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The biological outcome of CD40 signaling is dependent on the duration of CD40 ligand expression: reciprocal regulation by interleukin (IL)-4 and IL-12.

Lee BO, Haynes L, Eaton SM, Swain SL, Randall TD - J. Exp. Med. (2002)

Bottom Line: IL-4 represses, whereas IL-12 sustains CD154 expression.Consistent with this, Th1, but not Th2, cells express CD154 for extended periods.Thus, the differential ability of Th cells to sustain CD154 expression is an important part of their helper function and should influence the activities of other CD40-expressing cell types.

View Article: PubMed Central - PubMed

Affiliation: Trudeau Institute, Saranac Lake, NY 12983, USA.

ABSTRACT
CD40 ligand (CD154) expression on activated T cells can be separated into an early TCR-dependent phase, which occurs between 0 and 24 h after activation, and a later extended phase, which occurs after 24 h and is reciprocally regulated by the cytokines IL-4 and IL-12. IL-4 represses, whereas IL-12 sustains CD154 expression. Consistent with this, Th1, but not Th2, cells express CD154 for extended periods. Differences in the duration of CD154 expression have important biological consequences because sustained, but not transient, expression of CD154 on activated T cells can prevent B cell terminal differentiation. Thus, the differential ability of Th cells to sustain CD154 expression is an important part of their helper function and should influence the activities of other CD40-expressing cell types.

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Cytokines control the second phase of CD154 expression. (A) Purified naive AND T cells were stimulated with peptide-bearing CH12 cells in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression on Thy-1+ T cells was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (B) Purified naive AND T cells were cultured with plate-bound anti-CD3 in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (C) RNA was purified from AND T cells that had been stimulated by plate-bound anti-CD3 alone or with IL-4 or IL-12. 5 μg of purified total RNA was separated on a Northern blot, which was probed with the cDNA for CD154 and with the cDNA for a mitochondrial enzyme, CHO-B, as a loading control. (D) The amount of CD154 probe bound to the Northern blot was quantitated using a BioRad Molecular Imager FX PhosphorImager. The quantity shown represents the relative signal within the specific band minus the signal in an equivalent area adjacent to the specific band.
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fig2: Cytokines control the second phase of CD154 expression. (A) Purified naive AND T cells were stimulated with peptide-bearing CH12 cells in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression on Thy-1+ T cells was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (B) Purified naive AND T cells were cultured with plate-bound anti-CD3 in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (C) RNA was purified from AND T cells that had been stimulated by plate-bound anti-CD3 alone or with IL-4 or IL-12. 5 μg of purified total RNA was separated on a Northern blot, which was probed with the cDNA for CD154 and with the cDNA for a mitochondrial enzyme, CHO-B, as a loading control. (D) The amount of CD154 probe bound to the Northern blot was quantitated using a BioRad Molecular Imager FX PhosphorImager. The quantity shown represents the relative signal within the specific band minus the signal in an equivalent area adjacent to the specific band.

Mentions: To determine whether cytokines played a role in regulating the first or second phases of CD154 expression on activated T cells, purified naive AND T cells were stimulated with CH12 cells and PCCF in the presence of a variety of cytokines, and the kinetics of CD154 expression were followed over 72 h. We found that IL-2, IL-5, IL-6, IL-10, IL-13, and IFN-γ had no effect on either the levels or kinetics of CD154 expression (unpublished data). However, both IL-4 and IL-12 influenced the second phase of CD154 in the model of cognate T–B interaction (Fig. 2 A). Although IL-12 had minimal effect on the level of CD154 expression on peptide-stimulated T cells at 6 h, IL-4 slightly, but consistently, increased the percentage of cells expressing CD154 at this time (Fig. 2 A). Similar to what we observed in Fig. 1 B, there was a dramatic decrease in CD154 expression on T cells stimulated with peptide-presenting CH12 cells for 24 h, regardless of cytokine addition (Fig. 2 A). In striking contrast, however, the second phase of CD154 expression at 48 and 72 h was completely absent on T cells cultured with IL-4, whereas the second phase of CD154 expression remained elevated for extended periods on T cells cultured with IL-12 (Fig. 2 A). Furthermore, the inhibitory effects of IL-4 were dominant over the effects of IL-12 as T cells stimulated in the presence of both cytokines expressed CD154 in a pattern identical to that on T cells stimulated in the presence of IL-4 alone (Fig. 2 A). Similar results were seen using OTII transgenic T cells and with normal T cells (not depicted).


The biological outcome of CD40 signaling is dependent on the duration of CD40 ligand expression: reciprocal regulation by interleukin (IL)-4 and IL-12.

