Effects of leukotriene C4 and D4, histamine and bradykinin on cytosolic calcium concentrations and adhesiveness of endothelial cells and neutrophils

We compared the effects of leukotrienes B₄, C₄ and D₄ (LTB₄, C₄ and D₄) in vitro, as well as of histamine and bradykinin, on adhesive interactions between cultured umbilical vein endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) and on cytosolic calcium transients, [Ca²⁺]_i. in vitro. LTB₄, but not LTC₄ or LTD₄ (at 1-100 nM), increased HUVEC adhesiveness for PMN, maximally 2·8-fold; in addition, PMN adhesion was augmented by LTB₄ (but not by LTC₄ and LTD₄) to a plastic surface. Rapid, but smaller increments of HUVEC (but not of PMN) adhesiveness were induced by histamine and bradykinin (at 10 μM). Nonetheless, LTC₄ and LTD₄ (at 100 nM) induced rapid rises of [Ca²⁺]_i in HUVEC, whereas ⁺100-fold higher concentrations were needed of histamine and bradykinin for similar rises. In PMN LTD₄ (and LTB₄) induced rapid increases of [Ca²⁺]_i, whereas no significant effect was seen with LTC₄, histamine or bradykinin. The [Ca²⁺]_i responses to LTC₄ and LTD₄ were inhibited by the peptidoleukotriene receptor blocker SKF 104,353. Thus, LTB₄ and the peptidoleukotrienes display disparate profiles as inducers of adhesion and calcium transients in PMN and HUVEC, indicating discrete differences in the stimulus response coupling for these closely related leukotrienes.     

Leukotriene B4 production by blood neutrophils in allergic rhinitis—effects of cetirizine

Background: Mucosal inflammatory processes in late phase of allergic diseases involve cytokine production, cell adhesion molecule overexpression and release of inflammatory mediators with chemotactic activity, such as leukolriene B₄ (LTB₄), We had previously observed increased production of LTB₄ by neutrophils in patients with allergic rhinitis and discussed the role of granulocyte macrophage-colony stimulating factor (GM-CSF) priming. Some antihislaminic compounds were shown to diminish the production of leukotrienes by neutrophils. Objective: In a first step, we evaluated in ex vivo and in vitro studies, the effects of cetirizine on LTB₄ production by blood neutrophils from allergic and healthy subjects. In a second step, we studied the in vitro effect of cetirizine on LTB₄ production by neutrophils from healthy subjects during GM-CSF priming of these cells. Methods: Neutrophils from both populations were purified from venous blood and LTB₄ production was measured using high performance liquid cromatography (HPLC) method. Results: In ex vivo studies, cetirizine treatment induced a decreased LTB₄ production by neutrophils in allergic rhinitis. This effect of decreased LTB₄ production was reproduced in vitro with 10^?8–10^-6 cetirizine. Nevertheless, this anti-Hl compound had no effect on neutrophil priming with GM-CSF. Conclusion: As LTB₄ is an important chemotactic factor, Cetirizine could act on inflammatory cell recruitment by inhibiting LTB₄ production by neutrophils.     

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.     

Induction of plasma exudation and inflammatory cell infiltration by leukotriene C4 and leukotriene B4 in mouse peritonitis

Leukotriene induction of the fluid and cellular phases of the inflammatory response in the mouse was evaluated. Intraperitoneal injection of leukotriene C₄ (LTC₄ 250 ng) led to dye extravasation but not polymorphonuclear leukocyte (PMN) infiltration, whereas injection of leukotriene B₄ (LTB₄ 250 ng), led to PMN infiltration but not dye extravasation. The injection of both leukotrienes did not result in synergy. LTC₄ did not appear to induce significant release or formation of chemotactic mediators, but the dye extravasation induced by LTC₄ was inhibited by the vasoactive amine antagonist cyproheptadine and not by the eicosanoid inhibitors phenidone or naproxen. The response was markedly inhibited by the cytokine and eicosanoid inhibitors SK&F 86002 and SK&F 104493. PMN infiltration induced by LTB₄ was not inhibited by SK&F 86002 or phenidone but was abrogated by colchicine treatment. LTB₄ in this model did not appear to cause release or formation of vasoactive mediators. These leukotrienes appeared to be independent, complementary, and sufficient to mount a complete inflammatory response in the mouse.     

Leukotriene B4 metabolism in human leukocytes: Fact or artefact?

