Ultraviolet B irradiation selectively increases the production of interleukin-8 in human cord blood-derived mast cells

UVB irradiation modulates immune responses in the skin and is a major cause of sunburn, during which neutrophils accumulate in the skin. Because of their abundance in skin and ability to produce a variety of proinflammatory mediators, we propose that mast cells may play a key role in ultraviolet B (UVB)-induced skin inflammation. Cord blood-derived human mast cells were treated in vitro with varying doses of UVB and production of multiple cytokines was measured in culture supernatants. UVB exposure significantly increased the release of interleukin (IL)-8 and modestly increased IL-1alpha production, but cytokines such as IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma were unaffected. Cycloheximide reduced the UVB-mediated induction of IL-8 by 30-40%, suggesting that new protein synthesis contributed to IL-8 production. In line with this, UVB treatment of mast cells significantly increased IL-8 mRNA. In contrast to its effect on IL-8 production, optimal doses of UVB did not provoke histamine or tryptase release, indicating little effect on degranulation. Our data suggest that mast cells may play a major role during UVB-induced acute inflammation by selectively inducing cytokines involved in neutrophil recruitment.     

Regulation of eosinophil-active cytokine production from human cord blood-derived mast cells.

Human mast cells are multifunctional tissue-dwelling cells that play a crucial role in eosinophil-dependent disorders, such as asthma and parasitic diseases, by the secretion of eosinophil-active mediators. Mast cell-derived cytokines, generated in response to cross-linking of the high-affinity IgE receptor, can regulate eosinophil activation, survival, and chemotaxis. In this study, mast cells generated from human cord blood progenitors (stem cells) were studied for eosinophil-active inflammatory cytokine expression. Cord blood-derived mast cells (CBDMC) expressed typical intracellular scroll granules and microvilli-like structures on their cell surfaces, demonstrated the presence of tryptase, and elaborated prostaglandin D2 (PGD2) after cross-linkage of the high-affinity receptor for IgE (FcepsilonRI). CBDMC expressed tumor necrosis factor-alpha (TNF-alpha) and the eosinophil-active growth factors, interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after activation. (IL-1beta greatly enhanced IgE-dependent production of these cytokines in response to FcepsilonRI cross-linkage, suggesting a role for bystander/phagocytic cells in modulating mast cell function. In contrast, interferon-alpha (IFN-alpha) inhibited IL-5 and GM-CSF generation, and the glucocorticoid, dexamethasone (Dex), inhibited production of IL-5 and GM-CSF from CBDMC. A macrophage-mast cell-eosinophil axis may exist in vivo that may be susceptible to pharmacologic manipulation.     

Human umbilical cord blood-derived mast cells

Findings obtained using animal models have often failed to reflect the processes involved in human disease. Moreover, human cultured cells do not necessarily function as their actual tissue counterparts. Therefore, there is great demand for sources of human progenitor cells that may be directed to acquire specific tissue characteristics and be available in sufficient quantities to carry out functional and pharmacological studies. A case in point is the mast cell, well known for its involvement in allergic reactions, but also implicated in inflammatory diseases. Mast cells can be activated by allergens, anaphylatoxins, immunoglobulin-free light chains, superantigens, neuropeptides, and cytokines, leading to selective release of mediators. These could be involved in many inflammatory diseases, such as asthma and atopic dermatitis, which worsen by stress, through activation by local release of corticotropin-releasing hormone or related peptides. Umbilical cord blood and cord matrix-derived mast cell progenitors can be separated magnetically and grown in the presence of stem cell factor, interleukin-6, interleukin-4, and other cytokines to yield distinct mast cell populations. The recent use of live cell array, with its ability to study such interactions rapidly at the single-cell level, provides unique new opportunities for fast output screening of mast cell triggers and inhibitors.     

Interleukin-8 induces neutrophil transendothelial migration.

