Basic and clinical immunology
Regulation of TH2 responses by the pulmonary Clara cell secretory 10-kd protein

https://doi.org/10.1016/j.jaci.2004.05.042Get rights and content

Abstract

Background

Pulmonary Clara cell secretory 10-kd protein (CC10) is a steroid-inducible and potentially anti-inflammatory cytokine, but its direct involvement in the regulation of T-cell responses remains unknown.

Objective

The role of CC10 in the regulation of TH2 cytokine expression was investigated.

Methods

The levels of cytokine and GATA-3 expression were determined by ELISA and RT-PCR, respectively. Bronchoalveolar lavage fluid cell counts were also determined by using a standard protocol. CC10 expression in vivo was determined by immunocytochemistry and Western blotting.

Results

In vitro, a significant, dose-dependent suppressive effect of CC10 was found on TH2 cytokine expression, but not IFN-γ, in splenocytes of antigen-sensitized mice. A similar suppressive effect was also noted in polarized CD4+ TH2 cells, but not in naive CD4+ T cells. In contrast, CC10 was able to induce IFN-γ expression in naive CD4+ T cells, but not in polarized TH1 cells. Furthermore, the suppression of TH2 cytokine expression was concomitant with reduction of a critical transcription factor, GATA-3. Of significance was the finding that although no significant change was found in the decay kinetics of TH2 cytokine transcripts, a significant decrease in mRNA stability of GATA-3 was seen in CC10-treated cells. In vivo, reconstitution of the CC10 gene in CC10-deficient mice resulted in significantly lower levels of TH2 cytokines, concomitant with a decrease in GATA-3 expression, after challenge with Ag compared with those seen in mock-transduced mice, which are associated with reduced levels of pulmonary eosinophilia.

Conclusion

These results demonstrate, that CC10 plays a direct role in the regulation of T-cell–mediated inflammatory responses.

Section snippets

Cell culture, in vitro T-cell polarization, and cytokine analysis

Spleen cells were isolated from 8-week-old Balb/c mice after 2 weekly intraperitoneal sensitizations with 10 μg ovalbumin (OVA; grade V; Sigma, St Louis, Mo) emulsified in 2 mg alum (Pierce, Rockford, Ill). To obtain CD4+ T cells, splenocytes were isolated from naive Balb/c mice 8 to 12 weeks old to >97% purity with positive selection by using anti-CD4 magnetic beads (Miltenyi Biotec, Auburn, Calif) according to the manufacturer's instructions. For in vitro TH1 and TH2 polarization,12 CD4+ T

Results

CC10 gene deficiency exacerbates pulmonary inflammation,8 which is associated with increased levels of TH2 cytokines, whereas no significant change in the number of infiltrating T cells is seen, suggesting a modulating effect of CC10 on the activation and/or cytokine expression of T cells. To determine the effect of CC10 on Ag-induced T-cell responses in vitro, spleen cells from OVA-sensitized mice were treated with varying doses of CC10 for 2 hours, followed by the stimulation of the cells

Discussion

In this report, we provide evidence that rCC10 is able selectively to inhibit the expression of TH2 cytokines and a critical TH2 transcriptional factor, GATA-3. Also, the suppression of GATA-3 expression is mediated, at least in part, by CC10-mediated alteration of mRNA stability. In addition, the results of in vivo studies demonstrated that mucosal CC10 gene transfer was able to inhibit pulmonary TH2 cytokine production and eosinophilia seen in CC10-deficient mice, providing evidence for a

Acknowledgements

We thank Dr. Anil Mukherjee for scientific input and advice during the course of this study.

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    This work was supported, in part, by National Institutes of Health (ROI-40274), Philip Morris USA Inc, National Science Council (Taiwan:NSC92-2314-B-182A-111), and Chang Gung Memorial Hospital, Taiwan (CMRP 1359).

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    These authors contributed equally to this work.

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