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. 2000 Sep;101(1):30–37. doi: 10.1046/j.1365-2567.2000.00072.x

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

Y Sotsios *, P J Blair †, J Westwick *,‡, S G Ward *
PMCID: PMC2327056  PMID: 11012750

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 stumulated 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 4α-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-1α. 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.

Introduction

Two main T-cell activation pathways have been described: one is antigen dependent and involves the CD3/T-cell receptor (TCR) complex together with signals provided by costimulatory molecules such as CD28.1 The other is antigen independent and involves ligation of CD2 and CD28.2 The CD2 surface molecule is a 50 000 MW surface molecule present on 95% of T cells.3,4 Stimulation via CD2 requires a pair of CD2 antibodies directed against different epitopes,5,6 although activation requires the expression of the TCR/CD3 complex.7,8 CD28 is present on 90% of all CD4+ and 50% of CD8+ T cells and the natural ligands are B7.1 and B7.2.9 Curiously, these natural ligands for CD28 are also shared by the CD28 homologue cytolytic T lymphocyte-associated antigen-4 (CTLA-4), which provides important signals that negatively regulate T-cell activation.10,11 Proliferation of highly purified T cells in the absence of accessory cells that provide the second signal by cell contact or cytokine secretion, requires dual triggering by specific antibodies to either CD3 or CD2 in combination with CD28 ligation by either monoclonal antibodies (mAb) or the natural ligands B7.1/B7.2.2,12,13 Alternatively, phorbol esters such as phorbol 12-myristate 13-acetate (PMA) in combination with either calcium ionophores or antibodies/natural ligands to CD28 can support T-cell proliferation.2,12,13

Activation of T lymphocytes with CD2 and CD28 mAbs leads to T-cell proliferation that is independent of monocytes and is driven by high-level, long-lasting autocrine interleukin-2 (IL-2)-dependent CD4+ T-cell stimulation.2 This stimulation also induces secretion of other cytokines, either T-cell-specific such as interferon-γ (IFN-γ), or those normally synthesized by accessory cells such as tumour necrosis factor-α (TNF-α), colony-stimulating factor-1 (CSF-1) and IL-1α.14,15 A number of chemokines are also produced upon TCR/CD28-driven T-cell activation including RANTES, macrophage inflammatory protein-α (MIP-α) and MIP-1β as well as IL-8, and these chemokines have important roles to play in leukocyte migration and/or protecting against human immunodeficiency virus (HIV) entry.16–19 Strong up-regulation of the RANTES gene occurs 3–5 days after activation of resting peripheral blood T cells with either mitogen or antigen, whilst RANTES promoter activity in T cells has been shown to involve both early-acting and late-acting transcriptional regulatory events.20 However, in contrast to cytokines up-regulated relatively early during T-cell activation (e.g. IL-2) the precise stimuli and biochemical signals required for RANTES secretion by T cells, as well as the sensitivity of activated RANTES secretion to the inhibitory signals generated by CTLA-4 ligation have not been extensively investigated. We have therefore analysed the effect on RANTES secretion from purified human T cells, of distinct T-cell activating conditions which provide pharmacologically distinct biochemical signals for T-cell proliferation and IL-2 production. These stimuli include PMA in the absence or presence of either ionomycin or anti-CD28 antibodies, combinations of anti-CD3 or anti-CD2 antibodies plus anti-CD28 antibodies and combinations of anti-CD3, anti-CD28 and anti-CTLA-4 antibodies. Our results reveal that the stimulation conditions for RANTES secretion are disparate to those required for T-cell proliferation, although both functional responses are sensitive to inhibitory actions of CTLA-4 ligation.

