Studies on phospholipids of different mutants of Salmonella minnesota

Lipid composition of the cells of smooth (S form) and core-defective rough mutants (Ra, Rb & Re) of Salmonella minnesota has been studied. The readily-extractable lipids (RELs), acid-extractable lipids, and polar and nonpolar phospholipids have been analysed. Fatty acid composition of the different fractions containing phospholipids and other neutral lipids have been determined by GLC and GC-MS techniques. Phosphatidyl glycerol (PG), phosphatidylethanolamine (PE) and diphosphatidyl glycerol (DPG) were the major phospholipids present in all the strains. The major saturated fatty acid found was C16:0, and unsaturated fatty acids were, C16:1 and C18:1. Cyclopropane fatty acids, C17cy and C19cy, were also present in small amounts. Increased amounts of REL and unsaturated fatty acids were found in the mutants compared with the smooth strain. The amount of PG and PE decreased and DPG increased in the mutant strains.     

Comparative pharmacodynamics of flunixin, ketoprofen and tolfenamic acid in calves

The pharmacodynamics of the non-steroidal anti-inflammatory drugs flunixin, tolfenamic acid and ketoprofen were studied in calves after intravenous administration. An acute inflammatory reaction was induced in tissue cages by the intracaveal injection of the mild irritant carrageenan, and the inhibition of inflammatory mediators and enzymes was investigated. The substances measured in the exudate included the enzymes (active and total metalloproteases, serine and cysteine proteases, acid phosphatase [AP], lactate dehydrogenase [LDH] and beta-glucuronidase) and the eicosanoids (prostaglandin [PG]E2 and leukotriene [LT]B4). Studies were also made of inhibition of the synthesis of serum thromboxane (Tx)B2 ex vivo, of bradykinin-induced oedema in vivo and of the generation of superoxide anions (O2-) in vitro. None of the drugs affected the concentration of LTB4, or the activities of metalloproteases, cysteine and serine proteases, AP or LDH in the exudate. All the drugs inhibited the synthesis of serum TxB2 and exudate PGE2 and inhibited the release of beta-glucuronidase. They also decreased the oedematous response to intradermally injected bradykinin and inhibited the generation of O2- ions by neutrophils in vitro. These actions may contribute to the anti-inflammatory effects of the drugs and hence to their clinical efficacy.     

Intraspecies variability of cellular fatty acids among soil and intestinal strains of Desulfovibrio desulfuricans

A comparison of cellular fatty acid profiles of Desulfovibrio desulfuricans DSM 642 and 14 wild strains of this species, isolated from two completely different environments, soil and the human intestine, was carried out. All the D. desulfuricans strains grown on lactate and sulfate indicated the presence of considerable amounts of i-C15:0, i-C17:1 and C16:0. Although differences in the quantities of individual fatty acids present in each strain were clear in the group of soil strains (similarity, 67.6%), in contrast to almost identical fatty acid patterns (similarity, near 100%) in the intestinal strains, the results were variable within the limits acceptable for species demonstration. The higher similarity of the fatty acid profiles of intestinal strains may be a result of the similarity of biocenoses in the human digestive tract. The coefficients of variability of i-C17:1 and i-C15:0 (the major branched-chain fatty acids), as well as clustering of the investigated strains compared with strains described in the literature after plotting percentages of i-C17:1 fatty acid against i-C15:0 fatty acid, confirmed a certain heterogeneity of cellular fatty acid profiles within the group of soil strains, in contrast to almost ideal homogeneity within the group of intestinal isolates. Intestinal strains contained a higher ratio of saturated to unsaturated fatty acids (2.2 +/- 0.14) than did soil strains (1.6 +/- 0.2; in one case, 2.7). We propose that intestinal D. desulfovibrio bacteria should be assumed to be a highly homogeneous group and should be represented by the strain D. desulfuricans subsp. intestinus in collections of microbial cultures.     

