PMC

The assembly of a transcription factor complex occurs only in Il-4 expressing Th2 cells. A, Th2 cells polarized for 12 days were restimulated and sorted for IL-4 expression using the IL-4 cytokine secretion assay. B, Th2 cells isolated according to IL-4 secretion were lysed, and the Il4 mRNA expression was quantified in resting Th2 cells, Th2 cells not secreting IL-4, and Th2 cells secreting IL-4. C, Th2 cells isolated according to IL-4 secretion were lysed, and the GATA-3 protein expression was quantified by Western blot in resting Th2 cells, Th2 cells not secreting IL-4, Th2 cells secreting IL-4, and restimulated Th1 cells. D, Th2 cells polarized for 12 days were either fixed in a resting state or following restimulation and separation into IL-4 secreting and IL-4 non-secreting Th2 cells. ChIP was performed against the indicated transcription factors and probed for binding to regulatory regions in the Il4 locus. Binding to the Ifnγ promoter was used as negative control. Shown are the representative results of three independent experiments.

Effect of ramatroban on the ability of mast cell supernatant to cause chemotaxis of CRTH2⁺ CD4⁺ Th2 cells. Supernatants were collected from mast cells 1 hr after activation with IgE/anti-IgE. (a) Increasing concentrations of these supernatants were assayed for chemotactic activity in the presence and absence of ramatroban (1 μm). (b) Increasing doses of ramatroban were tested against a 1/10 dilution of the mast cell supernatant. (c) CRTH2⁺ CD4⁺ Th2 cells were stimulated with a 1/10 dilution of the supernatant with and without 1 μm ramatroban or 1 μm SQ29548. There was a significant difference between responses of untreated CRTH2⁺ CD4⁺ Th2 cells to control media and responses of untreated CRTH2⁺ CD4⁺ Th2 cells to supernatants from activated mast cells (P < 0·01 by anova) and a significant difference between responses of untreated CRTH2⁺ CD4⁺ Th2 cells and CRTH2⁺ CD4⁺ Th2 cells treated with ramatroban to activated mast cell supernatants (P < 0·01 by anova). There was no significant difference between the responses of untreated CRTH2⁺ CD4⁺ Th2 cells to control media and responses of ramatroban-treated CRTH2⁺ CD4⁺ Th2 cells to supernatants from activated mast cells (P > 0·05 by Newman–Keuls test). There was no significant difference between responses of untreated CRTH2⁺ CD4⁺ Th2 cells and responses of CRTH2⁺ CD4⁺ Th2 cells treated with SQ29548 to supernatants from activated mast cells. All data are presented as the mean ± SEM (n = 3–5).

PGE₂ inhibits AICD of Th2 cells derived from BALB/c but not C3H/OuJ mice. a, terminally differentiated Th1 and Th2 cells were activated with plate-bound anti-CD3 antibodies overnight. DNA fragmentation was measured by PI staining. Th1 and Th2 cells of these strains induced comparable AICD (left two sets of panels). Supernatant (Sup) from LPS-activated macrophages drastically inhibited AICD in Th2 cells of BALB/c but not C3H/OuJ origin (middle panels). Supernatant from LPS-activated macrophages generated in the presence of indomethacin (IM; 10 μm) did not alter the inhibition of AICD in Th2 cells of either strain (second set of panels from the right). The addition of PGE₂ to the culture medium (1 μm) dramatically inhibited AICD of Th2 cells of BALB/c but not C3H/OuJ origin (right panels). Numbers indicated in the figures are the percentages of apoptotic cells. b, expression of PGE₂ receptor EP2 in Th2 cells from BALB/c and C3H/OuJ mice. Th1 and Th2 cells from BALB/c and C3H/OuJ mice were activated with plate-bound anti-CD3 + anti-CD28 antibodies, and total mRNA was isolated. Quantitative real-time PCR was performed for quantifying expression of PGE₂ receptors. Relative expression was calculated using β-actin as an internal control. c, Western blot analysis of EP2 receptor (40 kDa) levels in Th1 and Th2 cells of BALB/c and C3H/OuJ mice. Terminally differentiated Th1 and Th2 cells were activated with plate-bound anti-CD3 + anti-CD28 antibodies. β-Actin (47 kDa) was used as an internal control. d, expression of GrB was measured in Th2 cells in the presence or absence of PGE₂ after activation with plate-bound anti-CD3 antibodies. The red line indicates GrB staining, whereas the blue area indicates isotype control. e, selective inhibition of EP2 by siRNA abrogates the AICD inhibitory activity of PGE₂. Terminally differentiated Th2 cells were activated on plate-bound anti-CD3 overnight. Supernatant from LPS-activated macrophages inhibited AICD in control and mock-treated Th2 cells but not in EP2-siRNA (1 μm)-treated cells. DNA fragmentation was measured by PI staining. f, PGE₂ is unable to inhibit AICD of Th2 cells derived from BALB/c mice in the presence of EP2 receptor antagonist AH6809 (100 μm). Numbers indicated in the figures are the percentage of apoptotic cells. Data presented here are representative of at least three independent experiments.

(A) CD40L mobilization to the cell surface in 5CC7 Th1, 5CC7 Th2, and anti-IL-4 treated 5CC7 Th2 cells following 30 minutes (left histogram) or 2 hours (right histogram) of PMA and ionomycin stimulation as detected by inclusion of PE-labeled anti-CD40L antibody during stimulation. Control, unstimulated Th2 cells stained with PE-labeled anti-CD40L is shown in gray. (B) Three representative 5CC7 Th1, AD10 Th2, and anti-IL-4 treated AD10 Th2 immunological synapse structures are shown. ICAM-Cy5 is shown in red. Unlabeled peptide-bound I-E^k was included in the lipid bilayer. Th2 cells and anti-IL-4-treated Th2 cells were scored for formation of a bull’s eye ring of ICAM-1 in three independent experiments: 82 Th2 cells and 50 anti-IL-4-treated Th2 cells were scored in Experiment 1, 650 Th2 and 278 anti-IL-4-treated Th2 in Experiment 2, and 255 Th2 and 237 anti-IL-4 treated Th2 in Experiment 3.