Misato Miyamasu

Inducible Expression of a Th2-Type CC Chemokine Thymus- and Activation-Regulated Chemokine by Human Bronchial Epithelial Cells

CCR4 is now known to be selectively expressed in Th2 cells. Since the bronchial epithelium is recognized as an important source of mediators fundamental to the manifestation of respiratory allergic inflammation, we studied the expression of two functional ligands for CCR4, i.e., macrophage-derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC), in bronchial epithelial cells. The bronchial epithelium of asthmatics and normal subjects expressed TARC protein, and the asthmatics showed more intense expression than the normal subjects. On the other hand, MDC expression was only weakly detected in the asthmatics, but the intensity was not significantly different from that of normal subjects. Combination of TNF-α and IL-4 induced expression of TARC protein and mRNA in bronchial epithelial A549 cells, which was slightly up-regulated by IFN-γ. The enhancement by IFN-γ was more pronounced in bronchial epithelial BEAS-2B cells, and a maximum production occurred with combination of TNF-α, IL-4, and IFN-γ. On the other hand, MDC was essentially not expressed in any of the cultures. Furthermore, expressions of TARC protein and mRNA were almost completely inhibited by glucocorticoids. These results indicate that the airway epithelium represents an important source of TARC, which potentially plays a role via a paracrine mechanism in the development of allergic respiratory diseases. Furthermore, the beneficial effect of inhaled glucocorticoids on asthma may be at least in part due to their direct inhibitory effect on TARC generation by the bronchial epithelium.

Expression of CXCR4 in eosinophils: functional analyses and cytokine-mediated regulation.

We examined the expression of transcripts of a panel of chemokine receptors in human eosinophils and found intense constitutive expression of CXCR4 mRNA. Although surface CXCR4 protein was hardly detectable in the peripheral blood or freshly isolated eosinophils, surface expression of CXCR4 became gradually apparent during incubation at 37°C. In contrast, the level of CCR3 expression was virtually unchanged during the incubation. Stromal cell-derived factor-1α (SDF-1α), the natural ligand of CXCR4, elicited an apparent Ca2+ influx in these cells and induced a strong migratory response comparable to that by eotaxin. The surface expression of CXCR4 in eosinophils was up-regulated by IFN-γ, TNF-α, and TGF-β while it was down-regulated by IL-4 and eosinophil-directed hemopoietins such as IL-5. The CXCR4 expression did not always parallel the apoptotic changes in cytokine-treated eosinophils. In contrast to IL-4 and IFN-γ, IL-5 potently reduced the level of CXCR4 mRNA. It seems unlikely that CXCR4 is fundamentally involved in the pathogenesis of allergic disorders by inducing the migration of eosinophils toward inflammatory sites, because a Th2-dominant state down-regulates eosinophil CXCR4 expression. However, CXCR4 may affect the size of the mobilizable pool by holding eosinophils at noninflamed tissues. Th2-dominant state may favor the liberation of eosinophils by down-regulating CXCR4 expression. The interplay between CXCR4 and SDF-1α in eosinophils potentially plays an important role in the accumulation of these cells at the allergic inflammatory sites.

Chemokine production by the BEAS-2B human bronchial epithelial cells: Differential regulation of eotaxin, IL-8, and RANTES by TH2- and TH1-derived cytokines

BACKGROUND Bronchial epithelial cells produce many types of chemokines and may contribute to lung inflammation by recruiting inflammatory cells. The CC chemokine eotaxin is a potent, eosinophil-specific chemoattractant that has been detected in the bronchial epithelium of patients with asthma. OBJECTIVES The aim of this study was to investigate the regulatory mechanisms of chemokine production from bronchial epithelium by inflammatory cytokines, especially T(H)2- and T(H)1-derived cytokines, in bronchial asthma. METHODS BEAS-2B human bronchial epithelial cells were cultured with TNF-alpha, IL-4, IL-13, and IFN-gamma alone or in combination, after which supernatants were assayed for eotaxin, IL-8, and RANTES proteins with ELISA. Reverse transcription-PCR was also performed. RESULTS TNF-alpha induced production of eotaxin, IL-8, and RANTES in a concentration-dependent manner. Both IL-4 and IL-13 synergistically enhanced TNF-alpha-induced eotaxin production, whereas IL-8 production induced by TNF-alpha was significantly down-regulated by the T(H)2-derived cytokines. IFN-gamma, a T(H)1 cytokine, counteracted the enhancing effects of IL-4 and IL-13 on eotaxin production. RANTES production by TNF-alpha was not affected by IL-4 and IL-13 but was markedly enhanced by IFN-gamma. CONCLUSIONS These results suggest that T(H)2 cytokines are involved in preferential recruitment of eosinophils in bronchial asthma by enhancing eotaxin and reducing IL-8 production from bronchial epithelial cells and that T(H)1 cytokines counteract the effects of T(H)2 cytokines by reducing eotaxin production.

