Marked improvement of the basophil activation test by detecting CD203c instead of CD63.

BACKGROUND: The flow cytometric basophil activation test by detection of CD63 expression has been developed as an alternative method for in vitro diagnosis of IgE-mediated reactions to various allergens. Despite promising initial studies, the test remains disappointing in terms of sensitivity. CD203c has recently been demonstrated as a specific activation marker of basophils that is rapidly up-regulated after allergen challenge in sensitized patients. OBJECTIVE: The goal of the present study was to compare basophil activation tests by using either CD203c or CD63 in the diagnosis of immediate-type allergy to latex. METHODS: Twenty-seven patients (health care workers of our institution) who developed clinical features evocative of allergy after contact with latex were included and classified into two groups. Group 1 (n = 16) comprised true allergic patients who presented with typical signs of immediate allergic reaction associated with a positive skin test (prick test). Group 2 (n = 11) consisted of patients whose clinical history was not typical and had negative skin test. Twelve healthy subjects were also studied as controls. We compared the sensitivity of two triple-staining flow cytometric protocols measuring basophil activation after latex stimulation: CD45-IgE-CD63 and CD45-IgE-CD203c. RESULTS: The CD203c protocol showed a higher sensitivity than the CD63 protocol (75% vs. 50%). In comparison, latex-specific IgE sensitivity was found to be 69%. Furthermore, the magnitude of the basophil response was significantly higher with CD203c in comparison with CD63. Specificity was 100% for both protocols. CONCLUSION: Due to superior gating of basophils and a higher range of activation in response to allergen, the basophil activation test is markedly improved by use of CD203c instead of CD63.

Prostaglandin D2 is a potent chemoattractant for human eosinophils that acts via a novel DP receptor.

Prostaglandin D2 (PGD2) is released following exposure of asthmatics to allergen and acts via the adenylyl cyclase-coupled receptor for PGD2 (DP receptor). In this study, it is reported that human eosinophils possess this receptor, which would be expected to inhibit their activation. In contrast, it was found that prostaglandin D2 is a potent stimulator of eosinophil chemotaxis, actin polymerization, CD11b expression, and L-selectin shedding. These responses are specific for eosinophils, as neutrophils display little or no response to prostaglandin D2. They were not due to interaction with receptors for other prostanoids, as prostaglandins E2 and F(2alpha), U46619 (a thromboxane A2 analogue), and carbaprostacyclin (a prostacyclin analogue) displayed little or no activity. Furthermore, they were not shared by the selective DP receptor agonist BW245C and were not prevented by the selective DP receptor antagonist BWA868C, indicating that they were not mediated by DP receptors. In contrast, the prostaglandin D2 metabolite 13,14-dihydro-15-oxoprostaglandin D2 induced eosinophil activation but did not stimulate DP receptor-mediated adenosine 3',5'-cyclic monophosphate (cAMP) formation. These results indicate that in addition to the classic inhibitory DP1 receptor, eosinophils possess a second, novel DP2 receptor that is associated with PGD2-induced cell activation. These 2 receptors appear to interact to regulate eosinophil responses to PGD2, as blockade of DP1 receptor-mediated cAMP production by BWA868C resulted in enhanced DP2 receptor-mediated stimulation of CD11b expression. The balance between DP1 and DP2 receptors could determine the degree to which prostaglandin D2 can activate eosinophils and may play a role in eosinophil recruitment in asthma.

Quantitative analysis of 5-oxo-6,8,11,14-eicosatetraenoic acid by electrospray mass spectrometry using a deuterium-labeled internal standard.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachidonic acid formed by the 5-lipoxygenase pathway, is a potent eosinophil chemoattractant that may be an important mediator in asthma. To further investigate the physiological and pathological roles of 5-oxo-ETE we have developed a mass spectrometric assay employing a tetradeuterated analog (5-oxo-[11,12,14,15-(2)H]ETE) as an internal standard. Collision-induced dissociation of the quasimolecular anion of 5-oxo-[11,12,14,15-(2)H]ETE (m/z 321) resulted in the formation of a major ion at m/z 207 that retained all four deuterium atoms. Measurement of the ratio of ions at m/z 203 (endogenous 5-oxo-ETE) and m/z 207 permitted quantitation of this compound by liquid chromatography-mass spectrometry-mass spectrometry using multiple reaction monitoring. The resulting assay was highly sensitive (< or =20 pg/sample) and selective, enabling detection of the amount of 5-oxo-ETE produced by as few as 10,000 neutrophils. This assay should permit measurement of 5-oxo-ETE in biological fluids, enabling evaluation of its role in asthma and other inflammatory diseases.