Lee BO, Haynes L, Eaton SM, Swain SL, Randall TD - J. Exp. Med. (2002)

Cytokines control the second phase of CD154 expression. (A) Purified naive AND T cells were stimulated with peptide-bearing CH12 cells in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression on Thy-1+ T cells was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (B) Purified naive AND T cells were cultured with plate-bound anti-CD3 in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (C) RNA was purified from AND T cells that had been stimulated by plate-bound anti-CD3 alone or with IL-4 or IL-12. 5 μg of purified total RNA was separated on a Northern blot, which was probed with the cDNA for CD154 and with the cDNA for a mitochondrial enzyme, CHO-B, as a loading control. (D) The amount of CD154 probe bound to the Northern blot was quantitated using a BioRad Molecular Imager FX PhosphorImager. The quantity shown represents the relative signal within the specific band minus the signal in an equivalent area adjacent to the specific band.
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Related In: Results  -  Collection

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fig2: Cytokines control the second phase of CD154 expression. (A) Purified naive AND T cells were stimulated with peptide-bearing CH12 cells in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression on Thy-1+ T cells was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (B) Purified naive AND T cells were cultured with plate-bound anti-CD3 in the presence of the indicated cytokines. Cells were removed from culture at the times indicated and CD154 expression was analyzed by flow cytometry. Open histograms represent control staining, whereas shaded histograms represent CD154 expression. (C) RNA was purified from AND T cells that had been stimulated by plate-bound anti-CD3 alone or with IL-4 or IL-12. 5 μg of purified total RNA was separated on a Northern blot, which was probed with the cDNA for CD154 and with the cDNA for a mitochondrial enzyme, CHO-B, as a loading control. (D) The amount of CD154 probe bound to the Northern blot was quantitated using a BioRad Molecular Imager FX PhosphorImager. The quantity shown represents the relative signal within the specific band minus the signal in an equivalent area adjacent to the specific band.
Mentions: To determine whether cytokines played a role in regulating the first or second phases of CD154 expression on activated T cells, purified naive AND T cells were stimulated with CH12 cells and PCCF in the presence of a variety of cytokines, and the kinetics of CD154 expression were followed over 72 h. We found that IL-2, IL-5, IL-6, IL-10, IL-13, and IFN-γ had no effect on either the levels or kinetics of CD154 expression (unpublished data). However, both IL-4 and IL-12 influenced the second phase of CD154 in the model of cognate T–B interaction (Fig. 2 A). Although IL-12 had minimal effect on the level of CD154 expression on peptide-stimulated T cells at 6 h, IL-4 slightly, but consistently, increased the percentage of cells expressing CD154 at this time (Fig. 2 A). Similar to what we observed in Fig. 1 B, there was a dramatic decrease in CD154 expression on T cells stimulated with peptide-presenting CH12 cells for 24 h, regardless of cytokine addition (Fig. 2 A). In striking contrast, however, the second phase of CD154 expression at 48 and 72 h was completely absent on T cells cultured with IL-4, whereas the second phase of CD154 expression remained elevated for extended periods on T cells cultured with IL-12 (Fig. 2 A). Furthermore, the inhibitory effects of IL-4 were dominant over the effects of IL-12 as T cells stimulated in the presence of both cytokines expressed CD154 in a pattern identical to that on T cells stimulated in the presence of IL-4 alone (Fig. 2 A). Similar results were seen using OTII transgenic T cells and with normal T cells (not depicted).

Bottom Line: IL-4 represses, whereas IL-12 sustains CD154 expression.Consistent with this, Th1, but not Th2, cells express CD154 for extended periods.Thus, the differential ability of Th cells to sustain CD154 expression is an important part of their helper function and should influence the activities of other CD40-expressing cell types.

View Article: PubMed Central - PubMed

Affiliation: Trudeau Institute, Saranac Lake, NY 12983, USA.

ABSTRACT
CD40 ligand (CD154) expression on activated T cells can be separated into an early TCR-dependent phase, which occurs between 0 and 24 h after activation, and a later extended phase, which occurs after 24 h and is reciprocally regulated by the cytokines IL-4 and IL-12. IL-4 represses, whereas IL-12 sustains CD154 expression. Consistent with this, Th1, but not Th2, cells express CD154 for extended periods. Differences in the duration of CD154 expression have important biological consequences because sustained, but not transient, expression of CD154 on activated T cells can prevent B cell terminal differentiation. Thus, the differential ability of Th cells to sustain CD154 expression is an important part of their helper function and should influence the activities of other CD40-expressing cell types.

Show MeSH