Cytokine-mediated regulation of CXCR4 expression in human neutrophils

Several lines of evidence have suggested that a CXC chemokine receptor 4 (CXCR4)/stromal cell‐derived factor‐1 [SDF‐1; CXC chemokine ligand 12 (CXCL12)] pair is involved in baseline trafficking of leukocytes into extravascular tissues and that modulation of surface CXCR4 expression may represent an alternative mechanism for control of cell‐specific biological responses to SDF‐1/CXCL12. We explored the regulation of CXCR4 expression by cytokines in polymorphonuclear neutrophils (PMNs). No significant surface expression of CXCR4 in freshly isolated PMNs was detected, but expression became apparent gradually during incubation. SDF‐1α/CXCL12 initiated Ca2+ mobilization and migratory responses in 20 h cultured PMNs. The surface CXCR4 expression was suppressed most potently by interferon‐γ (IFN‐γ). IFN‐α, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and G‐CSF also inhibited spontaneous CXCR4 expression. Real‐time, quantitative PCR experiments revealed that a spontaneous increase and an IFN‐γ‐mediated decrease in surface CXCR4 paralleled changes in the CXCR4 mRNA level. These results on PMNs support the argument that the SDF‐1 (CXCL12)/CXCR4 system is regulated by cell type‐specific mechanisms.

Intracellular localization and release of eotaxin from normal eosinophils

Eotaxin is a potent and selective CC chemokine for eosinophils and basophils. We established several monoclonal antibodies (Mabs) allowing the neutralization and measurement of human eotaxin. Using the Mabs as probes, we demonstrated that normal eosinophils contained intracellular granule‐associated eotaxin. Quantification of cell‐associated eotaxin in different leukocyte subsets revealed that it was principally expressed in eosinophils. Finally, we showed that normal eosinophils released eotaxin upon stimulation with either of two secretagogues, C5a or ionomycin. These findings raise the possibility that eosinophil‐derived eotaxin contributes to the local accumulation of eosinophils at the site of inflammation.

Chemokine receptors in human basophils: inducible expression of functional CXCR4

We examined the expression profile of chemokine receptors in humanbasophils and their regulation by cytokines. Basophils expressedtranscripts of CC chemokine receptors (CCR)1, CCR2, CCR3, and CCR5 andCXC chemokine receptors (CXCR)1, CXCR2, and CXCR4. In contrast to theother receptors, surface‐CXCR4 expression was not detected in fresh‐and whole‐blood basophils, but it became apparent gradually duringincubation. Among 16 chemokines tested, eotaxin induced the most potentbasophil migration. SDF‐1 also induced a strong, migratory responsecomparable with that induced by eotaxin in 24‐h, cultured basophils,but it failed to induce degranulation. IL‐3 abrogated CXCR4 expressioncompletely, and it only down‐regulated CCR2 and CCR3 expressionslightly. IL‐5, GM‐CSF, and IL‐4 also down‐regulated CXCR4 expression.Thus, expression of CXCR4 was the most strongly affected by cytokines,and this may represent an alternative mechanism for control ofcell‐specific, biological responses to SDF‐1.

Glucocorticoids inhibit chemokine generation by human eosinophils

Recent identification of eosinophils as a cellular source of various cytokines suggests that eosinophil-derived cytokines contribute to allergic inflammation through either an autocrine or a paracrine fashion. The profound inhibitory effects of glucocorticoids (GCCs) on the production of various cytokines have been well recognized, however, there has been no definitive evidence that GCCs in fact inhibit cytokine generation by eosinophils. To verify the inhibitory ability of GCCs on eosinophil cytokine generation, we studied the effect of GCCs by determination of IL-8 and monocyte chemoattractant protein-1 (MCP-1) as parameters. Dexamethasone (DEX) inhibited both generation and secretion of IL-8 in a dose-dependent fashion. DEX also dampened formyl-methionyl-leucyl-phenylalanine-or ionomycin-induced eosinophil IL-8 production. Furthermore, MCP-1 production was also inhibited by DEX. The slope and the shape of the dose-response curve of DEX were similar irrespective of either the input stimuli or the output cytokines; half-maximal inhibition was observed at 10(-8) mol/L, and nearly complete abolishment was observed at 10(-7) mol/L. The competitive polymerase chain reaction for IL-8 mRNA and semiquantitative polymerase chain reaction for MCP-1 mRNA revealed that the inhibition occurred at a level of pretranslation. These results indicate that the beneficial effect of GCCs in allergic inflammation might be related, at least in part, to a direct effect of the drugs on eosinophil cytokine synthesis.

