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.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

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.     

Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis.

The CD45 Ag family is a group of high m.w. glycoproteins that are expressed on the plasma membranes of all leukocytes. CD45 has protein tyrosine phosphatase activity and appears to regulate signal transduction and lymphocyte activation by specific association with receptor molecules on T and B lymphocytes. However, little is known about CD45 function in neutrophils (PMN). In this study, PMN were incubated with CD45 mAb and tested for their chemotactic responses to four unrelated chemo-attractants: FMLP, leukotriene B4 (LTB4), recombinant human C5a (C5a), and recombinant human neutrophil-activating protein-1, recently designated IL-8. A panel of CD45 mAb including an IgM mAb, AHN-12.1, and six IgG1 mAb, AHN-12, AHN-12.2, AHN-12.3, AHN-12.4, HLe-1, and KC56(T200), were tested for their effects on PMN chemotaxis. PMN chemotaxis was evaluated with two different membrane assays; one assay quantified the total number of migrating PMN and the other assayed the leading front of migrating PMN. AHN-12.1 and KC56(T200) significantly inhibited PMN chemotaxis to LTB4 and C5a. AHN-12.1 slightly inhibited PMN chemotaxis to FMLP, but KC56(T200) did not. In contrast, AHN-12 and HLe-1 did not significantly inhibit PMN chemotaxis to any of the chemoattractants. None of the CD45 mAb inhibited PMN chemotaxis to neutrophil-activating protein-1/IL-8. None of the CD45 mAb inhibited PMN superoxide production. These results suggest that PMN CD45 epitopes may interact with LTB4 and C5a receptor-associated molecules and regulate chemotactic responses.     

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.     

Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product

Leukotriene B5 (LTB5) and three stereoisomers were prepared biosynthetically from eicosapentaenoic acid and compared with the analogous derivatives of arachidonic acid for their chemotactic and aggregating effects on human neutrophilic polymorphonuclear leukocytes. Leukotriene B4 (LTB4), LTB5, and the 6-trans-diastereoisomers of each were generated by activating polymorphonuclear leukocytes with the calcium ionophore A23187 in the presence of 14C-labeled and unlabeled arachidonic acid or 14C-labeled and unlabeled eicosapentaenoic acid, respectively. The double lipoxygenase products, (5S,12S)-6-trans-8-cis-LTB4 and (5S,12S)-6-trans-8-cis-LTB5, were generated from 5S-hydroxyeicosatetraenoic acid and racemic 5-hydroxyeicosapentaenoic acid intermediates by incubation with platelet sonicates. The products of each reaction were isolated by reverse-phase-high performance liquid chromatography and identified by their retention times relative to the appropriate totally synthetic standards, ultraviolet absorption spectra, immunoreactivity in a radioimmunoassay for LTB4, and, for all but the double lipoxygenase products, by incorporation of radiolabel from the specific polyunsaturated fatty acid source. When the concentration of LTB5 eliciting maximum chemotactic response of human polymorphonuclear leukocytes, 50 ng/ml (1.5 X 10(-7) M), and that eliciting a maximum aggregation response, 20 ng/ml (5.9 X 10(-8) M), were compared with the interpolated values of LTB4 eliciting comparable effects, the potency of LTB5 relative to LTB4 was approximately 1:8 as a chemotactic agent and about 1:20 as an aggregating agent. The double lipoxygenase products and the resolved 6-trans-diastereoisomers of the pentaene and tetraene series were about 2 logs less active as chemotactic factors than LTB4 and only (5S,12S)-6-trans-8-cis-LTB4 had even minimal aggregating activity.     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B4 (LTB4) analog, 20-trifluoromethyl LTB4 (20CF3-LTB4), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF3-LTB4 was equipotent to LTB4 as a chemoattractant (EC50, 3 nM), produced 50% of maximal activity of LTB4, and competed with [H] LTB4 for binding to intact human neutrophil LTB4 receptors. In contrast to chemotactic activity, 20CF3-LTB4 in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 microM on LTB4-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB4, 20CF3-LTB4 induced neutrophil desensitization to degranulation by LTB4. The results indicate that hydrogen atoms at C-20 of LTB4 are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF3-LTB4 is a partial agonist for chemotaxis and an antagonist for degranulation suggests that different LTB4 receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB4 can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

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.     

