Stem cell factor plays a major role in the recruitment of eosinophils in allergic pleurisy in mice via the production of leukotriene B4

The understanding of the mechanisms underlying eosinophil migration into tissue is an essential step in the development of novel therapies aimed at treating allergic diseases where eosinophil recruitment and activation are thought to play an essential role. In this study, we have examined the effects of the in vivo administration of stem cell factor (SCF) on eosinophil recruitment and tested whether endogenous SCF was involved in mediating eosinophil recruitment in response to Ag challenge in sensitized mice. The intrapleural injection of SCF induced a time- and concentration-dependent recruitment of eosinophils in mice. In allergic mice, SCF message was expressed early after Ag challenge and returned to baseline levels after 8 h. In agreement with the ability of SCF to induce eosinophil recruitment and its expression in the allergic reaction, an anti-SCF polyclonal Ab abrogated eosinophil recruitment when given before Ag challenge. SCF increased the levels of leukotriene B4 (LTB4) in the pleural cavity of mice and an LTB4 receptor antagonist, CP105,696, abrogated the effects of SCF on eosinophil recruitment. Similarly, recruitment of eosinophils in the allergic reaction was virtually abolished by CP105,696. Together, our data favor the hypothesis that the local release of SCF following Ag challenge may activate and/or prime mast cells for IgE-mediated release of inflammatory mediators, especially LTB4. The mediators released in turn drive the recruitment of eosinophils. Inhibition of the function of SCF in vivo may reduce the migration of eosinophils to sites of allergic inflammation and may, thus, be a relevant principle in the treatment of allergic diseases.     

Stem cell factor-induced leukotriene B4 production cooperates with eotaxin to mediate the recruitment of eosinophils during allergic pleurisy in mice.

The understanding of the mechanisms underlying eosinophil recruitment in vivo may aid in the development of novel strategies for the treatment of allergic disorders. In this study, we investigated the role of chemokines in the cascade of events leading to eosinophil recruitment in a stem cell factor (SCF)- and leukotriene B(4) (LTB(4))-dependent allergic pleurisy model in mice. The intrapleural administration of the eosinophil-active chemokines eotaxin, RANTES, and macrophage-inflammatory protein 1alpha (MIP-1alpha) induced a time- and dose-dependent eosinophil recruitment. Pretreatment with anti-eotaxin, but not anti-RANTES or anti-MIP-1alpha, blocked the recruitment of eosinophils following Ag challenge of sensitized animals, and significant eotaxin immunoreactivity was detected in the pleural cavity of these animals. Similarly, only the anti-eotaxin inhibited the eosinophil recruitment induced by injection of SCF in naive animals. However, blockade of SCF did not inhibit the release of eotaxin after Ag challenge of sensitized mice. Akin to its effects on SCF and in the allergic reaction, eotaxin-induced eosinophil recruitment was blocked by the LTB(4) receptor antagonist CP105696. Nevertheless, SCF, but not eotaxin, appeared to regulate the endogenous release of LTB(4) after Ag challenge. Finally, we show that low doses of eotaxin synergized with LTB(4) to induce eosinophil recruitment in the pleural cavity. Overall, the present results show that eotaxin and SCF-induced LTB(4) cooperate to induce eosinophil recruitment into sites of allergic inflammation. Cooperation between inflammatory mediators must be an important phenomenon in vivo, explaining both the ability of lower concentrations of mediators to induce a full-blown functional response and the effectiveness of different strategies at inhibiting these responses.     

IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B4

IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-alpha induction, which drives the production of leukotriene B(4) (LTB(4)), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB(4) and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB(4) synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB(4) production could also be blocked by a neutralizing anti-TNF-alpha Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55(-/-) mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease severity and reduced articular inflammation and joint destruction. Furthermore, MK-886-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Finally, we showed that IL-18-activated human peripheral blood neutrophils produced significant amounts of LTB(4) that were effectively blocked by the MK. Together, these findings provide a novel mechanism whereby IL-18 can promote inflammatory diseases.     