A Functional Study on CysLT1 Receptors in Human Eosinophils

Background: The cysteinyl leukotrienes (CysLTs) mediate their biological actions through two receptors: CysLT₁ receptor and CysLT₂ receptor. Objective: This study was undertaken to examine the direct effects of CysLTs on eosinophils, such as chemotaxis and degranulation, focusing on CysLT₁. Methods: Eosinophils were isolated from venous blood from normal volunteers who had no history of allergy (purity >99%). They were subjected to reverse transcription-PCR analysis and flow-cytometric analysis for CysLT₁. Binding assays were performed with [³H]LTD₄. Purified eosinophils loaded with Fura-2 acetoxymethyl ester were stimulated with CysLTs, and Ca²⁺ influx was measured. Eosinophil migration in response to CysLTs was measured using a 96-well multiwell Boyden chamber. Eosinophils were treated with LTD₄ at 10^–6M for 60 min followed by incubation for 4 h at 37°C in the presence or absence of IL-5 and eosinophil-derived neurotoxin (EDN) release was evaluated. Results: The expression of the mRNA and protein of CysLT₁ on eosinophils and [³H]LTD₄-specific binding to eosinophils were observed. Neither Th1 cytokine (IFN-γ) nor Th2 cytokines (IL-4 or IL-5) affected CysLT₁ expression in eosinophils. CysLTs induced an increase in intracellular free Ca²⁺ in eosinophils via CysLT₁, as suggested by the efficient inhibition by a CysLT₁ antagonist, pranlukast, in addition to the rank order of potency being LTD₄, LTC₄ and LTE₄. LTD₄ stimulated eosinophils to migrate at 10^–6M via CysLT₁. LTE₄ also induced significant eosinophil migration at 10^–6M. LTD₄ enhanced EDN release induced by IL-5 via CysLT₁. Conclusion: CysLTs induce migration and enhance degranulation in eosinophils via CysLT₁. Accordingly, interaction of CysLTs and CysLT₁ on eosinophils has the potential to play a prominent role in the pathophysiology of asthma.

Regulation of Chemokine Receptor Expression in Eosinophils

Signals via chemokine receptors play an important role in the accumulation of eosinophils at allergic inflammatory sites. Eosinophils constitutively express CC chemokine receptor 3 (CCR3) and, to a lesser extent, CCR1. CCR3 is mainly responsible for migration of resting eosinophils, and its specific ligand, eotaxin, represents the most potent chemoattractant for eosinophils. Some reports also suggest the expression of CXC chemokine receptor 1 (CXCR1) and/or CXCR2 in eosinophils. In addition, we recently reported the functional expression of CXCR4. The ligand of CXCR4, stromal cell-derived factor-1 (SDF-1), was able to induce a strong migratory response comparable to that by eotaxin. In contrast to the CCR3/eotaxin system which is mainly regulated at the level of ligand production, the CXCR4/SDF-1 system is regulated at the level of receptor expression. CXCR4 expression was completely attenuated by IL-4 and IL-5 and upregulated by IFN-γ and dexamethasone, while CCR3 expression was only marginally affected. The balance between the biological effects of these chemokine systems may affect the distribution and migration of eosinophils.

Variations in the human Th2-specific chemokine TARC gene

Th2-specific chemokine thymus and activation-regulated chemokine (TARC)/CC chemokine ligand (CCL)17 is highly implicated in the pathogenesis of Th-2-dominated allergic diseases such as bronchial asthma (BA) and atopic dermatitis (AD). We performed polymorphism screening of the coding and promoter regions of the TARC gene. We found two rare variations in the coding region of exon 3 (2134C>T and 2037G>A) and a single nucleotide polymorphism (SNP) in the 5′-flanking region (-431C>T). Individuals carrying the 431T allele showed significantly increased serum levels of TARC compared with those not carrying the 431T allele, suggesting that this SNP has functional significance. However, when the genotypes at the SNP site were determined for 158 healthy individuals, 105 patients with BA and 148 patients with AD, we observed no significant association of the SNP with susceptibility to BA or AD.