Eotaxin and RANTES enhance 5-oxo-6,8,11,14-eicosatetraenoic acid-induced eosinophil chemotaxis.

BACKGROUND: The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of human eosinophils and, among lipid mediators, is the most active chemoattractant for these cells. Studies have demonstrated the importance of 5-lipoxygenase products in allergen-induced pulmonary eosinophilia. Because CC chemokines such as eotaxin and RANTES also play critical roles in this phenomenon, it would seem likely that members of both classes of mediators contribute to this response. OBJECTIVE: The study was designed to directly compare the effects of 5-oxo-ETE on eosinophils with those of eotaxin and RANTES and to determine whether these chemokines could enhance the chemotactic response to 5-oxo-ETE. METHODS: Eosinophil chemotaxis was measured with microchemotaxis chambers. CD11b, L-selectin, and actin polymerization were measured by flow cytometry. Calcium mobilization was measured by fluorescence. RESULTS: 5-Oxo-ETE stimulated eosinophil chemotaxis with a potency between those of eotaxin and RANTES and a maximal response about 50% higher than that of eotaxin. Threshold concentrations of eotaxin and RANTES increased the chemotactic potency of 5-oxo-ETE by more than 4-fold. 5-Oxo-ETE and eotaxin were approximately equipotent in mobilizing cytosolic calcium in eosinophils. Eotaxin was more potent in inducing CD11b expression and actin polymerization, but the maximal responses to 5-oxo-ETE were about 50% higher. 5-Oxo-ETE strongly induced L-selectin shedding, whereas eotaxin elicited only a weak and variable response. CONCLUSION: 5-Oxo-ETE is a strong activator of human eosinophils with a chemotactic potency comparable to those of eotaxin and RANTES, both of wwhich enhance 5-oxo-ETE-induced chemotaxis. 5-Oxo-ETE and CC chemokines may combine to induce pulmonary eosinophilia in asthma.

Inositol (1,4,5)trisphosphate metabolism and enhanced calcium mobilization in airway smooth muscle of hyperresponsive rats.

Airway hyperresponsiveness (AHR) is a phenotype of asthma and can be modeled by the inbred Fisher strain of rat, which is hyperresponsive in vivo relative to the Lewis strain. Enhanced airway smooth muscle (ASM) contractility and Ca(2+) mobilization are associated with the AHR observed in Fisher rats. In this study, we investigated whether the interstrain differences in Ca(2+) mobilization to serotonin (5HT) result from differences in inositol (1,4,5)trisphosphate (IP(3)) metabolism and/or IP(3) receptor (IP(3)R) sensitivity. Ca(2+) mobilization by 5HT in cultured ASM cells from both rat strains was phospholipase C (PLC) dependent. Inositol polyphosphate accumulation, and hence PLC activity, was similar in both rat strains, but a specific IP(3) transient was detectable only in Fisher myocytes in response to 5HT. These findings suggested that IP(3) degradation rather than production differed between the two strains. The Vmax and Michaelis constant (K(m)) of IP(3)-specific 5-phosphatase activity were higher in the particulate fraction of Lewis than in Fisher ASM cell homogenates and appeared to be related to a greater expression of two isoforms of 5-phosphatase (type I and type II) in Lewis cells as shown by Western blot analysis. The sensitivity of the IP(3)R to IP(3) was similar between Fisher and Lewis ASM cells, indicating that the interstrain intracellular Ca(2+) differences were unrelated to IP(3)R function. We propose that interstrain variations in 5-phosphatase activity and expression may give rise to the interstrain differences in IP(3)-mediated Ca(2+) release in ASM and may be a determinant of AHR.

Inhaled budesonide inhibits OVA-induced airway narrowing, inflammation, and cys-LT synthesis in BN rats.