MK886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5-lipoxygenase in ionophore-challenged leukocytes

Recently, we have shown that ionophore activation of human leukocytes results in leukotriene synthesis and a translocation of 5-lipoxygenase from the cytosol to cellular membrane. This membrane translocation was postulated to be an important early activation step for the enzyme. 3-[1-(p-Chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2- dimethylpropanoic acid (MK886) is a potent and specific inhibitor of leukotriene biosynthesis in vivo and in intact cells, but has no direct effect on 5-lipoxygenase activity in cell-free systems. In this report, we show that MK886 can both prevent and reverse the membrane translocation of 5-lipoxygenase, in conjunction with the inhibition of leukotriene synthesis. Similar compounds of the indole class could also inhibit the membrane translocation of 5-lipoxygenase in a rank order of potency that correlated with their potencies for leukotriene synthesis inhibition. In contrast L-656,224, a direct 5-lipoxygenase inhibitor, had no effect on the translocation of the enzyme. Attempts to demonstrate the effects of MK886 on the association of 5-lipoxygenase with membrane in cell-free preparations failed due to a nonspecific Ca2+-dependent sedimentation of the enzyme. The mechanism of action of MK-886 is therefore to block translocation, prevent subsequent activation of 5-lipoxygenase, and hence block cellular leukotriene biosynthesis.     

CCR8 is expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate Th2-mediated granuloma formation in mice

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4+ T cells. Populations of cytokine-secreting CD4+ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4+ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-gamma-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10+ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10+CCR8+ populations were CD25+ and CD44+ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10+ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8-/- mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4+CCR8+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10+ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8+ cells in granulomatous lungs. Thus, IL-10-producing, CCR8+CD4+CD25+CD44+ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.     

Inhibition of leukotriene biosynthesis abrogates the host control of Mycobacterium tuberculosis

Leukotrienes produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. Recently, it has been demonstrated that leukotrienes also play an important role in host defense against microorganisms. In vitro studies have shown that leukotrienes augmented the anti-mycobacterial activity of neutrophils. In this study, we examined the role of leukotrienes in regulating host response and cytokine generation in a murine model of tuberculosis. Administration of the 5-LO pathway inhibitor MK 886, which reduced lung levels of both the leukotriene B(4) and the anti-inflammatory substance lipoxin A(4) by approximately 50%, increased 60-day mortality from 14% to approximately 57% in Mycobacterium tuberculosis-infected mice, and increased lung bacterial burden by approximately 15-fold. Although MK 886-treated animals exhibited no reduction in pulmonary leukocyte accumulation, they did manifest reduced levels of nitric oxide generation and of the protective type 1 cytokines interleukin-12 and gamma interferon. Together our results demonstrate that 5-LO pathway product(s) - presumably leukotrienes - positively regulate protective Th1 responses against mycobacterial infection in vivo. Moreover, the immunosuppressive phenotype in infected mice observed with MK 886 is most consistent with inhibition of an activator (LTB(4)) rather than a suppressor (LXA(4)) of antimicrobial defense, suggesting the major effect of leukotrienes.     

Mast cell mediators prostaglandin-D2 and histamine activate human eosinophils.

