Toshiharu Nakajima

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.

Molecular Cloning of a Novel Human CC Chemokine (Eotaxin-3) That Is a Functional Ligand of CC Chemokine Receptor 3

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 ∼40 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 EC50 of 3 nm. Eotaxin-3 competed the binding of125I-eotaxin to CCR3-expressing L1.2 cells with an IC50 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 ∼10-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.

Thymic Stromal Lymphopoietin Gene Promoter Polymorphisms Are Associated with Susceptibility to Bronchial Asthma

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 β(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 = 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.

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.

Thrombospondin/CD47 Interaction: A Pathway to Generate Regulatory T Cells from Human CD4+CD25− T Cells in Response to Inflammation

Thymus-derived CD4+ CD25+ T regulatory cells (Tregs) are essential for the maintenance of self-tolerance. What critical factors and conditions are required for the extra-thymic development of Tregs remains an important question. In this study, we show that the anti-inflammatory extracellular matrix protein, thrombospondin-1, promoted the generation of human peripheral regulatory T cells through the ligation of one of its receptor, CD47. CD47 stimulation by mAb or a thrombospondin-1 peptide induced naive or memory CD4+CD25− T cells to become suppressive. The latter expressed increased amounts of CTLA-4, OX40, GITR, and Foxp3 and inhibited autologous Th0, Th1, and Th2 cells. Their regulatory activity was contact dependent, TGF-β independent, and partially circumvented by IL-2. This previously unknown mechanism to induce human peripheral Tregs in response to inflammation may participate to the limitation of collateral damage induced by exacerbated responses to self or foreign Ags and thus be relevant for therapeutic intervention in autoimmune diseases and transplantation.

Presence of eotaxin in tears of patients with atopic keratoconjunctivitis with severe corneal damage

The eotaxin concentration in tears from the erosion group (1475.4 ± 870.5 pg/mL [mean ± SD]) was significantly greater than that from the SPK group (163.4 ± 81.7 pg/mL), the clear group (24.3 ± 12.7 pg/mL), and the nonallergic control subjects (21.7 ± 9.7 pg/mL) (Fig 1, A). Greater numbers of eosinophils were found in tears from the erosion group (42.2 ± 52.2 cells/field, n = 9) than in tears from the SPK group (0.3 ± 0.4 cells/field, n = 5), the clear group (0 ± 0 cells/field, n = 8) and the nonallergic control subjects (0 ± 0 cells/field, n = 13) (Fig 1, B). Moreover, the number of eosinophils correlated with the eotaxin concentration in the tear samples (r = 0.603, P < .0004 as determined by Bartlett’s test; data not shown).

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.

Eotaxin in induced sputum of asthmatics: relationship with eosinophils and eosinophil cationic protein in sputum.

Background: Eosinophilic inflammation is a crucial aspect of allergic diseases such as bronchial asthma. An eosinophil‐active chemokine, eotaxin, may play a role in the pathogenesis of the tissue eosinophilia accompanying asthma.

Pepsin-resistant 16-kD buckwheat protein is associated with immediate hypersensitivity reaction in patients with buckwheat allergy.

Background: Buckwheat is becoming popular in many countries as a health food and the incidence of buckwheat allergy is increasing in Asia. The ingestion of small amounts sometimes provokes an anaphylactic reaction. However, it remains controversial which is the major allergen responsible for such reactions. Methods: The patients whose sera are positive for buckwheat-specific IgE antibody measured by the CAP system fluorescein-enzyme immunoassay (CAP-FEIA) were classified into two subgroups depending on the history of immediate hypersensitivity reactions (IHR). Major buckwheat allergens were identified with immunoblotting, ELISA and N-terminal amino acid sequencing. Various treatments such as pepsin digestion were added to characterize the proteins. Results: We found that the 24-kD protein that had previously been reported to be a major allergen reacted to IgE antibodies present in sera from almost all subjects (19/20) regardless of symptoms. On the other hand, 16- and 19-kD proteins were bound with IgE antibodies present in sera from 9 of the 10 patients with IHR including 8 patients with anaphylaxis but not in sera from buckwheat-specific IgE-positive subjects without IHR. After pepsin treatment, the 16-kD protein but not the 19- and 24-kD proteins remained undigested and preserved the capacity of IgE binding. This pepsin-resistant 16-kD protein had no homology with the 24-kD protein by the N-terminal amino acid sequencing. Conclusions: The 16-kD buckwheat protein was resistant to pepsin digestion and appeared to be responsible for IHR including anaphylaxis, while the pepsin-sensitive 24-kD protein was responsible for CAP-FEIA but not IHR.

Extremely Rapid and Intense Induction of Apoptosis in Human Eosinophils by Anti-CD30 Antibody Treatment In Vitro

Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival, whereas corticosteroids reduce their survival in vitro. CD30, a member of the TNFR family, is expressed on the surface of many cell types, including Hodgkin’s lymphoma cells. CD30 is capable of inducing apoptosis after Ab treatment in some cell lines. To determine whether this surface structure is involved in apoptosis of human eosinophils, we examined its expression and the effect of anti-CD30 Ab treatment on the viability of eosinophils. Purified human eosinophils expressed low, but consistently detectable, levels of CD30. Immobilized, but not soluble, forms of anti-CD30 Abs (HRS-4 and Ber-H8) or recombinant mouse CD30 ligand exhibited an extremely rapid and intense survival-reducing effect on the eosinophils in the presence of exogenous IL-5; this effect was both concentration and time dependent. Furthermore, high concentrations of IL-5 could not reverse the reduced survival rates. After treatment with anti-CD30 Ab, gel electrophoresis of DNA extracted from the eosinophils demonstrated changes consistent with apoptosis. The immobilized F(ab′)2 of the anti-CD30 Ab failed to induce eosinophil apoptosis. The addition of anti-CD18 Ab also completely abrogated the induction of eosinophil apoptosis. Further examination using specific signal transduction inhibitors suggested the involvement of p38, mitogen-activated protein kinase kinase 1/2, and specific tyrosine kinase, but not NF-κB, in the induction of CD30-mediated eosinophil apoptosis. These data demonstrate that CD30 can modify eosinophil survival by causing an extremely rapid and intense induction of apoptosis through a tightly regulated intracellular signaling pathway.