Materials and methods

Human T-cell isolation

Purified resting T lymphocytes were isolated from peripheral blood of healthy human donors. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation at 800 g for 30 min (Nycomed 1·077 g/ml) and the buoyant layer isolated. Following two washes with RPMI-1640 medium, the cells were further purified by adhering to plastic for 1 hr at 37° in complete medium (RPMI-1640 containing 10% fetal calf serum (FCS), 500 U/ml penicillin and streptomycin and 0·5 µg/ml amphotericin B). Non-adherent cells containing the T-cell population were recovered and incubated with mAbs against CD14 (UCHM1), human leucocyte antigen (HLA)-DR (L243) and CD19 (BU12). Stained cells were magnetically removed using sheep anti-mouse immunoglobulin G (IgG) magnetic beads (Dynal, Oslo, Norway). The remaining cells that comprised resting T cells were used in subsequent experiments. T-lymphocyte purity was typically ≥ 97% CD3+ T cells as assessed by flow cytometry (Becton Dickinson FACS Vantage, Becton Dickinson, San Jose, CA).

Reagents and antibodies

PMA, ionomycin, 4α-phorbol and Ro-32-0432 were all from Calbiochem (Nottingham, UK). T cells were stimulated with either humanized anti-CD3 mAb OKT3, anti-CD28 mAb 9.3 or anti-CD2 mAbs 6F103 and 39Ci5 (a generous gift from Daniel Olive, INSERM, Marseille, France) used in the combinations described. The anti-CTLA-4 mAb 3D6 was a kind gift to P. Blair from Beatriz Carreno at Genetics Institute. The anti-CD3, anti-CD2 and anti-CD28 mAbs were covalently attached (either alone or in the combinations indicated) to polyurethane-coated tosyl-activated Dynabeads (Dynal, Lake Success, NY) at 1 µg/ml each, whereas the anti-CTLA-4 mAb was used at 9 µg/ml. The Dynabeads were used at a bead : cell ratio of 1 : 1, as previously described.21,22

T-cell proliferation and chemokine enzyme-linked immunosorbent assay (ELISA) assays

Purified T cells were re-suspended in RPMI-1640 supplemented with 10% FCS and aliquoted (6 × 104 cells/well), in 96-well tissue culture plates and stimulated in quintuplicate as indicated. The plates were incubated at 37° for 72 hr and pulsed for the last 18 hr with 0·5 µCi/well [3H]thymidine (Amersham, Little Chalfont, UK). Cells were harvested onto 96-well filter plates (UnifilterTM, Packard Bioscience Ltd, Pangbourne, UK) and radioactivity was measured with a β-scintillation counter (TopCountTM, Packard Bioscience Ltd, Pangbourne, UK) according to manufacturer's specifications. Alternatively, purified T cells were resuspended at 5 × 105 cells/well in 24-well tissue culture plates and were stimulated appropriately. RANTES or MIP-1α were assayed in the supernatant by ELISA as previously described. Mouse monoclonal anti-RANTES and MIP-1α antibodies were used to coat the microtitre plates overnight at 4°. Polyclonal goat anti-RANTES and anti-MIP-1α antibodies, biotin conjugated to alkaline phosphatase were used for detection. ELISA were developed by incubation with streptavidin horseradish peroxidase (1 µg/ml for 15 min) followed by o-phenylenediamine dihydrochloride and H2O2 as the substrate. The sensitivity of the assays was 0·2 ng/ml.

Results

PMA stimulates RANTES secretion

The protein kinase C (PKC)-activating phorbol ester PMA elicited strong time-dependent secretion of RANTES in the absence of any other signal (Figs 1a and 2), yet elicited only slight increases above basal, of T-cell proliferation as assessed by [3H]thymidine incorporation (Fig. 1c). PMA-stimulated RANTES secretion exhibited a modest increase by day 2 with maximum levels of RANTES produced at 72 hr onwards (Fig. 1a). The effect of PMA on RANTES secretion was concentration-dependent and exhibited bell-shaped characteristics (Fig. 1b). In addition, PMA-stimulated RANTES secretion was unaffected by the non-PKC activating 4α-phorbol ester (Fig. 3), whilst the general PKC inhibitor Ro-32-0432 inhibited the PMA-stimulated RANTES secretion (Fig. 3). Moreover, the effect of PMA appears to be chemokine-specific because PMA was unable to increase levels of the related CC chemokine MIP-1α (Fig. 2).