Interleukin-8 (IL-8) is a potent neutrophil chemotactic stimulant. We have used chemically synthesized IL-8 to investigate its role in human neutrophil adhesion and transendothelial migration. IL-8 enhanced the adhesiveness of human neutrophils to plastic, and to both unstimulated and tumour necrosis factor (TNF)-stimulated endothelial monolayers in vitro. Using a two-compartment model separated by a confluent endothelial monolayer, we have shown that IL-8 chemotactic stimulation induced transmigration across the monolayer of up to 87.4 +/- 2.1% of added neutrophils (compared to random unstimulated transmigration of 2.2 +/- 0.7%), while chemokinetic stimulation led to transmigration of 21 +/- 3.8% of neutrophils. Preincubation of endothelium with TNF also induced transmigration in this model, and was additive when combined with an IL-8 chemotactic stimulus. Endothelial permeability was increased at maximal rates of chemotactic transmigration, which may correlate with increased permeability of vessels at inflammatory sites in vivo. The property of IL-8 to stimulate movement of neutrophils across endothelial monolayers in vitro supports the concept of a central role for this molecule in the accumulation of neutrophils at inflammatory lesions in vivo.     

Autocrine regulation of cord blood-derived human mast cell activation by IL-10.

BACKGROUND: Ligation of the high-affinity receptor for IgE on human mast cells (MCs) induces the release of proinflammatory mediators, including vasoactive amines and cytokines (TNF-alpha, IL-5, and IL-8). Moreover, we have recently shown that IL-10 inhibits the release of proinflammatory mediators by activated MCs. OBJECTIVE: We investigated whether human cord blood-derived MCs (CBMCs) could produce IL-10 and whether this production could inhibit their activation in an autocrine fashion. METHODS: IL-10 synthesis by resting or activated human MCs derived from cord blood progenitors was investigated in cell supernatants or by using immunostaining and RT-PCR methods. In addition, the effect of IL-4 on such synthesis was also studied. Anti-IL-10-neutralizing antibodies were used to investigate the validity of the hypothesis of an autocrine regulation of MCs by IL-10. Finally, the presence of specific receptors for IL-10 was searched on human CBMCs by using flow cytometric analysis. RESULTS: Human CBMCs spontaneously synthesize and release IL-10, and this synthesis is increased after IgE/anti-IgE stimulation. In addition, the presence of IL-10 in resting or in activated MCs was proved by immunostaining. Interestingly, the release of IL-10 was also increased after incubation of the cells with IL-4. Besides, the use of neutralizing antibodies against IL-10 confirmed that IL-10 released inhibited MC activation in an autocrine fashion. Finally, the presence of specific receptors for this cytokine was observed on the membranes of our population of human CBMCs. CONCLUSION: Taken together, our data are in favor of an autocrine regulation pathway through synthesis and release of IL-10 by human MCs. Such an autoregulatory mechanism is, to our knowledge, the first described for these elements.     

Dexamethasone inhibits maturation, cytokine production and FcεRI expression of human cord blood-derived mast cells

BACKGROUND: Mast cells are responsible for eliciting the early phase and for contributing to the development of the late phase of allergic reactions, through the release of cytokines and other inflammatory mediators. OBJECTIVE: To assess whether the glucocorticoid dexamethasone has a direct effect on mast cell progenitor maturation and on mature cord blood-derived mast cell properties. METHODS: Mast cells were obtained by culturing human umbilical cord blood mononuclear cells with stem cell factor, IL-6 and prostaglandin E2. Mast cell numbers were assessed by Toluidine Blue staining and immunocytochemistry of tryptase positive cells. The expression of Fc epsilon RI, CD49d and c-kit was assessed by flow cytometry. Histamine release was determined by a radioenzymatic assay. Cys-LT, GM-CSF and TNF-alpha production and release were determined by ELISA. RESULTS: Dexamethasone (10(-6) M-10(-9) M) time- and dose-dependently inhibited the maturation of the mast cell progenitors. Dexamethasone did not affect the basal expression of Fc epsilon RI, CD49d and c-kit, but it inhibited the IgE-dependent enhanced expression of Fc epsilon RI. Dexamethasone (10(-6) M-10(-9) M) had no significant effect on Fc epsilon RI-dependent histamine release or the synthesis and release of Cys-LT from the mature mast cells. However, pre-incubation of the mast cell cultures with dexamethasone for 1 h, prior to cross-linking of Fc epsilon RI, dose-dependently inhibited the production and secretion of both GM-CSF and TNF-alpha. CONCLUSIONS: From these in vitro data we propose that glucocorticosteroids are effective drugs in the management of allergic inflammation due to their capacity to inhibit mast cell development, IgE-dependent Fc epsilon RI expression and mast cell production of GM-CSF and TNF-alpha.     

