Interactions between synthesis of platelet-activating factor and leukotriene B4 in isolated human polymorphonuclear leukocytes

The aim of the present work was to study interactions between the synthesis of platelet-activating factor (PAF) and leukotriene B₄ (LTB₄) in human polymorphonuclear leukocytes (PMNs) in vitro. PAF, at nanomolar concentrations, stimulated calcium ionophore A23187-activated PMNs to release LTB₄ and 5-hydroxyeicosatetraenoic acid (5-HETE). This seems to be a receptor-mediated process as it was blocked by a PAF receptor antagonist WEB 2086 (IC₅₀ 6.6±3.9M). Moreover, LTB₄ stimulated the formation of PAF in activated PMNs. WEB 2086 did not, however, alter PMN migration towards either LTB₄ or the chemotactic peptide FMLP. This suggests that the enhancement of PAF synthesis in response to LTB₄ is a concomitant event rather than a mediating process in LTB₄-induced chemotactic movement of PMNs. These effects are implicated in the complex network of interactions between inflammatory mediators that results accumulation and activation of PMNs in the exacerbation of inflammatory processes.     

PAF and LTB4 biosynthesis in the human neutrophil: Effects of putative inhibitors of phospholipase A2 and specific inhibitors of 5-lipoxygenase

The effect of several putative phospholipase A₂ (PLA₂) inhibitors on [³H]-acetate incorporation into platelet-activating factor (PAF) upon calcium ionophore A23187 stimulation of purified human neutrophils (PMN) were evaluatedin vitro. PLA₂ inhibitors such asp-bromophenacyl bromide (pBPB), ellagic acid, aristolochic acid and gossypol were without effect or only weakly inhibited PAF biosynthesis. Luffariellolide, a potent PLA₂ inhibitor isolated from the marine spongeLuffariella sp., dose-dependently inhibited PAF production (IC₅₀=5 M). Due to the relationship between PAF and LTB₄ biosynthesis, the effect of inhibiting LTB₄ production on PAF biosynthesis was investigated. At concentrations which reduce LTB₄ production by >95%, Wy-50,295 tromethamine and A-64,077, specific 5-lipoxygenase (5-LO) inhibitors, did not significantly effect PAF production. In contrast, L-663,536, the 5-LO translocation inhibitor, was a potent inhibitor of PAF production (IC₅₀=1 M). This activity of L-663, 536 may contribute to its pharmacological profile at higher doses. These data also suggest that PAF biosynthesis in human PMNs is not dependent on the formation or continued presence of leukotrienes.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.     

PAF- and LTB4-induced migration of eosinophils and neutrophils isolated from horses with allergic skin disease

Platelet activating factor (PAF) and leukotriene (LT)B₄ cause thein vitro migration of equine granulocytes. In this study eosinophils and neutrophils have been prepared from horses showing clinical signs of the allergic skin disease sweet itch. Migratory responses have been compared to those of cells from normal animals. Cell migration was measured in a 48-well microchemotaxis chamber. PAF and LTB₄ caused the migration of neutrophils and eosinophils from both allergic and normal horses. However, responses of eosinophils, but not neutrophils, from allergic horses were significantly reduced (p<0.05,n=5). No difference was observed between the random movement of neutrophils or eosinophils from normal and allergic horses. These results suggest that the migratory responses to PAF and LTB₄ of eosinophils from allergic horses are down-regulated.

     

Human colonic (Na++K+)-ATPase and specific ouabain binding are not influenced by lipoxygenase products or superoxide radicals

Summary The effects of lipoxygenase products (5-, 12-, 15-HETE, LTB₄) and superoxide radicals on human colonic (Na⁺+K⁺)-ATPase and specific ouabain binding were measured. No significant inhibition in concentrations up to 3 × 10^–5 M was observed. The results are discussed with regard to a possible role of lipoxygenase products and radicals in the pathogenesis of water and electrolyte disturbances in various diarrheal states including inflammatory bowel disease.Abbrevations IBD Inflammatory bowel disease - HETE Hydroxytetraenoic acid - LTB₄ Leukotriene B₄ Supported by DFG (Er 65/4-4)     

