Abstract

Interleukin-4 (IL-4) is the main cytokine that polarizes activated naïve CD4+ T cells in the T helper 2 (Th2) direction. IL-4 also regulates the subsequent stages of Th2 cell-mediated diseases, such as allergies. We conducted a proteomics study to identify IL-4-induced differences during the initial stages of T helper cell differentiation. Primary CD4+ T lymphocytes were isolated from human cord blood, activated through CD3 and CD28, and cultured in the presence or absence of IL-4. Soluble proteins were separated by two-dimensional electrophoresis and visualized by staining with autoradiography, which indicated that at least 20 proteins might be regulated by IL-4. From this minimum of 20 stained proteins, altogether 35 proteins were identified using tandem mass spectrometry. Interestingly the fragmented form of GDP dissociation inhibitor expressed in lymphocytes/Rho GDP dissociation inhibitor 2 (Ly-GDI), a known target of Caspase-3, was observed to be down-regulated in IL-4-treated cells. It was shown in further studies that IL-4 decreases Caspase-3 activity and cell death in these cells. Neutralizing Fas-Fas ligand interaction led to decreased Caspase-3 activity and lowered Ly-GDI fragmentation. We further characterized the effects of IL-4 on the expression of main regulators in the Fas-mediated pathway. We demonstrated that IL-4 decreases expression of Fas receptor and increases expression of Bid, Bcl-2, and Bcl-xL. Importantly IL-4 significantly up-regulated the short form of c-FLIP, although the levels of c-FLIP long were unaltered after IL-4 induction. Taken together, our results indicate that IL-4 inhibits caspase activity during the initial stages of human Th2 cell differentiation by regulating expression of several key players in the Fas-induced pathway.

Highlights

  • Interleukin-4 (IL-4) is the main cytokine that polarizes activated naıve CD4؉ T cells in the T helper 2 (Th2) direction

  • IL-4-induced Differences in the Proteomes of Activated Naıve Human T Helper Cells—In this study we investigated the effect of IL-4 on the proteomes of freshly activated naıve human T helper cells

  • Increased I␬B-␣ levels thereby implicate decreased caspase activity in IL-4-treated cells, the effect of IL-4 on I␬B-␣ expression might be caused by other factors regulating the expression and turnover of this protein. In this proteomics study of IL-4-induced differences in activated primary CD4ϩ T cells, we observed reproducible differential expression in a minimum of 20 proteins by 2-DE after IL-4 stimulation; from these we identified 35 unique proteins

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Summary

EXPERIMENTAL PROCEDURES

Activation, and Culturing of CD4ϩ Cells—Human umbilical cord blood was obtained from Turku University Central hospital. Cells were cultured for 24 h in RPMI 1640 medium (Sigma) containing cross-linking dialyzed goat anti-mouse F(abЈ) (final concentration, 10 ␮g/ml; BIOSOURCE, Camarillo, CA) and 1% human AB serum (Finnish Red Cross). Cells were activated as above and cultured in methionine-free RPMI 1640 medium (Sigma) including [35S]methionine and [35S]cysteine (Redivue Pro-Mix L-[35S] in vitro cell labeling, Amersham Biosciences), goat dialyzed anti-mouse F(abЈ) (final concentration, 10 ␮g/ml; BIOSOURCE), and 1% human AB serum (Finnish Red Cross). For surface staining of the Fas receptor, cells were washed once with FACS buffer (2% FCS, 0.01% azide in D-PBS) and incubated for 15 min with 20 ␮l of mouse anti-Fas-FITC (ALX-805-010F-T100, Alexis Biochemicals) or isotype control mouse IgG2b-FITC (catalog number MG2b01, Caltag Laboratories, Burlingame, CA). Secondary antibodies used in this study were as follows: 1:10,000 HRP goat anti-mouse (sc-2005, Santa Cruz Biotechnology), 1:5000 HRP goat anti-rabbit (catalog number 554021, BD Pharmingen), 1:5000 HRP donkey anti-goat (sc-2020, Santa Cruz Biotechnology), 1:20,000 HRP goat anti-mouse IgG1 (catalog number 1070-05, Southern Biotech, Birmingham, AL), and 1:10,000 HRP goat anti-rat (catalog number 81-9520, Zymed Laboratories Inc., San Francisco, CA)

RESULTS
Serum albumin precursor
DISCUSSION

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