The object of this study was to investigate the importance of omega oxidation in regulating leukotriene B₄ (LTB₄) levels in man. In human polymorphonuclear leukocytes metabolism of LTB₄ was rapid but was critically dependent on PMN number: greater than 1.5×10⁶ PMN/ml were required. Metabolism of LTB₄ was blocked in the presence of plasma. In whole blood and in PMN-rich rheumatoid synovial fluids no significant metabolism of LTB₄ was detected within 30 min at 37°C. We conclude that LTB₄ metabolism at inflamed sites will be regulated both by cellular content and the degree of plasma exudation. In most pathological conditions rapid exchange with the micro-vasculature will be more important than metabolism in limiting LTB₄ levels.     

Effect of exogenous leukotriene B4 (LTB4) on BALB/c mice splenocyte production of Th1 and Th2 lymphokines

The effect of exogenous leukotriene B₄ (LTB₄) on the production of cytokines typical of Th₁ (interleukin-2 and interferon-γ) and Th₂ (interleukin-4 and interleukin-10) lymphocytes was studied. Splenocytes were stimulated with concanavalin A (ConA) with or without different concentrations of LTB₄ (3 × 10⁻¹⁰ to 3 × 10⁻⁷M) for various times in the presence of BW 755C to inhibit the endogenous synthesis of eicosanoids. LTB₄ was not able to induce cytokine secretion by itself. However, LTB₄ augmented ConA spleen cell production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) from Th₁ cells and interleukin-4 (IL-4) and interleukin-10 (IL-10) from Th₂ cells more than the controls treated with ConA alone. The pre-exposition of splenocytes to LTB₄ for 3 h made these cells more sensitive to ConA in terms of IL-2 and IL-10 production than those treated with LTB₄ at the onset of the incubation and maintained during the whole culture period. The results suggest that LTB₄ may participate as a component of the signal transduction process for ConA-induced Th₁ and Th₂ cytokine production in a timedependent manner.     

The effect of leukotriene-B4 receptor antagonist,SC-41930, on acetic acid-induced colonic inflammation

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, is a potentin vitro leukotriene-B₄ (LTB₄) receptor antagonist. LTB₄ levels are elevated in colonic tissue of inflammatory bowel disease (IBD) patients which may account for the high degree of neutrophil (PMN) infiltration. The guinea pig acetic acid-induced colonic inflammation model has characteristics of IBD including PMN infiltration, edema, ulceration and necrosis. The model was used to evaluate the effect of SC-41930. SC-41930 was given orally, 30 min before and after intrarectal administration of 3% acetic acid. The PMN marker enzyme, myeloperoxidase, was measured along with histological evaluation to assess inflammation. Both parameters showed significantly less inflammation in SC-41930 treated animals with an oral ED₅₀ of 20 mg/kg. These study results with an LTB₄ receptor antagonist indicate a role for LTB₄ in colonic inflammation and that an LTB₄ receptor antagonist may be beneficial for treatment of IBD.     

Granulocyte chemotaxis in the canine trachea: Inhibition by lipid mediator antagonists and systemic inhibitors

Inflammation of the airways contributes to the multicomponent disease known as asthma. The primary cells that infiltrate the airways in response to antigen exposure are PMNs and eosinophils, cells that can release cellular components, and damage the airways. We adapted a double-ballon endotracheal tube to study the cellular response to threede novo synthesized lipid mediators (LTB₄, PAF-acether and 15 HETE) found in respiratory fluids following antigen exposure.In random repeat challenges in groups of 7 dogs using mongrel dogs at 240 min following exposure to 10^–6 M agonists, the PMN content of the perfused fluid was 870±240, 1632±883, 515±395, and 1575±214 cells/ml/5 high power fields for vehicle, LTB₄, PAF, and 15 HETE respectively. Eosinophils that infiltrated the lumen at 240 min were 162±23, 608±287, 502±23, 115±14 cells/ml/5 HPF for vehicle, LTB₄, PAF, and 15 HETE respectively. Thus LTB₄ and PAF-acether significantly (p<0.05) increased eosinophils, and LTB₄ and 15 HETE increased PMNs (p<0.05). After determining the agonist response for the 3 agonists we included 2 specific antagonists in the perfusate. The LTB₄ antagonist U-75,302 10^–5 M, and the PAF antagonist L 652,731 10^–5 M in chambers containing LTB₄ and PAF-acether respectively blocked significantly the influx of PMNs and eosinophils compared to vehicle (p<0.01). Methylprednisolone 5 mg/kg i.m.-18 hrs blocked eosinophilia to PAF and LTB₄. Oral U-78,517F a Trolox amine lazaroid, active as an inhibitor of lipid peroxidation, 30 mg/kg-18 hrs significantly blocked eosinophilia to PAF-acether and LTB₄ directed chemotaxis compared to vehicle (p<0.05) but not 15 HETE. Specificity was shown for each antagonist since the PAF and LTB₄ antagonists did not block the opposite agonist. Use of this novelin vivo chemotaxis model allows the additional advantage of studying chemotaxis in living tissue.     