Release of granule proteins from human eosinophils stimulated with mast-cell mediators

Background It has been suggested that mast cells and eosinophils are major effector cells in the pathogenesis of allergic diseases. However, the interaction of these cells has not been thoroughly elucidated. We examined eosinophil cationic protein (ECP) release and cytosolic free calcium concentration ([Ca²⁺]) in human eosinophils induced by the major mast-cell mediators including cytokines. Methods Eosinophils from healthy donors were stimulated with the major mast-cell mediators for 20 min after preincubation with cytochalasin B for 10 min. ECP in supernatants was measured by radioimmunoassay. Moreover, t o examine changes of [Ca²⁺]i in eosinophils, Fura-2-loaded eosinophils were monitored for fluorescence changes after stimulus addition. Results Of the tested mediators (prostaglandin [PG]D₂, leukotriene (LT)B₄, platelet-activating factor (PAF), histamine, LTQ, and eosinophil chemotactic factor of anaphylaxis [ECF-A]), LTB₄ and PAF induced ECP release from eosinophils. Any cytokines produced by human mast cells, i.e., interleukin (IL)-4, IL-5, IL-8, tumor necrosis factor (TNF), or granulocyte-macrophage colony-stimulating factor (GM-CSF), did not induce ECP release in our system. ECP release triggered with LTB₄ and PAF occurred at concentrations of 10^?8-10^?6 M concentration-dependently. LTB4 and PAF also elicited a rise in [Ca²⁺]_i in eosinophils. Neither PGDj, histamine, nor LTC₄ induced ECP release, although they increased cytosolic calcium in eosinophils. Conclusions Of mast-cell mediators, LTB₄ and PAF induced eosinophil degranulation. The contribution of LTB4 and PAF from mast cells to eosinophil degranulation may be important in the pathogenesis of allergic inflammatory diseases.     

Decreased histamine content and metabolism in mammary cancer tissue from C3H mice

Interleukin-8 (IL-8), is a potent activator of polymorphonuclear leukocyte (PMN) functions including chemotaxis, superoxide anion production, and enzyme release and it is also chemotactic for lymphocytes. Additionally, it has recently been shown that IL-8 stimulates the formation of 5-lipoxygenase (LO) products of arachidonic acid (AA) by human PMNs. The purpose of the present study was to determine whether IL-8 also might affect the formation of 15-LO products from AA. Purified PMNs in phosphate buffered saline were preincubated with and without exogenous AA (10^–5–10^–4 M) for 10 min. Then IL-8 was added in biologically relevant concentrations ranging from 0.1 to 100 ng/ml and incubation was carried out for 5 min at 37°C. Lipids were then extracted from supernatants, and eicosanoids were determined by quantitative RP-HPLC. Compared with unstimulated cells, IL-8 resulted in a dose dependent increase in both LTB₄ and 15-HETE (up to 125% and 40% at 100 ng/ml, respectively). This increase in eicosanoid formation required the presence of exogenous AA. These results indicate that IL-8 is both a potent stimulator of 5-LO activity and of 15-LO activity. LTB₄ can induce both inflammation and contribute to hyperproliferation in the skin. 15-HETE in contrast has the ability to inhibit the effects induced by LTB₄. Because IL-8 is able to stimulate both LTB₄ and 15-HETE formation, the effect of IL-8 as a putative regulator of inflammatory processes may be dependent on the relative stimulation of 5-LO and 15-LO.Part of this work has been presented at the Annual Meeting of the European Society for Dermatological Research (E.S.D.R.), Turin, Italy, June, 1990.     

Specific binding of 1-[3H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl choline (platelet-activating factor,PAF) by human polymorphonuclear neutrophils

1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) is a potent activator of polymorphonuclear neutrophil (PMN) aggregation, exocytosis and chemotaxis. Specific desensitization of PMN to PAF suggests a receptor-mediated interaction. The binding of 1-[³H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (³H-PAF) to human PMN and platelets was analysed and compared. Binding was saturable at 0.6 nM and 0.1 nM for 2×10⁶ PMN and 5×10⁷ platelets, respectively. The time course of binding at 22°C and 37°C for both cell types reached the plateau at 2 min. The averageK _d was 45.0±1.7 nM (mean ±1 SD of 4 experiments) for PMN (27.391±1381 sites for PMN) and 20.1±6.3 nM (4 experiments) for platelets (1577±461 sites for platelets). The Scatchard plot analysis revealed two distinct binding sites both on PMN and platelets: a high affinity binding site and a non-saturable binding site.This work was supported by C.N.R. Rome grant no. 81.00089.04.     

