We investigated the expression of a panel of Toll-like receptors (TLRs) and their functions in human eosinophils. Eosinophils constitutively expressed TLR1, TLR4, TLR7, TLR9, and TLR10 mRNAs (TLR4 greater than TLR1, TLR7, TLR9, and TLR10 greater than TLR6). In contrast, neutrophils expressed a larger variety of TLR mRNAs (TLR1, TLR2, TLR4, TLR6, TLR8 greater than TLR5, TLR9, and TLR10 greater than TLR7). Although the expression levels in eosinophils were generally less prominent compared with those in neutrophils, eosinophils expressed a higher level of TLR7. Furthermore, among various TLR ligands (S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-Cys-Ser-(Lys)4, poly(I:C), LPS, R-848, and CpG DNA), only R-848, a ligand of TLR7 and TLR8, regulated adhesion molecule (CD11b and L-selectin) expression, prolonged survival, and induced superoxide generation in eosinophils. Stimulation of eosinophils by R-848 led to p38 mitogen-activated protein kinase activation, and SB203580, a p38 mitogen-activated protein kinase inhibitor, almost completely attenuated R-848-induced superoxide generation. Although TLR8 mRNA expression was hardly detectable in freshly isolated eosinophils, mRNA expression of TLR8 as well as TLR7 was exclusively up-regulated by IFN-γ but not by either IL-4 or IL-5. The up-regulation of the TLRs by IFN-γ had potentially functional significance: the extent of R-848-induced modulation of adhesion molecule expression was significantly greater in cells treated with IFN-γ compared with untreated cells. Although the natural ligands for TLR7 and TLR8 have not yet been identified, our results suggest that eosinophil TLR7/8 systems represent a potentially important mechanism of a host-defensive role against viral infection and mechanism linking exacerbation of allergic inflammation and viral infection.

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.

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.