Leukotriene B4 induces interleukin 5 generation from human T lymphocytes

Leukotriene B₄ (LTB₄) has been shown to affect several interleukin (IL)-linked functions of human lymphocytes. In this study, we investigated whether LTB₄ regulates IL-5 generation from human T cells and subsequently modulates eosinophil functions. Preincubation of T cells with very low concentrations (10^?12 to 10^?8 M ) of LTB₄ induced concentration-dependent IL-5 production, the event occurring after the first 24 h of cultivation. However, direct action of LTB₄ to IL-5 generation is strictly dependent on a preincubation with appropriate concentration of LTB₄. In contrast, the stereoisomer of LTB₄,5S,12S-dihydroxy-6,8,10,14-eicosatetraenoic acid showed no enhancement of IL-5 production. IL-5 released from LTB₄-primed T cells elicited sustained viability of mature eosinophils and reduced the content of eosinophil cationic protein in their crystalloid matrix by degranulation. These data suggest that LTB₄ induces bioactive IL-5 production from T cells and that the released IL-5 modulates eosinophil functions which might play a crucial role in eosinophil-linked allergic inflammatory process.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 μM). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.

     

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.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 M). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.     

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.     

Modulation by 310-1310-1310-1stimulation of IgE-mediated14C-serotonin release from rat mast cells

The formaldehyde method was used to examine the effects of clonidine and methoxamine on IgE-mediated¹⁴C-serotonin release from rat mast cellsin vitro. Clonidine (10^?11?10^?8 M) caused dose-related enhancement of the mediator release 7min after the antigen challenge yohimbine (10^?6 M) blocked this enhancement by clonidine (10^?6 M), but prazosin (10^?6 M) did not. Methoxamine did not enhance this immunological release reaction at concentrations up to 10^?6 M. PGE₁ (2×10^?8?2×10^?5 M), isoproterenol (10^?10?10^?8 M), dopamine (4×10^?8?4×10^?8 M) and aminophylline (6×10^?6?6×10^?4 M) caused dose-related inhibition of this mediator release 1 min after antigen challenge. Clonidine (10^?13?10^?12 M), but not methoxamine (10^?8?10^?6 M), reversed dose-dependently this inhibition of mast cells by PGE₁ (2×10^?6 M), isoproterenol (10^?8 M), dopamine (4×10^?6 M); yohimbine (10^?8 M) antagonized this reversing action of clonidine (10^?12 M), but prazosin (10^?10 M) did not. Neither clonidine (10^?14?10^?11 M) nor methoxamine (10^?8?10^?6 M) reversed the inhibitory action of aminophylline (2×10^?4 M). These results suggest that clonidine enhances IgE-mediated¹⁴C-serotonin release from rat mast cells and also antagonizes the inhibition of mast cells by PGE₁, isoproterenol and dopamine, but not by aminophylline in this immunological reaction through α₂-adrenergic receptors, and that the inhibition of adenylate cyclase of mast cells is one of the biochemical actions of α₂-adrenergic mechanisms.     

Histamine modification of spontaneous rate and rhythm in infarcted canine ventricle

1. Histamine (10^?3 M) increased the spontaneous rate similarly in isolated preparations of normal left ventricular tissue from control, i.e. normal and sham-operated, dogs (control preparations) and in preparations consisting of normaland contiguous infarcted left ventricular tissue from dogs with subacute, i.e. 24 hours after left coronary artery ligation, myocardial infarction (infarcted preparations).
2. Histamine (10^?3 M) markedly enhanced the irregular rhythm of infarcted preparations.
3. The H₁-receptor antagonist, chlorpheniramine (10^?4 M), and the H₂-receptor antagonist, cimetidine (10^?3 M), antagonized the effects of histamine (10^?3 M) on the spontaneous rate of both control and infarcted preparations.
4. The H₁-receptor agonist, 2-pyridyl ethylamine (PEA, 10^?4 M), increased the spontaneous rate of control and infarcted preparations; these effects were antagonized by chlorpheniramine (10^?4 M). The H₂-receptor agonist, dimaprit, had no effect.
5. Similar to histamine (10^?3 M), PEA (10^?4 M) enhanced the irregular rhythm of infarcted preparations; dimaprit had no effect.
6. High local concentrations of histamine may occur in poorly perfused ischemic tissue. The enhancement of irregular rhythm produced by histamine, and the specific H₁-receptor agonist, PEA, leads us to suggest its involvement in arrhythmias associated with subacute myocardial infarction.