Elevated Expressions of 15-Lipoxygenase and Lipoxin A4 in Children with Acute Poststreptococcal Glomerulonephritis

Anti-inflammatory effects of the 15-lipoxygenase (15-LO) derivatives lipoxin A₄ (LXA₄) and 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) have been documented in many experimental models of acute inflammation. However, the expression levels of 15-LO and its products in human renal diseases remain unknown. This study investigated the expression levels of LXA₄, leukotriene B₄ (LTB₄), and 15-LO in leukocytes and glomeruli obtained from 22 children with acute poststreptococcal glomerulonephritis (APSGN), and determined the modulatory effects of both 15-S-HETE and LXA₄ on LTB₄ synthesis in leukocytes and LTB₄-evoked chemotaxis of polymorphonuclear leukocytes (PMNs) obtained from children during the first 3 days after onset of APSGN. Expression levels of both LXA₄ and 15-LO in leukocytes and glomeruli were up-regulated during the acute phase of disease, further peaking between days 10 and 14, and remained increased after 6 to 8 weeks of APSGN onset. In contrast, blood and urinary levels of LTB₄ as well as the number of glomerular PMNs peaked during the acute phase of disease and then decreased during the resolution phase. Administration of both 15-S-HETE and LXA₄ in vitro inhibited LTB₄-induced chemotaxis of PMNs and production of LTB₄ from leukocytes obtained from patients with APSGN. The current study provides further support for an anti-inflammatory role for 15-LO products in human nephritis through both antagonism and inhibition of leukotriene synthesis and its biological activity.     

Preferential human eosinophil chemotactic activity of the platelet-activating factor (PAF) 1-0-hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC)

The chemotactic responses of human blood neutrophils and of eosinophils of two different densities, which were resolved by centrifugation on gradients of polyvinylpyrrolidone-coated silica gel (Percoll), were quantified in modified Boyden micropore filter chambers using highly purified synthetic 1-0-hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC or PAFacether) and leukotriene B₄ (LTB₄) as stimuli. Maximal chemotactic responses of the densest eosinophils, less dense eosinophils, and neutrophils were evoked by 1 nM, 100 nM, and 1 µM PAFacether, respectively, and by 30–100, 30–100, and 10 nM LTB₄. The magnitude of the maximal chemotactic response to PAFacether of the densest eosinophils was significantly greater than that of neutrophils. The eosinophil responses to PAFacether were chemotactic, as distinguished from chemokinetic, and were not influenced by the percentage of contaminating neutrophils. PAFacether is a more potent chemotactic factor for eosinophils than neutrophils and selectively attracts the densest population of human blood eosinophils.     