Airway damage secondary to eosinophil activation is thought to contribute to the development of asthma. Using the fluorescent dye FURA-2 to measure the concentration of cytosolic calcium, we found that supernatants from anti-IgE-stimulated human lung mast cells increased cytosolic calcium in human eosinophils. We then examined the major mast cell mediators (histamine, PGD2, platelet-activating factor (PAF), eosinophil chemotactic factor of anaphylaxis (ECF-A), leukotriene (LT)C4 and LTB4) for their ability to increase cytosolic calcium in eosinophils. We found that both PAF (5 x 10(-9) to 5 x 10(-6) M) and PGD2 (two of five donors responsive at 1 x 10(-9) M) were potent stimuli for calcium mobilization. LTB4 (10(-8), 10(-7) M) and histamine were also active, although higher concentrations of histamine were required to see a response (3 x 10(-7) to 10(-5) M). LTC4, val-ECF-A, and ala-ECF-A were inactive. The effects of PGD2 and histamine were specific for eosinophils, although LTB4 and PAF increased calcium in both neutrophils and eosinophils. The histamine-induced increase in intracellular calcium was not blocked by the H1 or H2 antagonists pyrilamine or cimetidine (10(-4) M), respectively; however, the response to 10(-6) M histamine was completely blocked by the specific H3 antagonist thioperamide (10(-6) M). To evaluate the relative contribution of these stimulatory mast cell mediators on the calcium mobilizing activity in supernatants from anti-IgE-stimulated human lung mast cell (HLMC), we examined the effect of supernatants from HLMC pretreated with indomethacin and/or the 5-lipoxygenase pathway inhibitor MK886. These supernatants were added to FURA-2-loaded eosinophils that had been preincubated with thioperamide and/or the PAF antagonist WEB-2086. We found that the increase in eosinophil calcium in response to supernatants from anti-IgE-stimulated-HLMC was totally inhibited only when the mast cells were challenged in the presence of indomethacin and MK886, and the eosinophils were preincubated with thioperamide. WEB-2086 had little effect. When we examined the effect of these mediators on eosinophil secretory function, we found that PGD2 (not histamine) primed eosinophils for enhanced release of LTC4 in response to the calcium ionophore A23187. We conclude that the activation of eosinophils by PGD2 and other mast cell products may contribute to airways inflammation that is characteristic of asthma.     

Prolonged pulmonary hypertension caused by platelet-activating factor and leukotriene C4 in the rat lung.

Platelet-activating factor (PAF) and leukotrienes (LTs) are potent pulmonary hypertensive and inflammatory mediators produced by the lung. Previously we showed that a rapid injection of PAF into the pulmonary artery of an isolated rat lung produced an extended elevation in mean pulmonary arterial pressure (PAP). The objective of the present study was to determine whether the extended pressor response induced by PAF was caused by prolonged activation of the 5-lipoxygenase pathway or slow clearance of LTs from the lung parenchyma. Rat lungs were perfused with a nonrecirculating physiological salt solution that contained indomethacin and albumin. Five minutes after a rapid injection of PAF into the pulmonary artery catheter, the following elevations (mean % above baseline) were observed: PAP (83%), LTB4 (3,260%), LTC4 (1,490%), LTD4 (970%), and LTE4 (1,500%). At 20 min these levels declined but were still significantly elevated above baseline. The 5-lipoxygenase inhibitor diethylcarbamazine (DEC), administered before the PAF injection, inhibited the elevations of PAP and all LTs. DEC administration that began 5 min after PAF reduced PAP and only LTC4 levels at 20 min in comparison to lungs with no DEC. The 5-lipoxygenase-activating protein inhibitor MK886, administered orally 2-6 h before perfusion, also inhibited the pressor response to PAF as well as LT production, as did DEC. We conclude that 1) the extended pulmonary hypertension induced by PAF was caused mainly by prolonged activation of 5-lipoxygenase with LTC4 production, 2) the relative overall lung clearance of LTB4, LTD4, and LTE4 was slower than that of LTC4, and 3) LTB4, LTD4, and LTE4 had no appreciable pressor effect.     

Leukotriene B4 mediates neutrophil migration induced by heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.     