     

Release of reactive oxygen intermediates by dengue virus-induced macrophage cytotoxin

Dengue type 2 virus (DV) induces a subpopulation of T lymphocytes of mice to produce a cytokine, cytotoxic factor (mCF), which induces H-2A positive macrophages to produce macrophage cytotoxin (CF2). The present study was undertaken to investigate the mechanism of cytotoxicity of CF2. It was observed that CF2 induced production of superoxide anion (O⁻₂) and hydrogen peroxide (H₂O₂) by the spleen cells of mice in vitro and in vivo. The maximum production of O⁻₂ (260 ±10 n M/4 × 10⁶ cells) was at 45 minutes while that of H₂O₂ was at 90 minutes after inoculation of CF2. Pretreatment of mice or spleen cells with anti-CF2-antisera inhibited O⁻₂ and H₂O₂ production in a dose-dependent manner. Superoxide dismutase (SOD) inhibited O⁻₂ production and cytotoxicity while H₂O₂ production was increased by increasing SOD concentration in the culture. This indicated that O⁻₂ production is necessary for the cytotoxic activity of CF2. Pretreatment of the cells with Ca²⁺ channel blocking drugs, nifedipine or verapamil, inhibited CF2-induced O⁻₂ and H₂O₂ production in a dose-dependent manner. We have shown earlier that the cytotoxic activity of CF2 is known to be Ca²⁺ dependent and CF2-induced production of nitrite and the cytotoxicity is inhibited by N^G-monomethyl- L-arginine. Thus, it is suggested that O⁻₂ and nitrite are necessary for cell killing by CF2 in a Ca²⁺ manner and the killing may possibly be by generation of peroxynitrite.     

LTD4 increases cytosolic free calcium and inositol phosphates in human neutrophils: inhibition by the novel LTD4 receptor antagonist,SR2640, and possible relation to modulation of chemotaxis

LTD₄ increased the level of free intracellular calcium ([Ca⁺⁺]_i) and stimulated the production of inositol phosphates (IP) in human polymorphonuclear neutrophils (PMN). Calcium was predominantly mobilized from intracellular pools. After a single stimulus, the cells were refractory to a second challenge with the same concentration of LTD₄, but the calcium response to LTB₄ was normal. The rise in [Ca⁺⁺]_i as well as the stimulated production of IP was inhibited by the novel LTD₄ antagonist, SR2640. SR2640 also abolished the attenuation by LTD₄ of LTB₄-directed PMN chemotaxis. The results suggest that human PMN contain specific LTD₄ receptor that trigger phosphatidyl inositol hydrolysis by activation of phospholipase C, leading to intracellular calcium mobilization, which may be involved in modulation of chemotaxis.     

Leukotriene B4 Receptor (BLT-1) Modulates Neutrophil Influx into the Peritoneum but Not the Lung and Liver during Surgically Induced Bacterial Peritonitis in Mice

Leukotriene B₄ (LTB₄) is a rapidly synthesized, early neutrophil chemoattractant that signals via its cell surface receptor, BLT-1, to attract and activate neutrophils during peritonitis. BLT-1-deficient (BLT-1^−/−) mice were used to determine the effects of LTB₄ on neutrophil migration and activation, bacterial levels, and survival after cecal ligation and puncture (CLP). Male BLT-1^−/− or wild-type (WT) BALB/c mice underwent CLP. Tissues were harvested for determination of levels of bacteria, myeloperoxidase (MPO), LTB₄, macrophage inflammatory protein 2 (MIP-2), and neutrophil (polymorphonuclear leukocyte [PMN]) numbers at 4 and 18 h after CLP. PMN activation was determined by an assessment of phagocytosis ability and CD11b expression. Survival was also determined. BLT-1^−/− mice had decreased numbers of PMNs in the peritoneum at both 4 and 18 h after CLP but increased numbers of PMNs in the blood at 18 h compared with WT mice. Liver and lung MPO levels were significantly higher in BLT-1^−/− mice at both 4 and 18 h after CLP, with increased bacterial levels in the blood, the liver, and peritoneal fluid at 4 h. Bacterial levels remained higher in peritoneal fluid at 18 h, but blood and liver bacterial levels at 18 h were not different from levels at 4 h. PMN phagocytosis and CD11b levels were decreased in BLT-1^−/− mice. LTB₄ levels were similar between the groups before and after CLP, but MIP-2 levels were decreased both locally and systemically in BLT-1^−/− mice. Survival was significantly improved in BLT-1^−/− mice (71%) compared with WT mice (14%) at 48 h post-CLP. Thus, LTB₄ modulates neutrophil migration into the mouse peritoneum, but not the lung or liver, after CLP. Despite higher bacterial and PMN levels at remote sites, there was increased survival in BLT-1^−/− mice compared to WT mice. Decreased PMN activation may result in less remote organ dysfunction and improved survival.     