The objective of the present investigation was to examine the effects of an inhaled glucocorticoid, budesonide, on antigen-induced production of cysteinyl leukotrienes (cys-LTs) and pulmonary inflammatory cell infiltration in the Brown Norway rat, an animal model of asthma. Two weeks after sensitization to ovalbumin, rats were treated with budesonide (2.5 mg/kg) 18 and 1 h before challenge with antigen. Budesonide abolished the late response to ovalbumin (P<0.02) and strongly inhibited the in vivo synthesis of N-acetyl-leukotriene E(4), an indicator of cys-LT synthesis, during this period (P<0.005). Both total bronchoalveolar lavage (BAL) cells (P<0.01) and BAL macrophages (P<0.005) were markedly reduced to approximately 25% of their control levels after treatment with budesonide. It can be concluded that inhibition of the antigen-induced late response in Brown Norway rats by budesonide is associated with reductions in both BAL macrophages and cys-LT synthesis. It is possible that the effect of budesonide on cys-LT synthesis is related to its effects on pulmonary macrophages.

Endotoxemia in transgenic mice overexpressing human glutathione peroxidases.

In response to endotoxemia induced by administration of lipopolysaccharide, a complex series of reactions occurs in mammalian tissues. During this inflammation response, cells produce different mediators, such as reactive oxygen species, a number of arachidonic acid metabolites, and cytokines. The reactive oxygen species thus generated have been suggested to produce tissue injury as a result of macromolecular damage or by interfering with regulatory processes. They may also act as important signaling molecules to induce redox-sensitive genes. We report here that transgenic mice overexpressing 2 major forms of human glutathione peroxidases (GPs), intra- and extracellular GP, are able to modulate host response during endotoxemic conditions. We show that these animals have a decreased hypotension and increased survival rate after administration of a high dosage of lipopolysaccharide. Overexpression of GPs alters vascular permeability and production of cytokines (interleukin-1 beta and tumor necrosis factor-alpha) and NO, affects arachidonic acid metabolism, and inhibits leukocyte migration. These results suggest an important role for peroxides in pathogenesis during endotoxemia, and GPs, by regulating their level, may prove to be good candidates for antioxidant therapy to protect against such injury.

Role of cysteinyl leukotrienes in CD4(+) T cell-driven late allergic airway responses.

Cysteinyl leukotrienes (cys LTs) play an important role in late responses to allergen challenge of actively sensitized rats. The aim of this study was to determine whether T cell-dependent late airway responses (LARs) also are mediated by cys LTs. To do this we tested the effects of the selective and potent LTD(4) antagonist pranlukast on airway responses to ovalbumin (OVA) challenge of naive recipients of CD4(+) T cells isolated from the cervical lymph nodes of OVA-sensitized donor rats. CD4(+) T cells (5 million) were purified by immunomagnetic separation and administered i.p. 2 days before OVA challenge. The pulmonary resistance was measured for 8 h after challenge and bronchoalveolar lavage (BAL) was performed for analysis of leukocytes and major basic protein-positive cells. The LAR, determined as the area under the curve of pulmonary resistance against time from 3 to 8 h after challenge, was 8.9 +/- 1.79 cm H(2)O/ml/s x min after OVA compared with 2.8 +/- 0.50 cm H(2)O/ml/s x min (P <.01) after OVA and pranlukast treatment. The total cell count in BAL was not significantly greater in the OVA challenged group (3.55 +/- 0.41 x 10(6) cells) compared with the OVA- and pranlukast-treated group (2.65 +/- 0.45 x 10(6) cells). However, lymphocytes and eosinophils were reduced in numbers by pranlukast. Interleukin-5 mRNA-positive cells were diminished by 50% in pranlukast-treated animals. In conclusion, pranlukast inhibits LAR, BAL eosinophilia, and Interleukin-5 expression in rats with adoptively transferred LAR, indicating an important role for cys LTs in these T cell-driven responses.

The beta(2)-agonist salbutamol inhibits bronchoconstriction and leukotriene D(4) synthesis after dry gas hyperpnea in the guinea-pig.

