose-dependently inhibited FMLP-stimulated chemotaxis in the presence of extracellular Ca2+; the maximum inhibition obtainable with NiCl2 was similar to that with the absence of extracellular Ca2+. These results suggest that FMLP-stimulated chemotaxis is, at least partially, dependent on stimulation of Ca2+ influx. The phorbol ester, PMA, dose-dependently inhibited chemotaxis; the response was almost completely inhibited by 10 nM PMA. This result indicates that activation of protein kinase C inhibits chemotaxis. These results are discussed in relation to the physiological responses of neutrophils.     

Granulocyte-macrophage colony-stimulating factor is a chemoattractant cytokine for human neutrophils: involvement of the ribosomal p70 S6 kinase signaling pathway

GM-CSF stimulates proliferation of myeloid precursors in bone marrow and primes mature leukocytes for enhanced functionality. We demonstrate that GM-CSF is a powerful chemotactic and chemokinetic agent for human neutrophils. GM-CSF-induced chemotaxis is time dependent and is specifically neutralized with Abs directed to either the ligand itself or its receptor. Maximal chemotactic response was achieved at approximately 7 nM GM-CSF, and the EC(50) was approximately 0.9 nM. Both concentrations are similar to the effective concentrations of IL-8 and less than the effective concentrations of other neutrophil chemoattractants such as neutrophil-activating peptide-78, granulocyte chemotactic protein-2, leukotriene B(4), and FMLP. GM-CSF also acts as a chemoattractant for native cells bearing the GM-CSF receptor, such as monocytes, as well as for GM-CSF receptor-bearing myeloid cell lines, HL60 (promyelomonocyte leukemic cell line) and MPD (myeloproliferative disorder cell line), following differentiation induction. GM-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in neutrophils, which are prerequisites for cell migration. The mechanism of GM-CSF-induced chemotaxis appears to involve the cell signaling molecule, ribosomal p70 S6 kinase (p70S6K). Both p70S6K enzymatic activity and T(421)/S(424) and T(389) phosphorylation are markedly increased with GM-CSF. In addition, the p70S6K inhibitor hamartin transduced into cells as active protein, interfered with GM-CSF-dependent migration, and attenuated p70S6K phosphorylation. These data indicate that GM-CSF exhibits chemotactic functionality and suggest new avenues for the investigation of the molecular basis of chemotaxis as it relates to inflammation and tissue injury.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB4 (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB4 and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo-LTB4. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB4 was at 10(-7)M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB4 was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at c-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB4. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta6 double bond are all important structural features for chemotactic activity with delta6 stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.     

Production of cyclooxygenase products and superoxide anion by macrophages in response to chemotactic factors

Mononuclear phagocytes are known to play a key role in various phlogistic reactions by synthesizing and releasing products that may potentiate or inhibit inflammatory processes. The expression of these products appears to be dependent on the source of the macrophage population as well as the stimulus employed. We have studied superoxide anion (O-2) production as well as the generation of PGE2, PGF2 alpha, and TXB2 from resident, oil-elicited and thioglycollate-induced peritoneal macrophages in mice in the presence and absence of chemotactic peptides. Production of O-2, occurred only in elicited macrophages stimulated with high concentrations of FMLP or C5a; resident cells stimulated with either of the chemotactic peptides were completely unresponsive. Although resident peritoneal macrophages incubated with chemotactic peptides did not generate O-2, these cells did secrete significant levels of PGE2, PGF2 alpha, and TXB2 in response to C5a. FMLP had no stimulatory effect. Elicited macrophages generated increased levels of PGE2 and PGF2 alpha when incubated with C5a. However, production of TXB2 was not stimulated. FMLP was inactive in stimulating PGE2, PGF2 alpha, and TXB2 in all types of macrophages studied. These studies indicate a heterogeneity in the production of inflammatory mediators from various macrophage populations in response to chemotactic factors.     

Influence of beta-radiation on human erythrocyte surface potential under eicosanoid metabolism inhibition

The aim of the research consisted in the study of influence of beta-radiation on response of erythrocyte surface potential to inhibitors of eicosanoid metabolism enzymes (cyclo-, lipoxygenase and phospholipase A2). It was shown, that inhibitors of phospholipase A2 (quinacrine, 10-100 microM), cyclooxygenase (aspirin, 10-100 microM) and cyclo- and lipoxygenase (BW755c, 1-100 microM) lowered electrophoretic mobility (EPM) of erythrocytes by 20-30%. An analogous effect can be exerted by beta-radiation. Nonradioactive leucine in the studied concentrations cannot simulate EPM erythrocytes. Response of cellular EPM to these inhibitors depended on their concentration in the incubation medium. Addition of 14C to the incubation medium changed response of EPM of cells to inhibitors of cyclo- and lipoxygenase but not to quinacrine. However beta-radiation fully abolished the stimulative action of nonspecific activator of phospholipase A2 (Ca-independent), H2O2, on cellular EPM. Under these conditions beta-radiation enhanced EPM response to aspirin only at concentration of 100 microM. The EPM response to BW755c is reduced by irradiation at all concentrations with the exception of equal-effective one (10 microM). Data obtained evidence for modification of eicosanoid metabolism by beta-radiation, probably, as a result of phospholipase A2 inhibition, as evident from elimination by radiation of stimulated action of hydrogen peroxide on EPM. The radiation action can also affect the cyclooxygenase lipoxygenase activity ratio, this activity being mediated by cellular membrane signaling systems.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB₄ (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB₄ and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo- LTB₄. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB₄ was at 10⁻⁷M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB₄ was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at C-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB₄. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta⁶ double bond are all important structural features for chemotactic activity with delta⁶ stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies. The data suggested that they acted at the same receptor since even the less active isomers were able to desensitive the neutrophils to LTB₄.     

Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice

A water-soluble oligosaccharide, N-acetyl chitohexaose (NACOS-6) was able to enhance the protecting effect of BALB/c male mice against Listeria monocytogenes infection, when administered intraperitoneally 24 hr before the challenge with this microbe. Significant decrease in number of microbes within the peritoneal cavity, spleen, and liver from the mice of NACOS-6-administered group was not observed 1 day after the infection but 4 days after the infection. Administration of NACOS-6 enhanced the delayed-type hypersensitivity response against sheep red blood cells (SRBC) or heat-killed L. monocytogenes. Splenic T lymphocytes from mice administered NACOS-6 released macrophage activating factor (MAF). These results suggested that NACOS-6 was also able to elevate the function of cellular immunity. Macrophages treated with a combination of NACOS-6 and the culture supernatant of splenic T lymphocytes from mice administered NACOS-6, "NACOS-6 sup," were found to exert a fairly strong growth-inhibitory effect on L. monocytogenes. Interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) were able to enhance the growth-inhibitory effect on L. monocytogenes by the NACOS-6-treated macrophages.     

Thrombin-induced chemotaxis and aggregation of neutrophils

Thrombin-induced neutrophil chemotaxis and aggregation were studied using cells isolated from either human or sheep blood. Sheep neutrophils (10(8) cells/ml) exhibited maximum chemotactic migration towards 10(-8)M human alpha-thrombin, 10(-8)M gamma-thrombin (which lacks the fibrinogen site), and 10(-12)MD-Phe-Pro-Arg-CH2-alpha-thrombin (catalytically inactive thrombin). Chemotactic responses of the same magnitude were obtained with human neutrophils (10(8) cells/ml). The chemotactic responses to thrombin were comparable to those obtained with diluted (1:200 v/v) zymosan activated serum (ZAS) and 10(-11)M FMLP. Premixing of the thrombin forms with hirudin in 1:1 stoichiometric amounts abolished the chemotaxis but not chemokinesis Aggregatory responses of human and sheep neutrophils were comparable for ZAS, alpha-thrombin, and gamma-thrombin. The responses of both human and sheep neutrophils to D-Phe-Pro-Arg-CH2-alpha-thrombin were attenuated, indicating that the proteolytic site may be involved in the aggregatory response. The results suggest that thrombin-induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, since chemotaxis is a catalytically independent response whereas aggregation is an active site independent response.     

Prostaglandins and inflammation: enhancement of monocyte chemotactic responsiveness by prostaglandin E2.

The effects of prostaglandins on human monocyte chemotaxis were studied in vitro. None of the prostaglandins tested, including members of the A, B, E or F series, were chemotactic for monocytes. Prostaglandin E2 however, enhanced the chemotactic responsiveness of monocytes to complement - activated human serum by almost 200%. The enhancement of chemotaxis was not directly related to the ability of PGE2 to raise intracellular cyclic AMP levels. These studies support a role for prostaglandins as modulators of the inflammatory response.     

PGF2α synthesis in isolated cerebellar glomeruli: Effects of membrane depolarization, calcium availability and phospholipase activity

The controlling factors for PGF2 alpha production were assessed in isolated cerebellar glomeruli, since this prostaglandin has been shown to stimulate the release of neurotransmitters from the mossy fiber terminals associated with this synaptic preparation. The metabolism of PGE2 was also examined, in order to determine the specificity of any treatment effects. It was observed that K(+)-dependent membrane depolarization or the activation of voltage-sensitive Na+ channels with veratradine stimulated the production of PGF2 alpha. The syntheses of both prostanoids were dependent on available calcium and were blocked by cyclooxygenase inhibitors. The lipoxygenase inhibitor NDGA also reduced the accumulation of PGE2 and PGF2 alpha. In addition, PGF2 alpha synthesis was stimulated by the phospholipase A2 activator melittin and was reduced due to phospholipase inhibition with dibucaine. These results are consistent with a role for PGF2 alpha in the evoked release of neurotransmitter from cerebellar mossy fiber terminals.