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.     

Polymorphonuclear leukocytes cap a derivative of wheat germ agglutinin upon stimulation with formyl peptide and C5a but not leukotriene B4

Previously, we reported that a derivative of wheat germ agglutinin (termed WGA-D) specifically inhibits human polymorphonuclear leukocyte (PMN) chemotaxis to FMLP by blocking reexpression (or recycling) of formyl peptide receptors. WGA-D (? formyl peptide receptor probe) binds to a protein on the PMN membrane that exhibits the same m.w. as the formyl peptide receptor. Since clustering (i.e., capping) of ligand-receptor complexes most likely precedes their internalization, we examined the ability of normal and stimulated PMN to cap fluoresceinated WGA-D. We found that, in contrast to capping of fluoresceinated Con A, PMN cap WGA-D in a chemotactic factor-specific fashion. Fluoresceinated WGA-D (5.0 to 20 micrograms/ml) alone did not induce either PMN shape changes (i.e., activation) or capping. Both FMLP (1 to 1000 nM) and human C5a (0.1 to 1.0 nM) induced PMN to polarize and to cap bound WGA-D, in a concentration-dependent fashion. Interestingly, leukotriene B4 (LTB4) (5.0 nM), while inducing the same degree of PMN polarization as FMLP (100 nM) and C5a (0.5 nM), failed to induce PMN to cap bound WGA-D. In contrast, FMLP (100 nM), C5a (0.5 nM), and LTB4 (5.0 nM) induced PMN to cap bound fluoresceinated Con A (10 micrograms/ml) to the same extent. The effect of suboptimal concentrations of FMLP and C5a on capping of WGA-D by PMN was additive. LTB4 did not enhance either FMLP or C5a-induced capping of WGA-D by PMN. Also, FMLP and C5a (but not LTB4) were capable of inducing both desensitization and cross-desensitization of WGA-D capping by PMN. Studies using rhodamine-labeled WGA-D and a fluoresceinated analog of FMLP revealed that both capped to the same place on the PMN membrane. Thus, the data suggest that WGA-D binds to a site on the PMN membrane that is either the FMLP receptor or very closely associated with it.     

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.     

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.     

Effect of structural modification at carbon atom 1 of leukotriene B4 on the chemotactic and metabolic response of human neutrophils

Human neutrophils biosynthesize the chemoattractant leukotriene B4 (LTB4) and metabolize LTB4 to omega oxidative products 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4). In this study, we prepared the C-1 methyl ester and N-methyl amide of LTB4 and then examined neutrophil chemotaxis and metabolism of these derivatives of LTB4. The results show that chemical modification of LTB4 at carbon atom 1 dramatically affects metabolism of the lipid molecule. The free acid form of LTB4 was taken up and metabolized by human neutrophils, while the methyl ester and N-methyl amide derivatives were poor substrates for omega oxidation. Although human neutrophils were poorly attracted to the methyl ester of LTB4, the amide derivative was a complete agonist of the neutrophil chemotactic response and displayed an ED50 for chemotaxis identical to that of LTB4. Therefore, we concluded that omega oxidation is not a requirement for the neutrophil chemotactic response induced by LTB4. These results also indicate that the N-methyl amide of LTB4 may be a useful ligand for the elucidation of molecular mechanisms operative in neutrophil chemotaxis to LTB4, since the C-1 derivative is not further metabolized. Two separate responses of human neutrophils are elicited by LTB4, resulting in both cellular activation and generation of omega oxidation products. It appears that putative receptors on the neutrophils can distinguish between LTB4 and certain derivatives that are structurally identical except for modification at the C-1 position (i.e., the methyl ester). LTB4 derivatives modified at the C-1 position do not undergo conversion to omega oxidation products by the neutrophil.     