Figure 1.

Figure 1

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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.

Figure 2

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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.

Figure 3

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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.

Elevation of [Ca2+]i down-regulates RANTES secretion

Interestingly, elevation of [Ca2+]i by the addition of the calcium ionophore ionomycin in combination with PMA, severely reduced the levels of RANTES secretion compared to the levels observed in response to PMA alone (Fig. 2). One possible interpretation of this data is that PKC-dependent pathways are sufficient to drive high-level RANTES secretion, whilst an as yet unidentified calcium-dependent factor is activated which negatively regulates either RANTES gene transcription and/or secretion. To further investigate this possibility we compared the levels of RANTES secretion stimulated by PMA with those induced by combinations of receptor stimuli in which at least one of the receptors is capable of eliciting an elevation of [Ca2+]i. Thus, ligation of CD3 is known to be a strong stimulus for mobilization of calcium, whilst in comparison CD28 elicits little if any elevation of [Ca2+]i, yet together these two receptors can effectively costimulate T-lymphocyte activation.23 As predicted, substantially lower levels of RANTES are obtained upon stimulation with CD3 in the presence of CD28 stimulation compared to levels obtained in response to PMA alone (Figs 1a and 2). Although much lower amounts of RANTES were produced in response to CD3/CD28 ligation compared to stimulation with PMA, the kinetics of CD3/CD28-stimulated RANTES secretion again exhibited a modest increase by day 2 with maximum levels of RANTES produced at 72 hr onwards (Fig. 1a).

CD28 costimulation of PMA-induced RANTES secretion is inhibited by the phosphoinositide 3-kinase inhibitor wortmannin

Ligation of CD28 with anti-CD28 mAb 9.3 in the presence of PMA resulted in enhanced RANTES secretion compared to the levels observed in the presence of PMA alone (Fig. 2). Once again, the kinetics of RANTES secretion in response to CD28/PMA stimulation, also exhibited a modest increase by day 2 with maximum levels of RANTES produced at 72 hr onwards (Fig. 4a). In contrast, the anti-CD28 antibody alone had no effect on either RANTES secretion (Fig. 2) or T-cell proliferation (Fig. 1c). It is interesting to note however, that CD28/PMA stimulated levels of T-cell proliferation were comparable to those stimulated by CD3/CD28 and PMA/ionomcyin (Fig. 1c). Pre-treatment with the phosphoinositide (PI) 3-kinase inhibitor wortmannin abrogated the costimulatory effect of CD28 ligation on PMA-stimulated RANTES secretion in a concentration-dependent manner. In contrast, pretreatment with wortmannin had no effect on the level of RANTES secretion in response to PMA alone (Fig. 4b)

Figure 4.

Figure 4

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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.

CD2 and CD28 ligation stimulates T-cell proliferation but not RANTES secretion

From the data presented, CD28 can support RANTES secretion in combination with either CD3 or PMA, albeit at different levels. Given that coligation of CD2 and CD28 leads to T-cell activation and induces production of cytokines,2,14,15 it was important to investigate whether CD28 and CD2 coligation can also support RANTES secretion. However, the combination of anti-CD2 and anti-CD28 antibodies, which was sufficient to drive T-cell proliferation at levels comparable to that elicited by CD3/CD28, PMA/CD28 and PMA/ionomycin (Fig. 1c), did not procure RANTES secretion above basal levels at any time point examined (Fig. 4).