IL-33 induces IL-13 production by mouse mast cells independently of IgE-FcepsilonRI signals

The IL-1-related molecules, IL-1 and IL-18, can promote Th2 cytokine production by IgE/antigen-FcepsilonRI-stimulated mouse mast cells. Another IL-1-related molecule, IL-33, was identified recently as a ligand for T1/ST2. Although mouse mast cells constitutively express ST2, the effects of IL-33 on mast cell function are poorly understood. We found that IL-33, but not IL-1beta or IL-18, induced IL-13 and IL-6 production by mouse bone marrow-derived, cultured mast cells (BMCMCs) independently of IgE. In BMCMCs incubated with the potently cytokinergic SPE-7 IgE without specific antigen, IL-33, IL-1beta, and IL-18 each promoted IL-13 and IL-6 production, but the effects of IL-33 were more potent than those of IL-1beta or IL-18. IL-33 promoted cytokine production via a MyD88-dependent but Toll/IL-1R domain-containing adaptor-inducing IFN-beta-independent pathway. By contrast, IL-33 neither induced nor enhanced mast cell degranulation. At 200 ng/ml, IL-33 prolonged mast cell survival in the absence of IgE and impaired survival in the presence of SPE-7 IgE, whereas at 100 ng/ml, IL-33 had no effect on mast cell survival in the absence of IgE and reduced mast cell survival in the presence of IgE. These observations suggest potential roles for IL-33 in mast cell- and Th2 cytokine-associated immune responses and disorders.     

Ion channel gene expression in human lung,skin, and cord blood-derived mast cells

Immunoglobulin E (IgE)-dependent activation of human mast cells (HMC) is characterized by an influx of extracellular calcium (Ca(2+)), which is essential for subsequent release of preformed (granule-derived) mediators and newly generated autacoids and cytokines. In addition, flow of ions such as K(+) and Cl(-) is likely to play an important role in mast cell activation, proliferation, and chemotaxis through their effect on membrane potential and thus Ca(2+) influx. It is therefore important to identify these critical molecular effectors of HMC function. In this study, we have used high-density oligonucleotide probe arrays to characterize for the first time the profile of ion channel gene expression in human lung, skin, and cord blood-derived mast cells. These cells express mRNA for inwardly rectifying and Ca(2+)-activated K(+) channels, voltage-dependent Na(+) and Ca(2+) channels, purinergic P2X channels, transient receptor potential channels, and voltage-dependent and intracellular Cl(-) channels. IgE-dependent activation had little effect on ion channel expression, but distinct differences for some channels were observed between the different mast cell phenotypes, which may contribute to the mechanism of functional mast cell heterogeneity.     

氧化型α1-抗胰蛋白酶对HBE细胞释放炎症因子的影响

 目的：探讨氧化型α1-抗胰蛋白酶（Ox-AT）对体外培养人支气管上皮(HBE)细胞炎症因子白细胞介素8（IL-8）和单核细胞趋化蛋白1（MCP-1）释放的影响及可能机制。方法：从人血浆中分离纯化获得天然构型AT（N-AT）,加入氧化剂获得Ox-AT;用不同浓度N-AT和Ox-AT分别作用于体外培养的HBE细胞,用ELISA方法检测不同时段培养上清液中IL-8和MCP-1的含量,同时观察NF-κB抑制剂Bay11-7082对Ox-AT引起HBE细胞炎症因子释放的影响。结果：Ox-AT可促进HBE细胞释放IL-8和MCP-1,其促进作用与Ox-AT的浓度及作用时间呈正相关;用0.5 g/L Ox-AT孵育HBE细胞4、10和24 h,其促进HBE细胞分泌IL-8和MCP-1的作用与10 μg/L肿瘤坏死因子 α（TNF-α）的作用基本一致,而 N-AT无刺激HBE细胞分泌IL-8和MCP-1的作用;Ox-AT 能显著增加NF-κB活性; Ox-AT的促炎症作用能被NF-κB抑制剂Bay11-7082抑制。结论：Ox-AT是人正常支气管上皮细胞的强致炎因子,机制可能与NF-κB信号通路激活有关。     