Potentiation of histamine release from human leucocytes by PAF

Studies on the effects of PAF on histamine release from human leucocytes have yielded conflicting results. We therefore investigated the effects of PAF on leucocytic histamine release (HR) focusing on direct as well as on modulating effects. Peripheral blood leucocytes of normal and atopic subjects were incubated with PAF, anti-IgE and FMP for 30 min at 37°C, and histamine was measured fluorometrically. Unlike anti-IgE (1/2000) and FMP (10^–5 M) which caused histamine release (HR) of 34±7% and 31±8%, respectively, PAF by itself (10^–11–10^–5 M) failed to induce any significant HR from human leucocytes (<3%) in normal (n=14) and atopic subjects (n=6). Nevertheless, in normals as well as atopics, PAF, but not lyso-PAF, enhanced anti-IgE (1/2000) and FMP (10^–5 M)-induced HR in a concentration-related manner. Maximal potentiation of histamine release caused by FMP and anti-IgE was achieved with PAF (10^–7) (mean±SEM: 26±5%,n=5,p<0.01) and PAF (10^–5) (mean±SEM: 20±7%,n=7,p<0.05), respectively. This potentiation was suppressed by WEB2086 (10^–5 M), a specific PAF antagonist. The time course of the enhancing effect produced by PAF was dependent on the type of secretagogue. The enhancement was nearly maximal when PAF and FMP were added simultaneously to the leucocytes, whereas a preincubation of 20 min with PAF was required to get maximal enhancement with anti-IgE. The enhancing activity of PAF on HR induced by both anti-IgE and FMP was reversed by washing the cells after preincubation. While PAF enhancement of FMP-induced HR persisted on mononuclear cell fraction containing basophils, that of anti-IgE-induced HR was considerably reduced under these conditions. Further, preincubation of mononuclear cells containing basophils with the supernatant of granulocytes stimulated by PAF resulted in a stronger potentiation of immunological histamine release than that afforded by the sole PAF on the mononuclear cell fraction. We conclude that PAF appears to be more an enhancing rather than a triggering agent for the basophil degranulation. The mechanism of this enhancing effect is different according to the secretagogue and probably involves an intermediate mediator regarding immunologically induced histamine release.     

Leukotriene C4 formation by purified human eosinophils can be induced by arachidonic acid in the absence of calcium-inophore A23187

Addition of arachidonic acid (50 M) to purified human eosinophils leads to the formation of considerable amounts of LTC₄ ((11.3±1.3)×10⁶ molecules/cell, mean±SEM,n=10), 15-HETE ((412±142)×10⁶ molecules/cell, mean±SEM,n=3) and 15-series leukotrienes ((35±15)×10⁶ molecules/cell, mean±SEM,n=3). The ratio of the amounts of LTC₄ and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. Platelet activating factor (1 M) was able to enhance significantly the production of LTC₄ but not that of 15-lipoxygenase products. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca²⁺ concentration, this explains, at least partly, its ability to induce the Ca²⁺-dependent formation of LTC₄.     

Antiinflammatory effects of second-generation leukotriene B4 receptor antagonist,SC-53228: Impact upon Leukotriene B4- and 12(R)-HETE- mediated events

Leukotriene B₄ (LTB₄) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] are proinflammatory products of arachidonic acid metabolism that have been implicated as mediators in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB₄ and 12(R)-HETE elicit a dose-dependent migration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxyl]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid}, a first-generation LTB₄ receptor antagonist, inhibited the chemotactic actions of LTB₄ when given orally with an ED₅₀ value of 1.7 mg/kg. The second-generation LTB₄ receptor antagonist, SC-53228 [(+)-(S)-7-(3-{2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)carbonyl]phenoxy} propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid], inhibited LTB₄-induced chemotaxis when given intragastrically with an ED₅₀ value of 0.07 mg/kg. Furthermore, SC-53228 inhibited 12(R)-HETE-induced granulocyte chemotaxis with an oral ED₅₀ value of 5.8 mg/kg. When dosed orally over a range of 0.03–100 mg/kg, SC-53228 gaveC _max plasma concentrations of 0.015–41.1g/ml. SC-53228 inhibited LTB₄-primed membrane depolarization of human neutrophils with an IC₅₀ value of 34 nM. As a potent LTB₄ receptor antagonist, SC-53228 may well have application in the medical management of disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease, contact dermatitis, and psoriasis, in which LTB₄ and/or 12(R)-HETE are implicated as inflammatory mediators.     

Synthesis and release of platelet-activating factor and eicosanoids in human endothelial cells induced by different agonists

Production of platelet-activating factor (PAF) and eicosanoids by human umbilical vein endothelial cells (HUVEC) after stimulation with different agonists has been studied. Significant amounts of PAF were measured in the cellular fraction after treatment with thrombin (2 NIHu/ml), calcium ionophore A23187 (2 M) and histamine (100 M) (110.3±14.3, 80.7±19.2 and 119.2±22.4 pg/10⁵ cells, respectively). Only thrombin caused a partial release of PAF into the supernatant. IL-1 (0.1 nM), TNF (1 nM), arachidonic acid (10 M) and endothelin (0.1 M) were not able to induce any PAF synthesis. High levels of 6-keto-PGF₁ were found after stimulation with thrombin and calcium ionophore A23187 (8641±2575 and 6715±3340 pg/10⁵ cells, respectively). Cytokines IL-1 and TNF were also able to stimulate PGI₂ synthesis, although to a lesser extent. PGE₂ production increased after treatment with thrombin and calcium ionophore A23187 three- and two-fold, respectively. Our results confirm that stimulated HUVEC are able to synthesize PAF and eicosanoids simultaneously, the relative amounts depending upon the agonist used. None of the agonists studied showed any significant effect on 15-HETE production.     