Isocapnic dry gas hyperpnea-induced bronchoconstriction (HIB) in the guinea-pig is mediated by both tachykinin release from airway sensory nerve C-type fiber terminals and secondary synthesis of cysteinyl-leukotrienes, in particular LTD(4). Beta (beta)(2)-agonists are potent bronchodilators but potentially could also inhibit the airway response to hyperpnea challenge via effects on the release of LTD(4)from airway cells in vivo. The purpose of this study was to test the hypothesis that beta(2)agonists attenuate HIB in guinea-pigs, in part, by reduction in LTD(4)release in vivo. Twenty-six guinea-pigs (400-550 g) were anesthetized with xylazine (7 mg/kg) and pentobarbital (65 mg/kg), tracheotomized and mechanically ventilated with a small animal ventilator using a tidal volume of 3 ml and a breathing frequency of 60 breaths/min. Dry gas (95%O(2)/5%CO(2)) with a 4 ml tidal volume and a breathing frequency of 150/min was used for hyperpnea challenge. Challenge with isocapnic dry gas triggered a significant increase in pulmonary resistance (0.3 +/- 0.02 vs. 0.57 +/- 0.06 cmH(2)O/ml per s; P=0. 017; n=13) and excretion of LTD(4)in the bile (baseline: 2.43 vs. HIB: 4.66 pmol/h; P=0.04). Salbutamol pretreatment completely blocked the airway response to the challenge (0.3+/-0.02 vs. 0.3+/-0. 05 cmH(2)O/ml per s; n=13) and reduced the biliary excretion of LTD(4)(baseline: 2.42 pmol/h; vs. HIB: 2.40 pmol/h). We conclude that salbutamol inhibited the airway responses to dry gas hyperpnea challenge and LTD(4)synthesis by the airway cells.

5-Oxo-6,8,11,14-eicosatetraenoic acid stimulates isotonic volume reduction of guinea pig jejunal crypt epithelial cells.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a recently discovered arachidonate metabolite that is a potent activator of eosinophils and neutrophils and may be an important mediator of inflammation. The objective of the present investigation was to determine whether 5-oxo-ETE affects the isotonic volume of Cl(-) secretory intestinal crypt epithelial cells. 5-Oxo-ETE caused rapid shrinkage of guinea pig jejunal crypt epithelial cells to a reduced but stable volume, which was measured electronically. This effect was prevented by Cl(-) and K(+) channel blockers and inhibitors of protein kinase C. 5-Oxo-ETE (EC(50) = 20 pM) was more potent than any of the other agonists tested, including its precursor, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (EC(50) = 5 nM); leukotriene D(4) (EC(50) = 1 nM); vasoactive intestinal peptide (EC(50) = 200 pM); and bradykinin (EC(50) = 50 nM). Leukotriene B(4) had no effect on crypt cell volume. In contrast to its effects on crypt cells, 5-oxo-ETE had no effect on the volume of jejunal villus cells. These results indicate that 5-oxo-ETE induces an isotonic volume reduction in intestinal crypt epithelial cells that appears to be dependent on Cl(-) secretion and activation of protein kinase C.

IL-2 enhances allergic airway responses in rats by increased inflammation but not through increased synthesis of cysteinyl leukotrienes.

BACKGROUND: IL-2 has been shown to increase allergic airway responses in rats. OBJECTIVE: The purpose of this study was to investigate whether induction of inflammation and enhancement of cysteinyl-leukotriene (cys-LT) synthesis were involved in the augmentation of airway responses caused by IL-2. METHODS: Brown Norway rats received human recombinant IL-2 or saline subcutaneously twice a day from day 9 to day 14 after sensitization to ovalbumin (OVA). On day 14, rats underwent either lung lavage or were challenged with an aerosol spray of OVA, the airway responses and biliary excretion of cys-LTs were measured for a period of 8 hours after challenge, and the lung leukocyte numbers were determined after enzymatic digestion of lung tissues. RESULTS: The early response after OVA increased from 184.2% +/- 13.5% in the animals receiving saline (n = 10) to 309% +/- 51% (baseline lung resistance) in IL-2-pretreated animals (n = 17; P <.05). The late response also increased from 19.6 +/- 4.5 (area under the curve of baseline lung resistance vs time) in the animals receiving saline to 37 +/- 5.4 after administration of IL-2 (P <.05). However, IL-2-treated animals had lower levels of biliary cys-LTs during the late response than saline-treated animals but similar levels during the early response. This difference could not be attributed to an increase in LT metabolism, which we assessed by the recovery of 3H-LTC4 instilled intratracheally in challenged or unchallenged rats. When compared with control animals, pretreatment with IL-2 increased all cell types retrieved from lung lavage fluid before OVA challenge (P <.05). After OVA challenge, the total cell yield from lung lavage fluid was also increased, mostly because of an increase in neutrophils (P <.05). Eosinophils and lymphocytes were greater in the lungs of IL-2-treated than vehicle-treated and OVA-challenged rats (P <.01), and IL-2-treated rats had a lower CD4(+)/CD8(+) ratio in the blood after challenge (P <.001). CONCLUSION: In conclusion, IL-2 increases early and late responses in rats, and it induces lung inflammation. Altered airway responses are not attributable to an increase in cys-LT production.