Helicobacter pylori induces transendothelial migration of activated memory T cells

Helicobacter pylori infection is associated with pronounced infiltration of granulocytes and lymphocytes into the gastric mucosa, resulting in active chronic gastritis that may develop into duodenal ulcer disease or gastric adenocarcinoma. Infiltrating T cells play a major role in the pathology of these diseases, but the signals involved in recruitment of T cells from blood to H. pylori-infected tissues are not well understood. We therefore examined H. pylori-induced T-cell transendothelial migration (TEM). The Transwell system, employing a monolayer of human umbilical vein endothelial cells, was used as a model to study TEM. H. pylori induced a significant T-cell migration, compared to spontaneous migration. CD4+ and CD8+ T cells migrated to the same extent in response to H. pylori, whereas there was significantly larger transmigration of memory T cells compared to naive T cells. Both H. pylori culture filtrate and urease induced migration, and the presence of the H. pylori cag pathogenicity island increased TEM. T-cell TEM was mediated by LFA-1-ICAM-1 interactions in accordance with an increased ICAM-1 expression on the endothelial cells after contact with H. pylori. Migrating T cells had increased expression of activation marker CD69 and chemokine receptors CXCR3, CCR4, and CCR9. Furthermore, T cells migrating in response to H. pylori secreted Th1 but not Th2 cytokines upon stimulation. In conclusion, our data indicate that live H. pylori and its secreted products contribute to T-cell recruitment to the gastric mucosa and that the responding T cells have an activated memory Th1 phenotype.     

Lysophosphatidic acid induces lymphangiogenesis and IL-8 production in vitro in human lymphatic endothelial cells

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.     

HLA-G inhibits the transendothelial migration of human NK cells

The expression of the non-classical MHC class I molecule HLA-G is normally restricted to the placenta during pregnancy, where it is found on fetal endothelial cells and on invasive cytotrophoblast cells, specifically those at the maternal / fetal interface. Its precise physiological role has yet to be defined. HLA-G may have nonimmune functions relating to angiogenesis and placentation, but most evidence suggests that it protects fetal cells from lysis by maternal uterine NK cells, which are found in large numbers around invading trophoblast cells. This effect is due to specific interaction with inhibitory receptors expressed on NK cells. We have examined the hypothesis that another function of HLA-G is to inhibit NK cell migration. Using an in vitro transmigration assay system, we present data to support this hypothesis. NK cell migration across porcine endothelial cells transfected with HLA-G1 was specifically inhibited compared to migration across HLA-A2-transfected monolayers. HLA- G1 had no influence on the migration of a control T lymphocyte line. These results support the idea that in vivo, HLA-G may inhibit NK cell traffic across the placenta.     

The establishment of a combined serum-free and serum-supplemented culture method of obtaining functional cord blood-derived human mast cells

BACKGROUND: Serum-free cultures supplemented with stem cell factor (SCF) and IL-6 is reported to support the extensive growth of less functional human cord blood-derived mast cells. OBJECTIVE: To obtain more functional mast cells from cord blood, we developed a culture system combining a serum-free condition for 0-8 weeks of culture, and followed by a serum-supplemented culture condition and examined the function of the cells compared to the cells cultured continuously in serum-free condition. METHODS: Human cord blood progenitors were purified with anti-CD133 antibody. They were cultured in a serum-free medium StemSpan supplemented with SCF at 100 ng/ml and IL-6 at 50 ng/ml for 8 weeks. Then, an aliquot of the cultured cells were cultured in the above condition but further supplemented with 10% fetal calf serum (FCS). RESULTS: The addition of FCS after 8 weeks of culture significantly increased the amount of histamine per mast cell (3.8 pg/cell) when compared to the serum-free condition (0.7 pg/cell). The cells cultured with FCS after 8 weeks expressed more FcvarepsilonRI alpha and released >30% of the histamine content upon anti-IgE stimulation than those cultured without serum. CONCLUSION: It is uncertain why FCS enhanced the functional maturation of mast cells when added after week 8 of culture but suppressed mast cell development when added at day 0 of culture. Yet, the present method combining a serum-free culture system with a serum-supplemented culture system seems to be beneficial for most of the laboratories to obtain functional human mast cells.     