Pharmacological profile of a novel,orally active leukotriene B4 antagonist,SM-15178

SM-15178, a new hydroxyacetophenone derivative, was evaluated to determine its antiinflammatory activity and antagonistic activity against leukotriene B₄ (LTB₄). SM-15178 inhibited [³H]LTB₄ binding to its receptors on human neutrophils (IC₅₀=0.30M). It inhibited LTB₄-induced chemotaxis of human neutrophils (IC₅₀ =0.72M) with little inhibitory effect against C5a or FMLP-induced chemotaxis at concentrations up to 30M. The compound alone did not cause human neutrophil chemotaxis at concentrations up to 10M. LTB₄-induced chemotaxis of mouse and rat neutrophils and guinea pig eosinophils was also inhibited by the compound, with IC₅₀ values of 0.55, 0.52, and 0.58 M, respectively. In an in vivo study, SM-15178, given orally, significantly prevented LTB₄-induced transient leukopenia. It also suppressed LTB₄-induced bronchoconstriction in the guinea pig almost completely when given orally at a dose of 40 mg/kg. Furthermore, orally given SM-15178 suppressed arachidonic acid-induced neutrophil infiltration in mouse ears and Arthus reaction-induced paw edema in the mouse in a dose-dependent manner. These results suggest that SM-15178 is a selective and orally active LTB₄ antagonist and that it might be effective for the treatment of some types of inflammatory diseases.     

Agonist-induced calcium flux,phosphoinositide metabolism,aggregation and enzyme secretion in human neutrophils

Formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and leukotriene B₄ (LTB₄) are potent activators of human neutrophils. Using human neutrophils prelabelled with the fluorescent indicator dye, Quin 2, or with [³²P]-orthophosphate, we examined the effects of these stimuli on intracellular free calcium concentration, [Ca²⁺]i, and, on various indices of phosphoinositide metabolism, including [³²P]-phosphatidic acid (PtdA) formation. The concentration-dependence of the observed changes in [Ca²⁺]i or [³²P]-PtdA were then compared to stimulus-induced aggregation and enzyme release (-N-acetylglucosaminidase (NAG) and lysozyme).FMLP, PAF and LTB₄ caused a concentration-dependent elevation of [Ca²⁺]i, aggregation and enzyme release. However, unlike FMLP and PAF, LTB₄ (2.5 M) did not cause significant formation of [³²P]-PtdA. The concentration response curves for agonist-induced elevation of [Ca²⁺]i lie to the left of those for aggregation and enzyme release. FMLP and PAF also caused an elevation of [Ca²⁺]i at concentrations lower than those required to elicit [³²P]-PtdA formation.These observations suggest that [Ca²⁺]i elevationper se cannot mediate human neutrophil functional responses to FMLP, PAF and LTB₄. Consequently there may exist other mediator(s) that act in concert with [Ca²⁺]i or are triggered by [Ca²⁺]i elevation to promote human neutrophil activation. Both the elevation of [Ca²⁺]i and the formation of these putative mediator(s) in response to LTB₄ apparently occur independently of inositol phospholipid hydrolysis.     

