Disparate effects of phorbol esters, CD3 and the costimulatory receptors CD2 and CD28 on RANTES secretion by human T lymphocytes

Abstract

This study has examined the stimuli required for secretion of regulated upon activation, normal T-cell expressed, presumed secreted (RANTES) from T lymphocytes and found that stimuli such as phorbol 12-myristate 13-acetate (PMA), which are unable to support T-cell proliferation and interleukin-2 (IL-2) production, are nevertheless able to elicit strong secretion of RANTES. Conversely, stimuli such as CD2 and CD28 ligation, which are able to support T-cell proliferation, are unable to elicit RANTES secretion. Coligation of CD3 and CD28 drives T-cell proliferation to a similar degree as CD2 and CD28 coligation, yet also supports modest RANTES secretion. Furthermore, CD28 ligation enhances the secretion of RANTES stimulated by PMA and this costimulatory effect is abrogated by the phosphoinositide 3-kinase inhibitor wortmannin. Our data also indicate that the observed effects of PMA on RANTES secretion are probably due to activation of protein kinase C (PKC) isoenzymes, since RANTES secretion was unaffected by the non-PKC activating 4alpha-phorbol ester, whilst the general PKC inhibitor Ro-32-0432 inhibits PMA-stimulated RANTES secretion. Moreover, the effect of PMA appears to be chemokine-specific because PMA was unable to increase secretion of the related CC chemokine MIP-1alpha. Under stimulation conditions where increases in [Ca2+]i occur (e.g. PMA plus ionomycin or CD3 plus CD28 ligation) RANTES secretion can be severely reduced compared with the levels observed in response to the phorbol ester PMA. Hence, whilst PKC-dependent pathways are sufficient for strong RANTES secretion, a calcium-dependent factor is activated which negatively regulates RANTES secretion. This correlates well with the observation that ligation of cytolytic T lymphocyte-associated antigen-4 (CTLA-4) (expression of which has been reported to be dependent on a sustained calcium signal), inhibits RANTES secretion induced by CD3/CD28, but has no effect on PMA-stimulated RANTES secretion.

Figure 1

Effect of PMA on RANTES secretion and [³H]thymidine incorporation. (a) Purified T lymphocytes were resuspended at 5 × 10⁵ cells/well in a 24-well tissue culture plates and were either left unstimulated (○) or stimulated with anti-CD3/CD28-coated beads at 1 bead/cell (▪) or 5 ng/ml PMA (□). (b) Alternatively, T lymphocytes were incubated with either vehicle or PMA at the concentrations indicated. At the times indicated (a) or at 72 hr (b), supernatants were removed and assayed for RANTES by ELISA as described in Materials and methods. Data are the means ± SEM of four separate experiments. (c) Purified T cells were aliquoted (6 × 10⁴ cells/well) in 96-well tissue culture plates and stimulated as indicated with anti-CD3/CD28, anti-CD2/CD28-coated beads at 1 bead/cell or 5 ng/ml PMA alone or in combination with 1 µm ionomycin or anti-CD28-coated beads as indicated. Proliferation was measured by [³H]thymidine incrporation as described in Materials and methods. Data are the means ± SEM of quintuplicate replicates from a single experiment representative of four others.

Figure 2

Divergent effect of PMA on RANTES and MIP-1α secretion by purified T lymphocytes. Purified T lymphocytes were resuspended at 5 × 10⁵ cells/well in 24-well tissue culture plates and were either left unstimulated or stimulated with 5 ng/ml PMA alone or in combination with 1 µm ionomycin or anti-CD28-coated beads. Alternatively, cells were stimulated with anti-CD3/CD28 or anti-CD2/CD28-coated beads as indicated at 1 bead/cell. After 72 hr at 37°, supernatants were removed and assayed for either RANTES (open histobars) or MIP-1α (shaded histobars) by ELISA as described in Materials and methods.

Figure 3

Effect 4α phorbol and Ro-32–0432 on PMA-stimulated RANTES secretion. Purified T lymphocytes were resuspended at 5 × 10⁵ cells/well in 24-well tissue culture plates and were treated with 5 ng/ml 4α-phorbol or incubated for 60 min either in the absence or presence of 10 µm of the PKC inhibitor Ro-32-0432 as indicated. Cells were then left unstimulated or stimulated with 5 ng/ml PMA alone or in combination with anti-CD28-coated beads, or with anti-CD3/CD28-coated beads at 1 bead/cell. After 72 hr at 37°, supernatants were removed and assayed for RANTES by ELISA as described in Materials and methods.

Figure 4

Effect of CD28 on PMA-stimulated RANTES secretion. Purified T lymphocytes were resuspended at 5 × 10⁵ cells/well in 24-well tissue culture plates and were (a) left unstimulated (○) or stimulated with either anti-CD2/CD28-coated beads (•) or 5 ng/ml PMA plus anti-CD28-coated beads (▪) as described in Materials and methods. (b) Alternatively, aliquoted T lymphocytes were incubated for 10 min with vehicle or wortmannin at the concentrations indicated prior to the addition of 5 ng/ml PMA either alone (open histobars) or in combination with anti-CD28-coated beads (solid histobars). Antibody-coated beads were used at 1 bead/cell. At the times indicated (a) or at 72 hr (b), supernatants were removed and assayed for RANTES by ELISA. Data are the means ± SEM of four separate experiments. Significant inhibition of vehicle-treated PMA/CD28 stimulated levels are denoted by *** (P < 0·001) ** (P < 0·01) and * (P < 0·05) using two-tailed Student's t-test.