Superoxide dismutase mimetics inhibit neutrophil-mediated human aortic endothelial cell injury in vitro

In this study, we evaluated the ability of low molecular weight manganese-based superoxide dismutase mimetics to attenuate neutrophil-mediated oxygen radical damage to human aortic endothelial cells in vitro. Human neutrophils, when exposed to tumor necrosis factor-alpha and the complement compound C5a, induced endothelial damage assessed by the release of 51Cr into the medium. This damage correlated with the amount of superoxide generated by neutrophils. Three superoxide dismutase mimetics, with catalytic rate constants for superoxide dismutation ranging from 4 to 9 x 10(7) M-1 S-1, inhibited neutrophil- or xanthine oxidase-mediated endothelial cell injury in a concentration-dependent manner. A similar manganese-based compound with no detectable superoxide dismutase activity was ineffective in inhibiting injury. Fluorescent studies of the neutrophil respiratory burst showed that the superoxide dismutase mimetics were protective without interfering with the generation of superoxide by activated neutrophils. Catalase, elastase inhibitors, and desferrioxamine mesylate (an iron chelator and hydroxyl radical scavenger) were not protective against cell injury. This investigation demonstrates that neutrophil-mediated human aortic endothelial cell injury in vitro is mediated by the superoxide anion and that low molecular weight manganese-based superoxide dismutase mimetics are effective in abrogating this damage.     

Cellular lipid and fatty acid compositions of Burkholderia pseudomallei strains isolated from human and environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and -2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1:1. Since snake venom phospholipase A2 digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16:0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16:0), 2-hydroxy hexadecenoic (2-OH-C16:1), 2-hydroxy octadecenoic (2-OH-C18:1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14:0) and 3-hydroxy palmitic (3-OH-C16:0) acids. There were significant differences in the concentration of hexadecenoic (C16:1), methylene hexadecanoic (C17CPA), octadecenoic (C18:1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated neutrophils: studies with selective inhibitors RO 31-8220 and calyculin A

Previously employed non-selective protein kinase inhibitors yielded inconclusive results regarding involvement of protein kinase C (PKC) in phosphorylation of 47 kDa protein (p47 phox) in intact neutrophils stimulated with physiologic agonists of superoxide generation. In the present study, phosphorylation of p47 phox in formylMet-Leu-Phe (fMLP) stimulated neutrophils was potently inhibited in the presence of 0.3 microM RO 31-8220, a selective inhibitor of PKC. These results provide experimental evidence in support of the currently considered essential involvement of PKC in p47 phox phosphorylation in response to physiologic stimulation of neutrophil surface receptors. The fMLP-induced phosphorylation of p47 phox was enhanced and prolonged by calyculin A, a specific inhibitor of protein phosphatases of types 1 and 2A, and such enhanced phosphorylation was also effectively inhibited by RO 31-8220. Our results suggest that the extent and duration of p47 phox phosphorylation in intact fMLP-stimulated neutrophils is probably controlled by a balance between the activities of PKC, on the one hand, and of protein phosphatase(s) of type(s) 1 and/or 2A, on the other. Effects of RO 31-8220 and of calyculin A on the fMLP-induced p47 phox phosphorylation were paralleled by similar effects on superoxide release. Calyculin A and RO 31-8220 were also used to study signal transduction by a post-receptor agonist of superoxide generation, a calcium ionophore A23187. The results of the latter study indicated that PKC was activated in A23187-stimulated neutrophils and was essentially involved in superoxide generation and p47 phox phosphorylation. Further, these results suggested that protein phosphatase(s) of type(s) 1 and/or 2A were also activated in A23187-signalling pathway, and limited the extent of superoxide release and p47 phox phosphorylation.     

Modification of mononuclear cell function after incubation with albumin-bound unsaturated fatty acids or soybean oil emulsion