Effect of inhibition of 5-lipoxygenase metabolism of arachidonic acid on response to endotoxemia in sheep

We studied the effects of a 5-lipoxygenase inhibitor, L-651,192, on the pulmonary dysfunction caused by endotoxemia in chronically instrumented unanesthetized sheep. The efficacy and selectivity of L-651,392 were tested by measuring in vivo production of leukotriene B4 (LTB4) and cyclooxygenase products of arachidonic acid after endotoxemia before and after pretreatment with L-651,392 and ex vivo from granulocytes and whole blood stimulated with calcium ionophore from sheep before and 24 h after pretreatment with L-651,392. A novel assay for LTB4 by high-performance liquid chromatography/gas chromatography/mass spectrometry techniques was developed as a measure of 5-lipoxygenase metabolism of arachidonic acid. L-651,392 proved to be an effective in vivo 5-lipoxygenase inhibitor in sheep. L-651,392 blocked the increase in LTB4 observed in lung lymph after endotoxemia in vivo in sheep as well as inhibited by 80% the ex vivo production of LTB4 by granulocytes removed from sheep treated 24 h earlier with L-651,392. Although L-651,392 blocked the increase in cyclooxygenase products of arachidonic acid observed in lung lymph after endotoxemia in vivo in sheep, the drug probably did not function directly as a cyclooxygenase inhibitor. L-651,392 did not attenuate the ex vivo production of thromboxane B2 by whole blood from sheep treated 24 h earlier with the drug. L-651,392 attenuated the alterations in pulmonary hemodynamics, lung mechanics, oxygenation, and lung fluid and solute exchange observed after endotoxemia in sheep. We speculate that 5-lipoxygenase products are a major stimulus for cyclooxygenase metabolism of arachidonic acid after endotoxemia in sheep.     

Radioimmunoassay of LTB4 in plasma from different species: a cautionary note

In clinical and pre-clinical research the pharmacodynamics of selective 5-lipoxygenase and dual 5-lipoxygenase/cyclo-oxygenase inhibitors may be studied by direct RIA of plasma LTB4. Although immunoreactive LTB4 in plasma from A23187 stimulated human blood has the characteristics of authentic LTB4 our results show, particularly in mice and rats, that exposure to A23187 produces large quantities of 12-HETE. Since in different species the levels of 12-HETE increase with platelet concentration we suggest that the 12(S)-HETE is produced by platelet lipoxygenase. However, we do not rule out the possibility that a proportion of 12-HETE may exist as the (R)-stereoisomer. The latter has greater potential for interference in the direct RIA of LTB4. Biosynthesis of 12-HETE may be measured either by RPHPLC/U.V. abs. (8) or by RIA (9) and LTB4 by a more specific antibody described in this report. We conclude that the combined ex vivo RIA of plasma TXB2, LTB4 and 12-HETE has utility in determining the selectivity of inhibitors of arachidonate metabolism and in distinguishing between selective 5-lipoxygenase inhibitors which interact directly with the enzyme and anti-oxidant or free radical scavenging types which may be less specific.     

Quantitation of leukotriene B4 in human serum by negative ion gas chromatography-mass spectrometry

Leukotriene B4 (LTB4) is a potent chemotactic agent formed via the 5-lipoxygenase pathway from arachidonic acid. To understand the role LTB4 plays in several pathological processes it is essential that endogenous concentrations of LTB4 be accurately quantitated. We have developed a method based on electron capture negative ion mass spectrometry for the analysis of LTB4 in serum at low picogram per milliliter concentrations. Blood is collected into the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) to suppress ex vivo formation. Serum is isolated, equilibrated with the internal standard [2H4]LTB4, and extracted using octadecyl-silica (C-18) cartridges. After conversion of the carboxylic acids to their pentafluorobenzyl esters the extract is purified by straight-phase HPLC. Gas chromatographic-mass spectrometric analysis is accomplished on the tert-butyldimethylsilyl ether derivatives using dual-selected ion monitoring of m/z 431 and 435. These ions correspond to loss of tert-butyldimethylsilanol from the (M-PFB)- ion of endogenous and [2H4]LTB4, respectively. The concentration of LTB4 in human serum samples was 10.0 +/- 4.0 pg/ml (n = 5). The assay exhibited satisfactory precision, with an intraassay coefficient of variation of 17% and a high degree of accuracy. The concentration of LTB4 in serum collected with (NDGA) was less than 10% of that observed in blood collected without the lipoxygenase inhibitor. Ex vivo formation can therefore be a major obstacle in assessing circulating levels of LTB4.     