Modulation of the heterogeneous membrane potential response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (FMLP) by leukotriene B4: evidence for cell recruitment

Individual human neutrophils (PMN) isolated by Hypaque-Ficoll gradient sedimentation, dextran sedimentation, or buffy coat preparation were assessed for the effects of leukotriene B4 (5S,12R dihydroxy 6,14-cis-8, 10 trans eicosatetraenoic acid (LTB4)-pretreatment on N-formylmethionyl-leucyl-phenylalanine (FMLP)-mediated membrane potential or oxidative responses by using flow cytometry and a lipophilic probe of membrane potential (di-pentyl-oxacarbocyanine, di-O-C(5)3), or the nitroblue tetrazolium dye (NBT) reduction test, respectively. Although exposure to LTB4 (10(-7) M) had no effect on the membrane potential of resting PMN and little effect on oxidant production, pretreating PMN with LTB4 followed by FMLP (10(-6) M) demonstrated a significant enhancement in the proportion of depolarizing PMN over that seen with FMLP alone (p = 0.0014, N = 9). This recruitment of previously unresponsive cells by LTB4 was dose and time dependent, with the maximal relative increase in the proportion of depolarizing cells occurring at LTB4 concentrations of 10(-8) to 10(-7) M and within 1 min of LTB4 addition. The recruitment effect persisted despite vigorous washing of the cells. LTB4 also increased the proportion of NBT-positive PMN in response to FMLP. Although LTB4 alone did not depolarize PMN it did induce a light scatter shift indicative of cell activation. 3H-FMLP binding studied at 0 degree C comparing buffer and LTB4-treated PMN indicated no significant change in the number or affinity of FMLP binding. The data provide evidence for the recruitment of a greater proportion of cells into a FMLP-responsive state as a mechanism for the enhanced functional response of PMN pretreated with LTB4, as well as for a dissociation of the membrane potential and light scattering responses of cells to this pro-inflammatory LT. The mechanism of recruitment remains unclear, but it most likely involves the modulation of a post-FMLP binding step.     

Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.     

Chemotactic response of human neutrophils to N-acetyl chitohexaose in vitro

N-Acetyl chitohexaose (NACOS-6) was able to display chemotactic response of human neutrophils in vitro. In order to analyze the mechanism, a series of chemotaxis studies by means of neutrophils treated with inhibitors of phospholipase A2, cyclooxygenase, or lipoxygenase to NACOS-6 was conducted. The treatment of neutrophils with inhibitors of phospholipase A2 or cyclooxygenase resulted in decrease of number of migrated cells. However, the lipoxygenase inhibitors did not exhibit the same effect. On the other hand, the treatment of neutrophils with inhibitors of phospholipase A2 or lipoxygenase resulted in decrease of chemotactic response to Formyl-Met-Leu-Phe (FMLP), although the cyclooxygenase inhibitors did not inhibit chemotaxis of neutrophils. Neutrophils added to exogenous prostaglandin E2 (PGE2) caused an enhanced chemotactic response to NACOS-6. These results indicate that the mechanism of chemotactic response to NACOS-6 was different from that of FMLP, and that the response was enhanced by PGE2 released from the neutrophils with stimulation of NACOS-6.     

Acetyl glyceryl ether phosphorylcholine (PAF-acether) and leukotriene B4-mediated neutrophil chemotaxis through an intact endothelial cell monolayer

Human endothelial cell monolayers were grown on nucleopore filters, and used to partition the two halves of a modified Boyden chamber. Human neutrophil chemotaxis through the monolayer was studied in response to leukotriene B4 and acetyl glyceryl ether phosphorylcholine (PAF-acether). Both leukotriene B4 and PAF-acether concentration-dependently stimulated neutrophil chemotaxis through intact monolayer. The biologically inactive lyso-PAF, and leukotriene C4 and D4 were inactive as chemotactic agents. Leukotriene A4 was weakly chemotactic. In the absence of chemotaxin, little penetration of the monolayer by neutrophils was observed. Agents that elevate neutrophil cyclic AMP levels inhibit both leukotriene B4 and PAF-acether-stimulated chemotaxis through the endothelial cell monolayer. The specific 5-lipoxygenase inhibitor, 6,8-de-epoxy-6,9-(phenylimino) delta 6,8-prostaglandin I1 (U-60257), inhibits PAF-acether, but not leukotriene B4-mediated chemotaxis. These data suggest that an intact 5-lipoxygenase may be required for normal PAF-acether-mediated chemotaxis, but leukotriene B4-mediated chemotaxis is independent of 5-lipoxygenase activity. This system may prove to be a useful model for the study of neutrophil-endothelial cell interactions.     