Administration of total parenteral nutrition (TPN) with soybean oil emulsion leads to a linoleic acid enrichment of the plasma membrane that may explain an in vivo activation of mononuclear cells (MNC) seen in our previous studies. Fatty acids from the lipid emulsion may have been accessible to MNC after endocytosis of lipid particles, or by direct uptake of fatty acids after lipoprotein lipase hydrolyzation of the emulsion triglycerides. To resemble the incorporation of fatty acids in vivo, we have modified MNC membrane lipid composition by incubation with different albumin-bound unsaturated fatty acids (UFA) or soybean oil emulsion. After incubation with albumin-bound linoleic and oleic acid, the unstimulated release of superoxide anion was unchanged, while zymosan-stimulated release was 140% (n.s) and 112% (p < 0.05) and phorbol-myristate-acetate (PMA)-stimulated release 148% (p < 0.05) and 124% (p < 0.05) of controls, respectively. Incubation with other UFAs or emulsion did not change superoxide anion release. Unstimulated lymphocyte proliferation increased 3 to 13-fold (p < 0.05) after incubation with all UFAs compared to controls, while UFA incubation did not change phytohemagglutinin (PHA) or PMA-stimulated proliferation. Unstimulated lymphocyte proliferation was decreased after incubation with emulsion, while PHA/PMA-stimulated proliferation was unchanged. Increase in membrane fluidity was detectable only after incubation with emulsion. The increased reactivity may have been caused by changes in the lipid environment surrounding membrane-bound enzymes important for signal transduction through the plasma membrane.     

Phospholipid regulation of a cyclic AMP-specific phosphodiesterase (PDE4) from U937 cells

The regulation of phosphodiesterase-4 (PDE4) by various phospholipids was explored using PDE4s partially purified from U937 cells. Preincubation (5 min, 4 degrees C) of the large molecular weight PDE4 denoted "Peak 2 PDE4" with mixed phosphatidic acids (PAs) produced a 2-fold increase in its Vmax without changing its Km (approximately 2 microM) for cyclic AMP. This "activation" was not limited to PAs with specific fatty acid substituents: Synthetic PAs containing saturated and/or unsaturated fatty acids 16-20 carbons long produced similar effects. Lysophosphatidic acids (LPAs) and phosphatidylserines (PSs) also induced PDE4 activation, whereas phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and diacylglycerol did not. Antibodies to a peptide region near the PDE4 catalytic site specifically inhibited PA-induced activation. The data demonstrate that anionic phospholipids can act as non-essential activators of a leukocyte PDE4, and suggest biochemical crosstalk between phospholipid-dependent and cyclic AMP-dependent signalling pathways in human leukocytes.     

Effects of PAF, FMLP and opsonized zymosan on the release of ECP, elastase and superoxide from human granulocytes

Platelet-activating factor (PAF) is a potent chemoattractant for human eosinophils and neutrophils and causes eosinophil and neutrophil recruitment into animal airways. Since eosinophils and eosinophil cationic proteins are thought to play an important role in the pathophysiology of asthma, we have examined the hypothesis that PAF may also stimulate eosinophil cationic protein (ECP) release from human granulocytes. Granulocytes (93% neutrophils, 3% eosinophils) were isolated from the blood of normal volunteers, using metrizamide density gradients, and stimulated in vitro with PAF, L-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or opsonized zymosan (OPZ). Superoxide generation was measured colorimetrically, granulocyte degranulation by a fluorimetric assay for elastase, and eosinophil activation by specific radioimmunoassay (RIA) for ECP. Granulocyte chemotaxis was also measured. Whilst both PAF and FMLP were potent chemoattractants for human mixed granulocytes (concentrations producing half the maximal effect (EC50s) ca 10 nM), PAF at concentrations below 10 microM was a poor stimulus to superoxide generation, elastase release or ECP release from the same cell population. In contrast, FMLP was a potent stimulus to both superoxide generation (EC50 48 nM) and ECP (EC50 ca 100 nM) and elastase release (EC50 ca 1 microM). OPZ was a potent stimulus to superoxide generation, but was a poor stimulus to ECP or elastase release. Thus, although PAF is a potent chemoattractant for human granulocytes, our results suggest that it alone may not stimulate their subsequent activation and release of cytotoxic products.     

3-Morpholino-sydnonimine-induced suppression of human neutrophil degranulation is not mediated by cyclic GMP, nitric oxide or peroxynitrite: inhibition of the increase in intracellular free calcium concentration by N-morpholino-iminoacetonitrile, a metabolite of 3-morpholino-sydnonimine

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.     

