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.

Thymic stromal lymphopoietin (TSLP) triggers dendritic cell-mediated T helper (Th) 2 inflammatory responses. A single-nucleotide polymorphism (SNP), rs3806933, in the promoter region of the TSLP gene creates a binding site for the transcription factor activating protein (AP)-1. The variant enhances AP-1 binding to the regulatory element, and increases the promoter-reporter activity of TSLP in response to polyinosinic-polycytidylic acid (poly[I:C]) stimulation in normal human bronchial epithelium (NHBE). We investigated whether polymorphisms including the SNP rs3806933 could affect the susceptibility to and clinical phenotypes of bronchial asthma. We selected three representative (i.e., Tag) SNPs and conducted association studies of the TSLP gene, using two independent populations (639 patients with childhood atopic asthma and 838 control subjects, and 641 patients with adult asthma and 376 control subjects, respectively). We further examined the effects of corticosteroids and a long-acting b 2 -agonist (salmeterol) on the expression levels of the TSLP gene in response to poly(I:C) in NHBE. We found that the promoter polymorphisms rs3806933 and rs2289276 were significantly associated with disease susceptibility in both childhood atopic and adult asthma. The functional SNP rs3806933 was associated with asthma (meta-analysis, P 5 0.000056; odds ratio, 1.29; 95% confidence interval, 1.14-1.47). A genotype of rs2289278 was correlated with pulmonary function. Moreover, the induction of TSLP mRNA and protein expression induced by poly(I:C) in NHBE was synergistically impaired by a corticosteroid and salmeterol. TSLP variants are significantly associated with bronchial asthma and pulmonary function. Thus, TSLP may serve as a therapeutic target molecule for combination therapy.Keywords: asthma; TSLP; bronchial epithelial cells; combination therapy; genetic polymorphisms Thymic stromal lymphoprotein (TSLP) is an epithelial cellderived cytokine that triggers dendritic cell-mediated T helper (Th) 2 inflammatory responses, and plays an important role in the initiation and maintenance of the allergic immune response (1-6). A recent study showed that TSLP is released by human epithelial cells in response to microbes, trauma, or inflammation, and potently activates mast cells (7). In humans, TSLP is highly expressed by airway epithelial cells during allergic inflammation (2), and the expression of the TSLP gene in asthmatic airways is correlated with both the expression of Th2-attracting chemokines and disease severity (3).Large numbers of association studies on asthma and asthmarelated phenotypes using genetic polymorphisms were performed in different populations (8). Recent studies showed roles of human genetic polymorphisms of the TSLP gene. A variant in TSLP was associated with reductions in IgE in response to cockroaches and total IgE in a sex-stratified analysis (9). A functional single-nucleotide polymorphism (SNP), rs3806933, was identified in the regulatory element of TSLP (10). The variant creates a binding site for a...

Eosinophils (Eos) and fibroblasts are known to play a major role in the pathogenesis of bronchial asthma and fibrotic lung disease. Therefore, we investigated whether Th1 and Th2 cytokines stimulate the production of Eo-activating chemokines by lung fibroblasts. Analyses of the culture supernatant using multiple steps of high-performance liquid chromatography demonstrated that interleukin (IL)-4 preferentially stimulates lung fibroblasts to secrete a peak of eosinophil chemotactic activity (ECA) which, upon N-terminal analyses, showed similar sequence to eotaxin, whereas interferon (IFN)-gamma had negligible effect on the release of this chemokine. In contrast, tumor necrosis factor (TNF)-alpha stimulated lung fibroblasts to release two peaks of activity that were found to correspond to eotaxin and regulated on activation, normal T cells expressed and secreted (RANTES), respectively. Interestingly, IL-4 synergized with TNF-alpha to increase greatly the production of three biochemically distinct eotaxin forms. In contrast, IFN-gamma synergized with TNF-alpha to increase RANTES production. Neither IL-2, IL-5, IL-6 nor IL-10 had an effect on lung fibroblasts' capacity to express or release eotaxin and RANTES. Upon appropriate cytokine stimulation, lung fibroblasts were also found to express messenger RNA for monocyte chemotactic protein (MCP)-3 and MCP-4 but not eotaxin-2. However, no ECA like MCP-3 or MCP-4 was detected. These observations suggest that the release of Th1 or Th2 cytokines in the lung tissue polarizes lung fibroblasts to produce either RANTES or eotaxin as major Eo attractants.

Previously, we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 (MPIF-2)/eotaxin-2 to chromosome 7q11.23 (Nomiyama, H., Osborne, L. R., Imai, T., Kusuda, J., Miura, R., Tsui, L.-C., and Yoshie, O. (1998) Genomics 49, 339 -340). Since chemokine genes tend to be clustered, unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci. Prompted by this hypothesis, we analyzed the genomic region containing the gene for MPIF-2/ eotaxin-2 (SCYA24) and have identified a novel CC chemokine termed eotaxin-3. The genes for MPIF-2/ eotaxin-2 (SCYA24) and eotaxin-3 (SCYA26) are localized within a region of ϳ40 kilobases. By Northern blot analysis, eotaxin-3 mRNA was constitutively expressed in the heart and ovary. We have generated recombinant eotaxin-3 in a baculovirus expression system. Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 (CCR3)-expressing L1.2 cells with an EC 50 of 3 nM. Eotaxin-3 competed the binding of 125 I-eotaxin to CCR3-expressing L1.2 cells with an IC 50 of 13 nM. Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations. Collectively, eotaxin-3 is yet another functional ligand for CCR3. The potency of eotaxin-3 as a CCR3 ligand seems, however, to be ϳ10-fold less than that of eotaxin. Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena. The strategy used in this study may also be applicable to identification of other unknown chemokine genes.