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Suppressors of cytokine signaling 3 expression in eosinophils: Regulation by PGE2 and th2 cytokines

Clinical and Developmental Immunology (2011) 2011
DOI: 10.1155/2011/917015
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Abstract

Asthma and nonasthmatic eosinophilic bronchitis (NAEB) are respiratory disorders characterized by a predominance of Th2 cells and eosinophilic inflammation. Suppressors of cytokine signaling (SOCS) proteins play an important role in Th2-mediated allergic responses through control of the balance between Th1 and Th2 cells, particularly, SOCS3 and SOCS5. The aim of this study was to analyze SOCS expression in human peripheral blood eosinophils from patients with asthma, NAEB and healthy controls. SOCS expression in eosinophils from subjects was demonstrated by different techniques. Results showed that expression of SOCS3 in eosinophils and CD4 T cells from patients was higher than in healthy subjects. In addition, we demonstrated that prostaglandin E 2 (PGE2) and Th2 cytokines are able to upregulate SOCS3 production in eosinophils and attenuate its degranulation. In conclusion, eosinophils are able to transcribe and translate SOCS3 protein and can contribute to the regulation of the Th1/Th2 balance through SOCS3 production. Copyright 2011 Esther Lpez et al.

Figures

  • Table 1: Clinical characteristics of study subjects.
  • Figure 1: Semiquantitative expression of SOCS3 and SOCS5 genes in peripheral blood purified CD4 T cells and eosinophils. Relative mRNA levels of SOCS3 and SOCS5 in CD4 T cells ((a) and (b)), and eosinophils ((c) and (d)) of healthy controls, asthmatic and NAEB patients were determined by real-time quantitative PCR. Values were normalized with rRNA gene used as an endogen. Black boxes represent the healthy control group (mean ± SD, n = 9), white boxes represent the asthmatic patient group (mean ± SD, n = 6), and lineated boxes represent the NAEB group (mean ± SD, n = 8). Significant differences in the levels of SOCS3 expression in CD4 T cells were obtained for asthmatic and NAEB patients versus the healthy control group (∗P < .05 and ∗∗P < 0.01, resp.). In eosinophils, the SOCS3 level was significantly higher in NAEB patients than in healthy controls (∗∗P < .01). In the case of SOCS5, no significant differences were found between the groups. (e) SOCS3 mRNA levels in bronchial biopsies from healthy controls (mean ± SD, n = 2), asthmatics (mean ± SD, n = 2), and NAEB patients (mean ± SD, n = 4). Significant differences in the levels of SOCS3 expression were obtained for asthmatic versus NAEB patients (∗P < .05). The results represent relative gene expression, as determined by the ΔΔCT method.
  • Figure 2: SOCS3 expression in peripheral blood eosinophils from Th2 patients analyzed by immunohistochemical, immunofluorescence, and Western blot techniques. Eosinophils from asthmatic and NAEB patients within healthy controls were adhered to slides and incubated with peroxidase-conjugated goat antirabbit IgG against SOCS3 antibody ((b), (c), and (d)) or rabbit IgG as a control (a), and Texas Red conjugated goat antirabbit IgG against SOCS3 antibody ((f), (g), and (h)) or rabbit IgG as a control (e). The slides were observed by optical ((a), (b), (c), (d)) or confocal ((e), (f), (g) and (h)) microscopy. Western blot analysis of the cytosolic extract of purified eosinophils was achieved using antibody against SOCS3 (i). Lane 1: recombinant SOCS3 was loaded as a positive control; lane 2: eosinophil lysate from NAEB patients; lane 3: eosinophil lysate from healthy control patients; lane 4: isotype negative control. The picture is a representative example of 5 individuals, all displaying similar results. (j): SOCS3 bands were quantified by densitometry and corrected by actin expression; data are expressed as the mean ± SD, n = 5, ∗P < .05 (2: NAEB; 3: healthy patients).
  • Figure 3: SOCS3 expression in peripheral blood eosinophils treated with Th2 cytokines and IFN-γ. Purified eosinophils from healthy donors were cultured with 10 ng/mL of IL-4 (a), IL-5 (b), IL-13 (c), or IFN-γ (d) for different periods of time. Dose-response curves were performed with IL-4, IL-5, IL-13, or IFN-γ (e)–(h) at maximal time response; SOCS3 mRNA was measured by real-time quantitative PCR. The results are expressed as a fold induction relative to untreated eosinophils, and significant differences are indicated. Data represent the geometric mean ± SD from four individuals, and each measure was performed in triplicate.
  • Figure 4: SOCS3 mRNA expression in Th2-stimulated eosinophils from healthy controls, asthmatics, and NAEB patients. Eosinophils from healthy controls, asthmatics, and NAEB patients were cultured with 10 ng/mL of IL-4 or IL-5, or IL-13 during 60 min. SOCS3 mRNA was measured by real-time quantitative PCR. The results are expressed as a fold induction relative to untreated eosinophils, and significant differences are indicated. Data represent the geometric mean ± SD from three individuals, and each measure was performed in triplicate.
  • Figure 5: PGE2 stimulation of SOCS3 mRNA expression. Purified eosinophils from healthy donors were cultured with 10−4 M of PGE2 for up to 120 min (a), and with increasing doses of PGE2at 120 min (b), and SOCS3 mRNA was measured by real-time quantitative PCR. The results are expressed as a fold induction relative to untreated eosinophils, and significant differences are indicated. Data represent the geometric mean ± SD from four individuals, and each measure was performed in triplicate.
  • Figure 6: Th2 cytokines and PGE2 inhibit eosinophil degranulation. Purified human eosinophils were pretreated with vehicle, IL-4 (10 ng/mL), IL-5 (10 ng/mL), IL-13 (10 ng/mL), and PGE2 (10−6 M) for 1 or 2 hours and then stimulated with C5a (300 nM) for 30 min at 37◦C. The release of EPO activity into supernatants was determined by photometry. Data were expressed as a percentage of the maximal control response (300 nM) and are shown as the mean ± SD; n = 5–9; ∗∗P < .005, ∗P < .05 versus C5a alone (vehicle).

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CITATION STYLE

APA

Del Pozo, V., López, E., Zafra, M. P., Sastre, B., Gámez, C., Fernández-Nieto, M., … Quirce, S. (2011). Suppressors of cytokine signaling 3 expression in eosinophils: Regulation by PGE2 and th2 cytokines. Clinical and Developmental Immunology, 2011. https://doi.org/10.1155/2011/917015

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