Effect of CTLA-4 on RANTES secretion

CTLA-4 ligation has previously been reported to deliver a unique signal to resting human T cells that inhibits IL-2 secretion but allows CD28-induced Bcl-xL induction.21 Hence, given our observations that RANTES secretion can occur under conditions which are unable to support T-cell proliferation, it was important to establish whether the activating signals that control RANTES secretion were subject to the same negative-regulating signals provided by CTLA-4. We therefore investigated the effect of the anti-CTLA-4 3D6 mAb on RANTES secretion because it has been previously reported to negatively regulate IL-2 expression and secretion.21 Indeed, stimulation of T cells with anti-CTLA-4 mAb 3D6 in combination with anti-CD3 and anti-CD28 antibodies, resulted in reduced RANTES secretion and T-cell proliferation compared to that induced by anti-CD3 and anti-CD28 antibodies (Table 1). However, the anti-CTLA-4 mAb 3D6 had no major effect on the RANTES secretion or T-lymphocyte proliferation stimulated by PMA either alone or in combination with CD28.

Table 1.

Effect of CTLA-4 on CD3/CD28 and PMA-stimulated [3H]thymidine incorporation and RANTES secretion

Treatment [3H]thymidine incorporation (c.p.m. ± SEM) RANTES production (ng/ml)
Basal 1259 ± 600 1·46 ± 0·5
CD3/CD28 23652 ± 3500 25·59 ± 2·3
CD3/CD28/CTLA-4 6018 ± 1500 10·97 ± 1·6
PMA 3630 ± 700 41·16 ± 7
PMA/CTLA-4 3446 ± 680 39·83 ± 5·8
PMA/CD28 31029 ± 4800 69·51 ± 6·8
PMA/CD28/CTLA-4 29653 ± 4000 65·56 ± 5
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Purified T lymphocytes were resuspended at 5 × 105 cells/well in 24-well tissue culture plates or at 6 × 104 cells/well in 96-well tissue culture plates. Cells were left unstimulated or stimulated with 5 ng/ml PMA, 5 ng/ml PMA plus anti-CD28-coated beads or anti-CD3/CD28-coated beads in the absence or presence of anti-CTLA-4 3D6 antibody. Antibody-coated beads were used at 1 bead/cell. [3H]thymidine incorporation and RANTES secretion were assayed as described in Materials and methods. Data are the means ± SEM of four separate experiments.

Discussion

This study has examined the stimuli required for secretion of RANTES from T lymphocytes and found that the phorbol ester PMA, which is unable to support T-cell proliferation, is 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, whereas coligation of CD3 and CD28 drives T-cell proliferation to a similar degree as CD2 and CD28 coligation, yet supports only modest RANTES secretion.

CD3 can support RANTES secretion from highly purified T cells in the presence of CD28 stimulation. However, it appears that CD2 is unable to provide a key signal(s) that can complement those provided by CD28 in order to support RANTES production. It is surprising that CD2 ligation is unable to support RANTES secretion in the presence of CD28 stimulation since previous observations are consistent with CD2 generating similar intracellular biochemical signals to those generated by the TCR/CD3 complex. For instance, they both stimulate Ras24 activate phospholipase C and elevate [Ca2+]i25,26 and activate PKC,27 stimulate PI 3-kinase and the accumulation of D-3 phosphoinositide lipids28 as well as stimulate tyrosine phosphorylation of CD3ζ29 and Lck.30 However, whilst it has been demonstrated that CD3 and CD2 stimulate the tyrosine phosphorylation of a similar pattern of polypeptides,31 they may not be identical. Indeed, CD2 ligation unlike CD3, is unable to induce tyrosine phosphorylation of HS1, a signalling protein of unknown function.32