Recombinant interleukin-1β interacts with high-affinity receptors to activate neutrophil leukotriene B4 synthesis

The capacity of interleukin-1 (IL-1) to function as a neutrophil (PMN) activator has been the subject of controversy. While IL-1 purified from mononuclear cell supematants induced PMN activation, these observations have not been confirmed with recombinant IL-1. To document a cellular basis for a putative PMN-IL-1 interaction, we investigated the presence of IL-1 receptors on the PMN. Using an [³⁵S]methionine-labeled preparation, specific binding of IL-1 to PMNs was demonstrated. Through Scatchard analysis PMNs were calculated to have a mean of 469 ±337 receptors per PMN with an affinity(K _d) of 0.32±0.09 nM. As IL-1 frequently activates arachidonic acid metabolism in other cell types, we investigated eicosanoid production as a putative consequence of the IL-1-PMN interaction. HPLC analysis of extracted supernatants of IL-1-treated PMNs demonstrated the release of leukotriene B₄ (LTB₄), its oxidative products, and 5-hydroxyeicosatetraenoic acid (5-HETE). Production of LTB₄ was quantified using a commercial RIA. LTB₄ secretion increased from 17.2±1.1 to 96.7+-16.4 ng, also with 10.0 ng of IL-1. In time-course studies, it was shown that maximal eicosanoid secretion required a 30-min incubation with IL-1. These observations confirm the proinflammatory activity of IL-1 on neutrophils and resolve the controversy concerning a direct effect of IL-1 on neutrophils. In conclusion, recombinant IL-1 interacts with neutrophils through the presence on the PMN of a high-affinity receptor and results in the secretion of arachidonate metabolites.This work was presented in part at the American Federation for Clinical Research National Meeting, Washington, D.C., May 1988 (Clin. Res. 36:436A). This work was supported by USPHS NIH grants A125173 (L. J. R.), HL33961 (L. J. R.), and A124843 (L. B.). L. J. R. is the recipient of an Allergic Diseases Academic Award (A100595). L. B. is the recipient of a Burroughs Wellcome Developing Investigator Award.     

Neutrophil migration,oxidative metabolism,and adhesion in elderly and young subjects

Objective. To evaluate neutrophil functions in the elderly.Methods. We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects.Results. No difference was found in PMN migration in vivo (62.9±21.3×10⁶ and 65.5±9.1×10⁶ PMN/cm²/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6±2.7 and 9.3±3.3 nMOLES O ₂ ^– /10⁶ PMN respectively, p<0.0001) and by exudate PMNs (13.6±4.3 and 19.4±6 nMOLES O _2– ^{10 6} PMNs respectively, p<0.005).Conclusion. Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.     

Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells

The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB₄, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1M), in the presence of calcium. These cells produce 12.94±1.8 ng/10⁶ cells of LTC₄ and 3.8±0.4 ng/10⁶ cells of LTB₄. LTC₄ and LTB₄ are also synthesized in the undifferentiated cell when stimulated with 1M A23187 and 1 mM Ca²⁺, but in much smaller quantities, i.e., 1.91±0.42 ng/10⁶ cells of LTC₄ and 0.41 ng±0.06/10⁶ cells of LTB₄. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC₄ and LTB₄ in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5±0.2 ng of LTC₄ and 1.45±0.2 ng of LTB₄ per 10⁶ cells. The observation that DMSO-differentiated HL60 cells produce LTC₄, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.     

Effect of BRL-35135 on LTB4-induced guinea pig eosinophil chemotaxis

The effect of an atypical -adrenoceptor agonist, BRL-35135 on leukotriene B₄-induced-guinea pig eosinophil chemotaxis was studied. BRL-35135 and SC-41930 (leukotriene B₄-antagonist) inhibited the chemotaxis in a concentration-dependent manner (IC₅₀=9.0×10^–6 and 2.6×10^–7 M, respectively). However, isoproterenol, fenoterol and another atypical -agonist, BRL-37344 had no effects. The inhibitory effect of BRL-35135 was not affected by (±)-propranolol (10^–4 M). In contrast, the nonselective -adrenoceptor antagonist, (–)-alprenolol (10^–4 M) dextrally shifted the inhibitory curve of BRL-35135. The response to BRL-35135 was antagonized in a competitive manner by (–)-alprenolol, with the slope of the Schild plot close to unity, and a pA ₂ value of 5.62. These findings suggest that guinea pig eosinophils possess an atypical receptor, which differs from either ₁, ₂ or atypical -adrenoceptor on guinea pig ileum, and through which eosinophil chemotaxis can be modulated by BRL-35135.