IL-3 does not affect the allergic airway responses and leukotriene production after allergen challenge in rats.

T cell cytokines are important in asthma. Interleukin (IL)-3, an important growth factor for mast cells and eosinophils has been shown to be increased in the airways of asthmatic subjects, but its precise functions are uncertain. The aim of this study was to determine whether recombinant human (rh) IL-3 affected airway responses, inflammation and leukotriene production after antigen challenge in Brown Norway (BN) rats. Having established that rhIL-3 (>12.5 microg subcutaneously b.i.d. for 4 days) caused a doubling of mast cell numbers in the airways of BN rats, sensitized rats were pretreated with rhIL-3 (50 microg) or vehicle subcutaneously b.i.d. for 4 days. Ovalbumin (OA) challenge was performed and the early (EAR), and late (LAR) airway response and the associated biliary leukotriene (LT) excretion measured. The pulmonary cellularity was evaluated by means of lung digestion 8 h after challenge. IL-3 increased the number of eosinophils isolated from the lungs after antigen challenge (0.77+/-0.23 versus 0.38+/-0.12 x 10(6) cells, p=0.03). However, there were no effects on the numbers of neutrophils, lymphocytes and macrophages. Neither the EAR nor the LAR after OA challenge were altered by IL-3. Likewise biliary cysteinyl-LT excretion was similar in IL-3-treated animals and controls after challenge. In conclusion, interleukin-3 caused an increase in the numbers of mast cells and eosinophils around the airways without affecting the magnitude of either early or late airway responses or mediator release after antigen challenge. The present results suggest that airway inflammation can occur in rats without increasing the allergic asthmatic response.

Strain dependence of the airway response to dry-gas hyperpnea challenge in the rat.

The aim of the study was to investigate strain dependence and mechanisms of airway responses to dry-gas hyperpnea challenge in the rat. We studied responses in a strain that is hyperresponsive to methacholine, Fischer 344 (F-344); in two normoresponsive strains, Lewis and ACI; and in an atopic but normoresponsive strain, Brown Norway (BN). We examined the effects of a neurokinin (NK) 1-receptor (CP-99994), an NK2-receptor (SR-48968), and a leukotriene D4 (LTD4)-receptor antagonist (pranlukast) on responses to hyperpnea challenge in BN rats. The animals were ventilated with a tidal volume of 8 ml/kg and a frequency of 150 breaths/min with either a dry or humidified mixture of 5% CO2-95% O2 for 5 min for hyperpnea challenge, whereas responses to challenge were measured during spontaneous breathing. Pulmonary resistance increased after dry-gas challenge in BN and ACI but not in F-344 and Lewis rats. CP-99994, SR-48968, and pranlukast significantly attenuated the increase in pulmonary resistance after dry-gas challenge. There were no significant differences in responsiveness to airway challenge with LTD4 among the BN, F-344 and ACI rats. We conclude that responses to dry-gas hyperpnea challenge are strain dependent in rats and are mediated by NKs and LTD4.

Biological inactivation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human platelets.

Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of neutrophils and eosinophils. In the present study we examined the biosynthesis and metabolism of this substance by platelets. Although platelets contain an abundant amount of 5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the formation of 5-oxo-ETE, they synthesize only very small amounts of this substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless endogenous NADPH is converted to NADP+ by addition of phenazine methosulfate. Similarly, relatively small amounts of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and neutrophils, which instead formed substantial amounts of two 12-hydroxy metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, which were identified by comparison with authentic chemically synthesized compounds. These metabolites were also formed from 5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast, unstimulated platelets converted 5-oxo-ETE principally to 5-HETE. Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects on neutrophil calcium levels or platelet aggregation at concentrations as high as 10 micromol/L, but both blocked 5-oxo-ETE-induced calcium mobilization in neutrophils with IC50 values of 0.5 and 2.5 micromol/L, respectively. We conclude that platelets can biologically inactivate 5-oxo-ETE. Unstimulated platelets convert 5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas stimulated platelets convert this substance to 12-hydroxy metabolites, which possess antagonist properties.

5-oxo-6,8,11,14-eicosatetraenoic acid is a potent stimulator of L-selectin shedding, surface expression of CD11b, actin polymerization, and calcium mobilization in human eosinophils.