Leukotrienes play a role in the control of parasite burden in murine strongyloidiasis

It is clear that leukotrienes mediate inflammatory response; new aspects of leukotriene function have recently been described. In this study, we demonstrate that leukotrienes are key chemical mediators in the control of parasite burdens in mice infected with Strongyloides venezuelensis. High leukotriene levels were detected in the lungs and small intestines of Swiss mice. In infected Swiss mice treated with MK886, a leukotriene synthesis inhibitor, numbers of adult worms, and eggs/g/feces were greater than in infected-only animals. The MK886 treatment inhibited leukotriene B(4) production in the lungs and small intestines, albeit on different postinfection days. Similarly, parasite burdens and eggs/g/feces were greater in 5-lipoxygenase(-/-) mice than in wild-type animals. These observation were confirmed by histopathological study of the duodena. We subsequently observed significant lower numbers of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid of Swiss mice treated with MK886. In the lung parenchyma of infected animals, MK886 significantly inhibited synthesis of IL-5 at the beginning of infection, whereas levels of IL-12 increased progressively throughout the postinfection period. However, levels of leukotriene C(4), PGE(2), TNF-alpha, IL-3, IL-4, IFN-gamma, and IL-10 were comparable between the treated and untreated groups. Nevertheless, IgE and IgG1 (but not IgG2a) synthesis was also significantly inhibited by MK886 administration. Therefore, in S. venezuelensis-infected mice, adult worm and egg burdens are leukotriene dependent. These findings indicate potential immunostimulatory strategies involving leukotriene administration, and may serve as an alert to physicians treating Strongyloides stercoralis-infected patients presenting asthma-like symptoms because use of 5-lipoxygenase inhibitors may worsen the infection.     

Expanding expression of the 5-lipoxygenase/leukotriene B4 pathway in atherosclerotic lesions of diabetic patients promotes plaque instability

Emerging evidence now indicates that the 5-lipoxygenase (5-LO) pathway play a role in the pathogenesis of atherosclerosis and restenosis. The expression of 5-LO by activated macrophages in symptomatic plaques leads to leukotriene B(4) (LTB(4)) accumulation and enhanced synthesis and release of matrix metalloproteinases (MMPs) that can promote plaque rupture. However, the role of 5-LO pathway in diabetic vascular disease has not been previously reported. Thus, the present study was designed to analyze the expression of 5-LO in carotid plaques of diabetic patients and to investigate the possible role of 5-LO pathway in the pathogenesis and progression of diabetic atherosclerosis. Atherosclerotic plaques from 60 patients undergoing carotid endarterectomy were divided into non-diabetic and diabetic group. Plaques were analyzed for 5-LO, MMP-2 and MMP-9 by immunohistochemical, Western blot, and densitometric analyses, whereas zymography was used to detect MMP activity. Immunocytochemistry was also used to identify CD68+macrophages, CD3+T-lymphocytes, and HLA-DR+inflammatory cells. LTB(4) were quantified by enzyme-linked immunosorbent assay. 5-LO showed abundant immunoreactivity in human atherosclerotic carotid lesions, and was colocalized with macrophage infiltrates in atherosclerotic intima. 5-LO expression was higher in diabetic compared with non-diabetic plaques and was associated with increased MMP-2 and MMP-9 expression. Follow-up analyze with zymography assay revealed MMP activity was elevated in diabetic compared with non-diabetic plaques. Notably, in contrast to non-diabetic plaques, LTB(4) levels were significantly increased in diabetic plaques by enzyme-linked immunosorbent assay. These results suggest that overexpression of 5-LO and LTB(4) in atherosclerotic plaques possibly promote MMP-induced plaque rupture in diabetes. Hence, anti-LTs may be useful, not only in reducing atherogenesis, but also in the prevention and treatment of acute atherothrombotic events in diabetic patients.     