Decreased Leukotriene Release from Neutrophils after Severe Trauma: Role of Immature Cells

Polymorphonuclear granulocytes (PMN) play a key role in host defense against microbial infections. After severe trauma PMN show cellular dysfunctions including chemotactic migration, phagocytosis, and bacterial killing. In these settings the contribution of the cellular matruation stage compared to functional activities has not been investigated. Polymorphonuclear granulocytes are potent producers of lipid mediators via the 5-lipoxygenase (5-LO) pathway (leukotrienes, LTs) which exert important proinflammatory and immunoregulatory activities. We analyzed leukotriene generation from PMN-fractions (N = 23) of 15 polytrauma patients in comparison to 17 healthy donor cell fractions and correlated this lipid mediator release to the hematopoietic maturation stage of respective PMN. Polymorphonuclear granulocytes were isolated from EDTA-anticoagulated peripheral blood employing a one step procedure based on a discontinuous double Ficoll-gradient. Cells (5 × 10⁶/500 l phosphate-buffered saline) were stimulated for 20 min at 37°C with 1 M Ca-ionophor A23187 in the presence of 1 mM Ca⁺⁺ and 0.5 mM Mg⁺⁺. Leukotrienes were analyzed by reversed-phase HPLC. Expression of 5-lipoxygenase (5-LO) was additionally determined by Western blot. Maturation stage of PMN was quantitated by Pappenheim-staining of cell smears. After polytrauma the generation of leukotrienes from PMN was individually diminished. Synthesis of enzymatically formed metabolites (LTB₄, OH-LTB₄ and COOH-LTB₄) was concomitantly reduced. The decresaed leukotriene synthesis strongly correlated (r² = 0.907, P < 0.0001) to the occurrence of immature PMN (mostly band cells). The expression of 5-lipoxygenase in PMN fractions consisting mainly of band cells was decreased. Our results provide evidence that posttraumatic granulocyte dysfunction is partly due to immature functional cell capacities.     

Enhancement of polymorphonuclear leukocyte (PMN) function by OK-432

The influence of OK-432, a streptococcal preparation, on human polymorphonuclear leukocytes (PMN) was examined. OK-432 increased O₂⁻ generation by PMN in oral cancer patients, and sustained the production of O₂⁻ in patients on chemoradiotherapy. Enhanced O₂⁻ generation was also observed when PMN were cultured with 10⁻² KE/ml OK-432 for 1 h and then stimulated with phorbol myristate acetate or formyl-metionyl-leucil-phenylalanine (FMLP). In addition, PMN O₂⁻ generation was promoted by culture supernatants of peripheral blood mononuclear cells (PBMC) incubated with 10⁻³ or 10⁻² KE/ml OK-432. Furthermore, OK-432 (10⁻³−10⁻² KE/ml) enhanced the chemiluminescence of FMLP- and PMA-stimulated PMN. However, nitroblue tetrazolium reduction and myeloperoxidase activity were only minimally enhanced. Not only the candidacidal activity of PMN but also antibody-dependent cell-mediated cytotoxicity against Candida and Raji cells were enhanced in correspondence with the increased generation of reactive oxygen species.Culture of PMN or PBMC for 24 h with OK-432 resulted in a concentration-dependent increase in the substantial production of interleukin-1β, interleukin-6 and tumor necrosis factor-α. OK-432 also enhanced granulocyte-macrophage colony stimulating factor and gamma-interferon generation by leukocytes in a dose-dependent manner. Our research indicates that OK-432 enhances PMN function directly as well as via the promotion of cytokine production, and suggests that these effects of OK-432 could be beneficial in immunosuppressed patients.