Our data indicates a strong up-regulation of RANTES secretion by PMA alone that is probably due to activation of PKC isoenzymes, as RANTES secretion was unaffected by the non-PKC activating 4α-phorbol ester, whilst the general PKC inhibitor Ro-32-0432 inhibited PMA-stimulated RANTES secretion. Moreover, the effect of PMA appears to be chemokine-specific since PMA was unable to increase levels of the related CC chemokine MIP-1α, except in the presence of CD28 ligation. The reasons for the bell-shaped concentration-dependent effects of PMA on RANTES secretion are not clear at present. However, this phenomenon may indicate that at lower concentrations, PMA is able to stimulate distinct PKC isozymes that support RANTES secretion, whereas higher concentrations stimulate additional PKC isozymes that operate negative regulatory pathways for RANTES secretion. The maximum response of RANTES secretion in response to PMA and CD3/CD28 occurs at 72 hr onwards and correlates well with reports describing the late (e.g. day 3–5) up-regulation of transcription factors controlling the RANTES gene in T cells.20 The significance of the delayed kinetics of RANTES secretion compared to IL-2 secretion20 is not entirely clear, but may be an important process related to subsequent differentiation changes within the T-cell pool.

PKC can regulate a number of T-cell activation genes via control of transcription factors and the RANTES promoter region contains PKC responsive elements such as sites for nuclear factor κB (NFκB), AP-1 and NF-AT.18 Mutation of these sites has been found to disrupt PMA–ionomycin stimulated RANTES promoter activity.18 Interestingly, CD3 and CD28 have both been reported to activate PKC,23,27 although ligation of either receptor in the absence of any other stimuli, is unable to support RANTES production. This may suggest that PKC activation has to occur at critical thresholds of stimulation to enable RANTES secretion. Hence, whilst stimulation of PKC by PMA is sufficient to cross this threshold, CD3 or CD28 ligation alone provide insufficient activation of PKC to support RANTES secretion. It is also possible that other elements within the RANTES promoter may respond to PMA and previous studies have indicated that phorbol esters can activate the 9E3/cCAF chemokine gene via multiple signal transduction pathways which are predominantly PKC-independent and which converge on mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK1/ERK2) and activate the Elk1 transcription factor.33

PMA-stimulated RANTES secretion can be further enhanced by CD28 ligation. CD28 has also been demonstrated to enhance PMA-stimulation of NFκB as well as AP-134 and one of the NFκB binding sites serves as a CD28RE.18,20 A major biochemical event elicited by CD28 is activation of the PI 3-kinase-dependent signalling cascade.23 Downstream effectors of this pathway include PKB,35 which has been shown to be involved in activation of NFκB in several systems including T cells.36–38 Pharmacological studies revealed that a functional PI 3-kinase-dependent signalling mechanism is indeed required for the CD28-mediated enhancement of PMA-stimulated RANTES secretion. However, whilst CD28-stimulated AP-1 generation is inhibited by PI 3-kinase inhibitors, it should be noted that CD28-mediated induction of NFκB is resistant to PI 3-kinase inhibitors in a T lymphoblast cell model.39 So, the contribution of other signalling pathways to the CD28 costimulatory effect on PMA-stimulated RANTES secretion cannot therefore be discounted.