Fatty acid composition of myelin isolated from the brain of a patient with cellular deficiency of co-enzyme forms of vitamin B12

Myelin fractionation and subsequent lipid isolation have been carried out on a brain from a patient who suffered from a cellular deficiency of the adenosylcobalamin and methylcobalamin co-enzyme forms of vitamin B12. Examination of the fatty acid composition of choline and ethanolamine glycerophospholipids indicated a relative enrichment of odd-chain fatty acids which were identified by gas-liquid chromatography-mass spectroscopy as C15, C15:1, C17 and C17:1. A mixture of methyl branched C17 fatty acids was also identified. Odd-chain fatty acids accounted for 9.8% of the total fatty acid in the myelin choline phospholipid conpared to control values of 1.2%. The affected brain myelin phospholipids had a lower unsaturated fatty acid content. Examination of the myelin sphingolipids, sphingomyelin, cerebroside and sulfatide, yielded abnormal fatty acid profiles. The sphingomyelin contained only small amounts of C24:1 fatty acid. Both normal and hydroxy fatty acid containing cerebroside and sulfatide had reduced levels of C24 fatty acid. Determination of the relative hydroxy and normal fatty acid content of the galactolipids indicated an abnormally high hydroxy fatty acid level. Abnormal fatty acid profiles of brain cerebral sphingolipids have not been previously described in cases of vitamin B12 deficiency. Whether or not these alterations are characteristic will only be established by estimating sphingolipids in other such cases.     

Distinct phospholipases A2 regulate the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils

Arachidonic acid (AA) released from membrane phospholipids by phospholipase A2 (PLA2) is important as a substrate for eicosanoid formation and as a second messenger for superoxide anion (O2-) generation in neutrophils. Different isoforms of PLA2 in neutrophils might mobilize AA for different functions. To test this possibility, we sought to characterize the PLA2s that are activated by the neutrophil stimuli, Aroclor 1242, a mixture of polychlorinated biphenyls, and A23187, a calcium ionophore. Both Aroclor 1242 and A23187 caused release of [3H]AA; however, O2- production was seen only in response to Aroclor 1242. Eicosanoids accounted for >85% of the radioactivity recovered in the supernatant of A23187-stimulated cells but <20% of the radioactivity recovered from cells exposed to Aroclor 1242. Omission or chelation of calcium abolished A23187-induced AA release, but did not alter AA release in Aroclor 1242-stimulated neutrophils. AA release and O2- production in response to Aroclor 1242 were inhibited by bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2. BEL, however, did not alter Calcium-independent activity was inhibited >80% by BEL, whereas calcium-dependent activity was inhibited <5%. Furthermore, calcium-independent, but not calcium-dependent, PLA2 activity was significantly enhanced by Aroclor 1242. These data suggest that Aroclor 1242 and A23187 activate distinct isoforms of PLA2 that are linked to different functions: Aroclor 1242 activates a calcium-independent PLA2 that releases AA for the generation of O2-, and A23187 activates a calcium-dependent PLA2 that mobilizes AA for eicosanoid production.     

Differential regulation of early phase and late phase responses in human neutrophils by cAMP

The elevation of intracellular levels of cyclic AMP by forskolin stimulation of adenylate cyclase regulates early and late phase neutrophil responses differentially. Early phase neutrophil responses as measured by shape change in response to chemotactic factors, transmigration across a polycarbonate membrane and priming were unaffected by forskolin-induced elevation of intracellular cAMP. Late phase neutrophil responses such as release of superoxide anions, activation of phospholipase A2 and platelet activating factor (PAF) synthesis were inhibited by increasing intracellular cAMP through the addition of 10 microM forskolin for 10 min prior to stimulation. N-Formyl-methionyl-leucyl-phenylalanine-stimulated arachidonic acid release fell from 9.3% (untreated cells) to 4.6% in forskolin-treated cells. PAF generation was also inhibited from 430 pg/10(6) cells in untreated cells to background levels in forskolin-treated cells (110 pg/10(6) cells). Also, the reduction of cytochrome c by superoxide anions fell from 4.2 nmol/10(6) cells in the absence of forskolin to 2.0 nmol/10(6) cells following forskolin treatment. These results indicate that in neutrophils the elevation of cAMP acts differentially on cellular responses, not affecting early activation events, but markedly inhibiting late events such as the release of inflammatory mediators.     