5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of arachidonic acid formed by the oxidation of 5-hydroxy-6,8,11, 14-eicosatetraenoic acid by a highly specific dehydrogenase. 5-oxo-ETE is a chemoattractant for both neutrophils and eosinophils. Although it is not as effective as leukotriene B4 (LTB4) and platelet-activating factor (PAF) in stimulating neutrophil migration, we found that it is considerably more active than these and a variety of other lipid mediators as an eosinophil chemoattractant. Moreover, low concentrations of 5-oxo-ETE appear to enhance the responsiveness of these cells to PAF. The objectives of the current investigation were to identify rapid responses induced in eosinophils by 5-oxo-ETE that might be related to the infiltration of these cells into tissues. We found that 5-oxo-ETE is more effective than PAF and LTB4 in inducing both L-selectin shedding and actin polymerization in human eosinophils, whereas PAF is the most active of these mediators in stimulating calcium mobilization. The complementary effects of 5-oxo-ETE and PAF on actin polymerization and calcium mobilization may explain their synergistic effect on eosinophil migration. 5-oxo-ETE and PAF were equipotent in stimulating the surface expression of the beta2-integrin CD11b, but were slightly less potent than LTB4. 5-oxo-ETE- induced actin polymerization was subject to homologous but not heterologous desensitization. It was not prevented by incubation of eosinophils with inhibitors of protein kinase C (staurosporine), mitogen-activated protein kinase kinase (PD98059), or phosphatidylinositol-3-kinase (wortmannin). In conclusion, 5-oxo-ETE is a potent activator of human eosinophils and may be an important regulator of tissue infiltration of these cells.

5-oxo-ETE induces pulmonary eosinophilia in an integrin-dependent manner in Brown Norway rats.

We have shown previously that the 5-lipoxygenase product 5-oxo-6,8, 11,14-eicosatetraenoic acid (5-oxo-ETE) is a highly potent eosinophil chemoattractant in vitro. To determine whether this substance can induce pulmonary eosinophil infiltration in vivo, it was administered to Brown Norway rats by tracheal insufflation. Eosinophils were then counted in lung sections that had been immunostained with an antibody to eosinophil major basic protein. 5-Oxo-ETE induced a dramatic increase in the numbers of eosinophils (ED50, 2.5 microg) around the walls of the airways, which reached maximal levels (five times control levels) between 15 and 24 h after administration, and then declined. LTB4 also induced pulmonary eosinophil infiltration with a similar ED50 but appeared to be somewhat less effective. In contrast, LTD4 and LTE4 were inactive. 5-Oxo-ETE-induced eosinophilia was unaffected by the LTB4 and PAF antagonists LY255283 and WEB 2170, respectively. However, it was inhibited by approximately 75% by monoclonal antibodies to CD49d (VLA-4) or CD11a (LFA-1) but was not significantly affected by an antibody to CD11b (Mac-1). In conclusion, 5-oxo-ETE induces pulmonary eosinophilia in Brown Norway rats, raising the possibility that it may be a physiological mediator of inflammation in asthma.

Calcium/calmodulin-dependent conversion of 5-oxoeicosanoids to 6, 7-dihydro metabolites by a cytosolic olefin reductase in human neutrophils.

We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils. The first step in the formation of these metabolites is oxidation of the 5-hydroxyl group by 5-hydroxyeicosanoid dehydrogenase. The objective of the present investigation was to characterize the second step in the formation of the dihydro metabolites, reduction of an olefinic double bond. We found that the olefin reductase reduces the 6,7-double bond of 5-oxoeicosanoids, is localized in the cytosolic fraction of neutrophils, and requires NADPH as a cofactor. Neutrophil cytosol converts a variety of both 5-oxo- and 15-oxoeicosanoids to dihydro products. However, conversion of 5-oxoeicosanoids to their 6,7-dihydro metabolites is inhibited by EGTA and a calmodulin antagonist and stimulated by the addition of calcium and calmodulin, whereas the reduction of 15-oxoeicosanoids to their 13,14-dihydro metabolites is slightly inhibited by calcium. Furthermore, eicosanoid Delta6- and Delta13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Delta6-reductase to 6,7-dihydro-5-oxo-ETE, which is 1000 times less potent than 5-oxo-ETE in mobilizing calcium in neutrophils. We conclude that neutrophils contain both 5-oxoeicosanoid Delta6-reductase and prostaglandin Delta13-reductase. Metabolism of 5-oxo-ETE by the Delta6-reductase results in loss of its biological activity.