Grain dust induces IL-8 production from bronchial epithelial cells: the effect of dexamethasone on IL-8 production.

BACKGROUND: Recent publications have suggested an active participation of neutrophils to induce bronchoconstriction after inhalation of grain dust (GD). OBJECTIVE: To further understand the role of neutrophils in the pathogenesis of GD-induced asthma, this investigation was designed to determine whether human bronchial epithelial cells could produce IL-8 production and to observe the effect of dexamethasone on IL-8 production. MATERIALS AND METHODS: We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four concentrations (1 to 200 microg/mL) of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant, which was derived from the culture of PBMC from a GD-induced asthmatic subject under the exposure to 10 microg/mL of GD, and compared with those cultured without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. To evaluate the effect of dexamethasone on IL-8 production, four concentrations (5 to 5000 ng/mL) of dexamethasone were pre-incubated for 24 hours and the same experiments were repeated. RESULTS: There was significant production of IL-8 from bronchial epithelial cells with additions of GD in a dose-dependent manner (P < .05), which was significantly augmented with additions of PBMC supernatant (P < .05) at each concentration. Compared with the untreated sample, pretreatment of dexamethasone could induced a remarkable inhibitions (15% to 55%) of IL-8 production from bronchial epithelial cells in a dose-dependent manner. CONCLUSION: These results suggest that IL-8 production from bronchial epithelial cells may contribute to neutrophil recruitment occurring in GD-induced airway inflammation. The downregulation of IL-8 production by dexamethasone from bronchial epithelial cells may contribute to the efficacy of this compound in reducing cellular infiltration and ultimately to its anti-inflammatory property.     

SDF-1/CXCR4轴活化诱导内皮祖细胞增殖、迁移及管型形成

目的观察趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成的作用。方法用免疫细胞化学检测内皮祖细胞SDF-1和CXCR4表达;用MTT法、Millicell趋化法及Matrigel体外三维成型法分别检测不同浓度的趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成。并应用CXCR4受体抑制剂AMD3100观察上述指标的变化。结果免疫细胞化学显示内皮祖细胞表达SDF-1和CXCR4蛋白。SDF-1可促进内皮祖细胞的增殖、迁移和体外小管样结构的形成。AMD3100可抑制SDF-1的诱导作用。结论SDF-1/CXCR4轴在内皮祖细胞参与血管新生中可能发挥重要作用。     

IL-15 induces mast cell migration via a pertussis toxin-sensitive receptor

IL-15 induces proliferation, inhibits apoptosis and increases IL-4 production in murine mast cells. There is evidence that these activities are mediated via the uncharacterised receptor, IL-15R-X, rather than the classical three-chain IL-15 receptor. Effects of IL-15 on important aspects of mast cell biology, such as migration and degranulation, are unknown. We report that IL-15 induces migration of murine and human mast cells in a dose-dependent and biphasic manner, with peaks of migration occurring at approximately 10(-15) and approximately 10(-9) M. The potency of the response was similar to that induced by other well-established mast cell chemoattractants. Competition assays performed with murine and human mast cells indicate that both peaks of migration are due to chemotaxis. Pre-treatment of cells with pertussis toxin (PTX), a guanine nucleotide-binding regulatory protein (G-protein) inhibitor, resulted in complete inhibition of murine mast cell migration at approximately 10(-15) M IL-15, and human mast cell migration at approximately 10(-15) and approximately 10(-9) M. This demonstrates that murine and human mast cells express a PTX-sensitive receptor, activated in response to IL-15. Additionally, IL-15 did not induce degranulation in murine mast cells. Locally-produced IL-15 may contribute to mast cell recruitment during inflammatory responses, thereby acting as a linking cytokine between innate and adaptive arms of the immune system.     