The proportion of fatty acids in mammary secretion of cows lactating after calving and hormonal induction of lactation

The changes in the proportion of fatty acids in lacteal fat of cows induced into lactation by 7-day treatment with oestradiol and progesterone were characterized by an increases in the proportion of fatty acids C6--C13: the proportions of C14, C15, C17, C19 fatty acids changed slightly if at all; the proportions of C16, C20 and C22 fatty acids decreased during the first 28 days of lactation. Similar changes in fatty acid composition were found in lacteal fat of cows lactating after calving. These results demonstrated that the hormonal induction of lactation in nonpregnant cows initiates mammary secretion similar to the fatty acid composition in normal cow milk.     

Contrasting effects of two arachidonate 5-lipoxygenase inhibitors on formyl-methionyl-leucyl-phenylalanine (fMLP) and complement fragment 5a induced human neutrophil superoxide generation

SC-45662 and SC-41661A, selective arachidonate 5-lipoxygenase (5-LO) inhibitors, had markedly different effects on formyl-methionyl-leucyl-phenylalanine (fMLP) and complement fragment 5a (C5a) induced superoxide release from human neutrophils (PMNs). SC-45662 inhibited superoxide generation induced by fMLP and C5a with IC50 values of 12 and 5 microM, respectively. Furthermore, SC-45662 was capable of inhibiting fMLP and C5a induced superoxide release in PMNs primed with bacterial lipopolysaccharide, tumor necrosis factor-alpha and other priming agents. SC-41661A, a compound from the same chemical series as SC-45662, did not inhibit or induce superoxide generation, but instead primed PMNs for fMLP and C5a induced superoxide generation. The induced superoxide release was concentration dependently enhanced 2 to 4-fold at 5-50 microM. Superoxide release induced by phorbol myristate acetate or serum-activated zymosan was unaffected by either SC-45662 or SC-41661A. The regulation of superoxide generation by these compounds, both of which have the identical oxidation-reduction pharmacophore, was clearly independent of their effects on 5-LO activity. Furthermore, the mechanism by which SC-45662 and SC-41661A alter superoxide generation did not appear to depend on inhibition of xanthine oxidase, catalase or superoxide dismutase. These new compounds provide effective tools for further investigation of the relationship of these two biochemical oxidative systems.     

Accelerated healing of gingival incisions by the helium-neon diode laser: a preliminary study

Platelet-activating factor (PAF) concordantly primes neutrophils (PMNs) for superoxide generation and elastase release. beta-Adrenergic stimulation of PMNs enhances cAMP-dependent protein kinase A (PKA) activity and has been shown to inhibit PAF-mediated NADPH-oxidase activity. PMN superoxide generation is thought to play a predominate microbicidal role, whereas elastase is known to mediate untoward PMN-endothelial interactions. We hypothesized that beta-adrenergic neutrophil stimulation has disparate effects on PAF-mediated PMN superoxide generation versus elastase release. Human PMNs were isolated using a standard Ficoll/Hypaque gradient. PMNs were then primed with PAF (200 nM) and activated with fMLP (1 microM). Subsets of PMNs were pretreated for 5 min with a beta agonist (10(-4) M isoprotereno) or an adenylate cyclase agonist (10(-5) M forskolin). Superoxide generation was determined by superoxide dismutase inhibitive cytochrome c reduction. Elastase activity was measured by the cleavage of n-methoxylsuccinyl-A-A-P-V-p-nitroanilide. Pretreatment with isoproterenol and forskolin yielded superoxide generation of 3.2 +/- 0.6 and 3.1 +/- 1.2 nmole/2.5 x 10(5) PMN/min compared to 9.0 +/- 0.6 nmole/2.5 x 10(5) PMN/min for PAF/fMLP alone, whereas isoproterenol and forskolin did not significantly affect PAF-mediated neutrophil elastase release, 22.4 +/- 5.3 and 24.0 +/- 3.6%, respectively, compared to 39.4 +/- 9.1% for PAF/fMLP alone. Disparate PMN signal transduction for superoxide generation versus elastase release may explain the SICU clinical paradox, in which patients are both susceptible to infection and vulnerable to PMN-mediated multiple organ failure.     