Evidence for local eosinophil differentiation within allergic nasal mucosa: inhibition with soluble IL-5 receptor

Eosinophil differentiation occurs within the bone marrow in response to eosinopoietic cytokines, particularly IL-5. Recently, however, eosinophil precursors (CD34/IL-5Ralpha+ cells) and IL-5 mRNA+ cells have been identified within the lungs of asthmatics, indicating that a population of eosinophils may differentiate in situ. In this report, we examined the presence of eosinophil precursors within allergic nasal mucosa and examined whether they undergo local differentiation following ex vivo stimulation. We cultured human nasal mucosa obtained from individuals with seasonal allergic rhinitis with either specific allergen, recombinant human IL-5 (rhIL-5), or allergen + soluble IL-5Ralpha (sIL-5Ralpha), shown to antagonize IL-5 function. Simultaneous immunocytochemistry and in situ hybridization demonstrated that there were fewer cells coexpressing CD34 immunoreactivity and IL-5Ralpha mRNA following culture with allergen or rhIL-5, compared with medium alone. Immunostaining revealed that the number of major basic protein (MBP) immunoreactive cells (eosinophils) was higher within tissue stimulated with allergen or rhIL-5, compared with unstimulated tissue. In situ hybridization detected an increase in IL-5 mRNA+ cells in sections from tissue cultured with allergen, compared with medium alone. These effects were not observed in tissue cultured with a combination of allergen and sIL-5Ralpha. Colocalization analysis indicated this expression to be mainly, but not exclusively, T cell (44%) and eosinophil (10%) derived. Our findings suggest that a subset of eosinophils may differentiate locally within allergic nasal mucosa, in what appears to be a highly IL-5-dependent fashion, and imply that this process might be regulated in vivo by endogenous production of sIL-5Ralpha.     

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.     

Role of CCL5 (RANTES) in viral lung disease

CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+ and CD8+ cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.     

Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein''s expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), the product of 5-lipoxygenase, or 5-HPETE''s downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of the TRPM7 channel capable of attenuating TRPM7''s function during cell stress, making them effective tools for the biophysical characterization and suppression of TRPM7 channel conductance in vivo.     

Inhibition of airway inflammation by amino-terminally modified RANTES/CC chemokine ligand 5 analogues is not mediated through CCR3

Chemokines play a key role in the recruitment of activated CD4(+) T cells and eosinophils into the lungs in animal models of airway inflammation. Inhibition of inflammation by N-terminally modified chemokines is well-documented in several models but is often reported with limited dose regimens. We have evaluated the effects of doses ranging from 10 ng to 100 micro g of two CC chemokine receptor antagonists, Met-RANTES/CC chemokine ligand 5 (CCL5) and aminooxypentane-RANTES/CCL5, in preventing inflammation in the OVA-sensitized murine model of human asthma. In the human system, aminooxypentane-RANTES/CCL5 is a full agonist of CCR5, but in the murine system neither variant is able to induce cellular recruitment. Both antagonists showed an inverse bell-shaped inhibition of cellular infiltration into the airways and mucus production in the lungs following allergen provocation. The loss of inhibition at higher doses did not appear to be due to partial agonist activity because neither variant showed activity in recruiting cells into the peritoneal cavity at these doses. Surprisingly, neither was able to bind to the major CCR expressed on eosinophils, CCR3. However, significant inhibition of eosinophil recruitment was observed. Both analogues retained high affinity binding for murine CCR1 and murine CCR5. Their ability to antagonize CCR1 and CCR5 but not CCR3 was confirmed by their ability to prevent RANTES/CCL5 and macrophage inflammatory protein-1beta/CCL4 recruitment in vitro and in vivo, while they had no effect on that induced by eotaxin/CCL11. These results suggest that CCR1 and/or CCR5 may be potential targets for asthma therapy.