From the above data, it is apparent that the biggest increases in RANTES secretion were detected following PMA stimulation either alone or in combination with CD28 ligation. However, markedly reduced levels of RANTES secretion were observed when activation conditions capable of eliciting elevation of [Ca2+]i were employed (e.g. ligation of CD3 or CD2 in the presence of CD28 stmulation or use of calcium ionophores in combination with PMA). One possible interpretation of this data is that PKC-dependent pathways are sufficient to ensure major RANTES secretion, whilst an as yet unidentified calcium-dependent factor is activated that negatively regulates RANTES gene transcription/secretion. Certainly, a negative regulatory region of the RANTES promoter has been described.18 Whilst there may well be an intracellular calcium-dependent factor(s) that can modulate RANTES gene transcription, we considered that an alternative explanation for this consistent observation may revolve around the fact that mRNA induction and surface expression of CTLA-4 expression is dependent on a sustained calcium signal.40,41 In addition, repeatedly activated T cells are known to up-regulate expression of firstly B7.2 followed by B7.142,43 and the B7.2 on T cells has been suggested to preferentially bind CTLA-4.44 Whilst B7.1 and B7.2 up-regulation has been reported to occur much later (e.g. 10 days) than the time courses examined in this study, it is possible that these molecules are expressed at low, undetectable levels. Given the very high affinity of CTLA-4 for its B7 ligands,23 this low expression of B7.1 and/or B7.2 may be sufficient to exert functional effects via CTLA-4. Certainly, inhibitory anti-CTLA-4 antibodies abrogated CD3/CD28-driven RANTES secretion in this study. Hence, the lower levels of RANTES secretion observed in response to CD3/CD28 ligation compared to PMA stimulation, may indeed reflect an inhibitory signal that is initiated following CTLA-4 binding B7 which is expressed on activated T cells. This explanation does not correlate too well with the observation that CD3 plus CD28 ligation is able to initiate robust T-cell proliferation. However, T-cell proliferation is driven by IL-2 production, which is initiated within 6 hr of stimulation,12 whilst RANTES gene up-regulation occurs much later at 72 hr.20 It is certainly possible therefore, that the mechanisms for IL-2 and RANTES gene transcription, mRNA stability, translation and/or secretory processes have different thresholds for regulation by CTLA-4. That is to say, inhibition of IL-2 secretion may occur at very robust levels of CTLA-4 activation, whilst inhibition of RANTES secretion may occur at quantitatively lower levels of CTLA-4 activation. The specificity of the anti-CTLA-4 antibody inhibitory effect on CD3/CD28 stimulated RANTES secretion was confirmed by the fact that it had no effect on RANTES secretion in response to PMA, which has previously been reported to be unable to induce detectable CTLA-4 expression in this cell model.45

As a conclusion, we propose that RANTES secretion can occur under stimulation conditions which are not sufficient to drive T-cell activation and proliferation (e.g. in response to PMA alone). Conversely, certain stimuli sufficient to drive T-cell proliferation and IL-2 production (e.g. CD2 plus CD28 ligation) are not able to elicit RANTES secretion. Moreover, RANTES secretion is also subject to inhibitory modulation by appropriate antibody ligation of CTLA-4 as has been previously reported for IL-2 secretion and T-cell proliferation.21 However, this particular inhibitory modulation of RANTES can be by-passed if cells are activated under conditions in which CTLA-4 expression cannot occur (e.g. PMA stimulation). It is important to note that at present, we are unable to distinguish between the effects of the various activating stimuli on RANTES mRNA, protein synthesis and/or release of RANTES from intracellular stores and this will be the focus of future work. Nevertheless, selective induction of RANTES secretion in the absence of T cell proliferation and vice versa, may offer novel and selective targets for therapeutic intervention in a number of disease settings where it may be beneficial to regulate chemokine secretion without necessarily altering overall immune capabilities. For instance, targeted up-regulation of RANTES secretion from candidate cells such as T lymphocytes, monocytes or macrophages, may be beneficial during the early stages of M-tropic HIV infection, where it may be possible to compete out the M-tropic HIV binding to CC chemokine receptor-5 (CCR5) by autocrine ligand-induced CCR5 down-regulation. Moreover, T helper 1 (Th1) cells have been reported to express the chemokine receptor CCR5.19 Hence, it is possible that abnormal autocrine over-production of RANTES secretion may contribute to excessive Th1-dependent immune responses in disease states such as rheumatoid arthritis. One important goal of future studies will be to gain a greater understanding of the biochemical signals required for RANTES gene expression and secretion, as these may offer potential routes for manipulation of RANTES bioavailability.

Acknowledgments

We thank Peter Nelson and Melanie Welham for critical reading of the manuscript. This work was supported by the Wellcome Trust (S.G.W.).

Glossary

Abbreviations

PKC

protein kinase C

PI 3-kinase

phosphoinositide 3-kinase

References

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