Studies of leukotriene B4-specific binding and function in rat polymorphonuclear leukocytes: absence of a chemotactic response

Rat PMN isolated from peripheral blood show a small amount of high-affinity (specific) binding of [3H]-LTB4 at nanomolar concentrations. This binding is reversible and has a stereospecificity similar to rat PMN aggregation in response to several LTB4 analogs. This population of binding sites shares many characteristics with a population of high-affinity binding sites in human PMN; however, human PMN bind a significantly greater amount of [3H]-LTB4 to a second population of specific binding sites that is not present in rat PMN. The aggregation responses of human and rat peripheral blood PMN to LTB4 are similar in magnitude and specificity, but unlike human PMN, LTB4 fails to elicit a chemotactic response in rat PMN at concentrations from 10(-10) M to 10(-6) M. Rat PMN also fail to metabolize exogenous LTB4 when compared with human PMN. These data suggest that different PMN functions, such as chemotaxis and aggregation, may involve different classes of specific receptors. The finding that rat PMN do not exhibit chemotaxis to LTB4 calls for a reevaluation of the relevance to inflammation in humans of studies of inflammation performed in rat models.     

Some newly synthesized leukotriene B4 analogs inhibit LTB4-induced lysozyme release from rat polymorphonuclear leukocytes

Antagonistic activities of newly synthesized LTB4 analogs, which possess the same olefinic geometry and the same hydroxyl group stereochemistry as natural LTB4, were investigated by studying lysozyme release from rat polymorphonuclear leukocytes (PMNLs). 14,15-Dihydro LTB4(LTB3) induced lysozyme release from PMNLs as well as LTB4, while 14,15-dehydro LTB4 did not cause lysozyme release but instead clearly inhibit LTB4-induced lysozyme release. Compounds containing aminocarbonyl groups partially retained lysozyme releasing activity. A displacement of the hydrocarbon chain at C13-20 by cyclohexenyl and beta-cyclohexylethyl groups had little effect on lysozyme release, but did strongly inhibit release induced by LTB4. These results may be useful in developing LTB4 antagonists.     

Stereochemical requirements for substrate specificity of LTB4 20-hydroxylase

LTB4 20-hydroxylase (P-450LTB) is the cytochrome P-450 in the microsomes of human polymorphonuclear leukocytes that catalyzes the omega-oxidation of leukotriene B4 (LTB4) to 20-OH LTB4. The activity of P-450LTB for LTB4 compared to isomers and analogs of LTB4 at a concentration of 0.3 microM revealed a preference of P-450LTB for both the triene bond configuration of LTB4 and for the chirality of the 5S and 12R hydroxyl groups. 15S-Hydroxyeicosatetraenoic acid, 8(R/S), 15S-dihydroxy-5-cis-9,11,13-trans-eicosatetraenoic acid, 8R,15S-dihydroxy-5,13-cis-9,11-trans-eicosatetraenoic acid, and 5S,15S-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid were each not subject to omega-oxidation, indicating a negative effect of the presence of a 15-hydroxyl group on substrate recognition. At a concentration of 1.5 microM, 12R- and 12S-hydroxyeicosatetraenoic acid were converted to their respective 20-OH derivatives at rates that were 34.2 +/- 11.6% (mean +/- S.D., n = 3) and 3.5 +/- 4.3% (mean +/- S.D., n = 4), respectively, of that of LTB4 to 20-OH LTB4, further indicating that P-450LTB can distinguish the chirality of the 12-hydroxyl group. The lower Km of LTB4 (2.0 microM), as compared to those of its 6-trans-12-epi isomer (3.8 microM) and 5-epi-LTB4 (6.6 microM) confirmed the preference of P-450LTB for the specific triene bond structure of LTB4 and its preference for the chirality of the hydroxyl groups of LTB4 within this structurally related class of molecules. At equal 1.5-microM concentrations, LTB4 completely inhibited the omega-oxidation of all other substrates and partially suppressed that of leukotriene B5, consistent with the lower Km of LTB4 and indicating that P-450LTB catalyzed the omega-oxidation of all substrates. Thus, P-450LTB is a novel cytochrome P-450 of human polymorphonuclear leukocytes with substrate recognition determined by the triene bond configuration and the chirality of the hydroxyl groups.