Recombinant human interleukin-11 does not affect functions of purified human neutrophils in vitro

Recombinant human interleukin-11 (rHu-IL-11) is a multifunctional cytokine with thrombopoietic activity and demonstrated clinical efficacy in treating chemotherapy-induced thrombocytopenia. rHu-IL-11 also exhibits anti-inflammatory activity and is currently in clinical trials for the treatment of several inflammatory diseases. As neutrophils are involved in both innate immunity and an acute inflammatory response, the effect of rHU-IL-11 on the function of human peripheral blood neutrophils in vitro was examined. rHu-IL-11 was not cytotoxic and did not induce superoxide anion production or the release of granular enzymes from resting neutrophils. Phagocytosis and chemotaxis were unaffected. rHu-IL-11 treatment did not block the response of neutrophils to stimulation. Pretreatment with rHu-IL-11 did not reduce production of IL-8 following activation with lipopolysaccharide (LPS) or zymosan A particles. Pretreatment with rHu-IL-11 did not affect the release of lysozyme and beta-glucuronidase in response to A23187 or PMA-stimulated production of superoxide anion. These results indicate that rHu-IL-11 does not directly modulate key functions of neutrophils in vitro.     

The immediate activator of the NADPH oxidase is arachidonate not phosphorylation

Superoxide generation is rapidly triggered following the addition of a stimulus to neutrophils. The signal-transduction pathway culminates in the activation of protein kinase C, whose phosphorylation of a protein component is considered to activate the oxidase. Arachidonate stimulated the oxidase in a concentration-dependent manner but, unlike phorbol-12-myristate-13-acetate (PMA), was not inhibited by staurosporine, a protein kinase inhibitor. Increase protein phosphorylation, apparent with PMA, was not observed when superoxide generation was triggered by arachidonate. Inhibitors of phospholipase A2 inhibit the PMA activation of the oxidase. Therefore, we propose that arachidonate and not phosphorylation is the immediate stimulus for superoxide generation.     

Differential superoxide anion generation by equine eosinophils and neutrophils

Equine eosinophils and neutrophils are believed to play an important part in the protection of horses against parasitic and bacterial invasion. Eosinophils may also play a key role in the pathogenesis of equine inflammatory conditions such as the allergic skin disease, insect hypersensitivity. The factors which stimulate the respiratory burst of equine eosinophils and neutrophils are poorly understood. The first aim of the present study was to determine the effects of the phorbol ester, phorbol myristate acetate (PMA), which is believed to activate intracellular protein kinase C, and opsonised particles of serum-treated zymosan (STZ), on the production of superoxide anions by equine eosinophils and neutrophils. Since histamine has been detected after antigen challenge in the skin of horses with insect hypersensitivity, the second aim was to establish the effects of this mediator on superoxide anion production by equine eosinophils and the receptor sub-type(s) that mediate histamine-induced responses. For comparison, responses of neutrophils from the same horses were also examined. PMA and STZ induced significant increases in superoxide anion generation by equine eosinophils and neutrophils. The estimated maximum (EMAX) superoxide anion production by eosinophils in the presence of PMA was significantly greater than that of neutrophils; the estimated concentration of PMA inducing 50% of the maximum response (EC50) by eosinophils was significantly less. The EMAX values for superoxide anion production by neutrophils in the presence of STZ were significantly greater than those for eosinophils. Histamine induced superoxide anion generation by equine eosinophils which was inhibited by the histamine-1 receptor antagonists chlorpheniramine and mepyramine, but not the histamine-2 and histamine-3 receptor antagonists, cimetidine and thioperamide, respectively. Histamine did not cause superoxide anion production by equine neutrophils. These studies demonstrate that equine granulocytes vary in their ability to produce a respiratory burst in the presence of different stimuli, with eosinophils being more responsive to protein kinase C activators and neutrophils to opsonised particles. They also show that histamine selectively induced the generation of superoxide anions by equine eosinophils via histamine-1 receptor activation. Thus, in horses with insect hypersensitivity, histamine released from cutaneous mast cells after antigen challenge could activate eosinophils which have migrated into the dermis.