Nerve growth factor prevents apoptosis of cord blood-derived human cultured mast cells synergistically with stem cell factor

BACKGROUND: Stem cell factor (SCF) has been identified as a critical survival factor of human mast cells. Other cytokines which possess survival promotion activity on human mast cells are less known. OBJECTIVE: We examined the survival promotion activity of nerve growth factor (NGF) on cord blood-derived human cultured mast cells. METHODS: Expression and function of NGF receptors on the mast cells were examined by RT PCR, flowcytometric analysis, immunoprecipitaion and western blotting. The survival promotion activity of NGF to the mast cells was examined. To evaluate the proliferating activity of NGF on the human cultured mast cells, flow cytometric analysis with propidium iodide staining was applied. To confirm whether the human mast cell growth activity of NGF was caused by a suppression of apoptosis, the proportion of the cells containing in situ DNA fragmentation was counted. RESULTS: The human cultured mast cells expressed the high affinity receptor p140trk but not the low affinity receptor p75LNGFR. NGF induced the phosphorylation of p140trk. NGF alone could not support the survival of the mast cells, however, the addition of NGF to the culture medium containing recombinant SCF led to a significant increase of the number of survival mast cells. No significant changes of the cell cycle from G0/G1 phase to the S/G2 + M phases were observed by NGF. In contrast, the addition of NGF to the medium with SCF showed a significant inhibitory effect on the apoptosis of the mast cells. CONCLUSION: NGF may act as a key factor to promote the survival of human mast cells synergistically with SCF through the prevention of apoptosis.     

Double-stranded RNA induces production of RANTES and IL-8 by human nasal fibroblasts

Double-stranded RNA (dsRNA) and the viral RNA mimic, polyinosine-polycytidylic acid (poly(I:C)), are recognized by toll-like receptor 3 (TLR3) that mediates the innate immune response to viral infections. In this study, we investigated the effects of poly(I:C) on the production of chemokines (IL-8, RANTES, and eotaxin), Type I IFNs (IFNalpha and IFNbeta), Th1-cytokines (IL-12 and IFNgamma), and pro-inflammatory cytokines (TNF-alpha and IL-1beta) by human nasal mucosa-derived fibroblasts. Human nasal fibroblasts were treated with poly(I:C), and levels of cytokines and chemokines were measured by ELISA. Incubation with poly(I:C) significantly enhanced the secretion of RANTES and IL-8. However, eotaxin, IL-1beta, TNF-alpha, IFNalpha, IFNgamma, and IL-12 were not secreted from nasal fibroblasts stimulated with poly(I:C). The JNK inhibitor SP600125 and the PI3-kinase inhibitor LY294002 significantly blocked the poly(I:C)-induced release of RANTES and IL-8, whereas the p38 MAP kinase inhibitor SB203580 suppressed poly(I:C)-induced secretion of IL-8, but not RANTES. Nasal fibroblasts play an important role in initiating antiviral responses and inflammation of the nasal cavity by producing chemokines leading to enhanced inflammatory cell recruitment.     

Cysteinyl leukotrienes induce IL-4 release from cord blood-derived human eosinophils

BACKGROUND: Eosinophils contain preformed stores of IL-4 within their cytoplasmic granules, but physiologic stimuli to release IL-4 from eosinophils are not yet defined. OBJECTIVE: We evaluated whether cysteinyl leukotrienes (CysLTs) could elicit IL-4 release from eosinophils. METHODS: We used a dual-antibody capture and detection assay (EliCell) for IL-4 release and used eosinophils differentiated in vitro from human cord blood-derived progenitors. RESULTS: Leukotriene (LT) C4, LTD4, and LTE4 each elicited the rapid, vesicular transport-mediated, dose- and time-dependent release of IL-4 from eosinophils. Both LTD4 and LTE4 evoked similar and earlier IL-4 release than LTC4. LTC4 did not act directly but only after conversion to LTD4 because an inhibitor of gamma-glutamyl transpeptidase, acivicin, blocked LTC4-induced IL-4 release. MK571 and LY171833, receptor antagonists for CysLT1 and not CysLT2, and pertussis toxin inhibited LTC4-, LTD4-, and LTE4-induced IL-4 release. Cord blood-differentiated eosinophils contained CysLT1 protein detectable by means of immunoblotting. CONCLUSION: CysLTs acting through G(i) protein-coupled and MK571- and LY171833-inhibitable receptors on cord blood-derived human eosinophils can act as autocrine or paracrine mediators to stimulate the rapid, nonexocytotic release of preformed IL-4.