RANTES activates antigen-specific cytotoxic T lymphocytes in a mitogen-like manner through cell surface aggregation

Victor Appay, P. Rod Dunbar, Vincenzo Cerundolo, Andrew McMichael, Lloyd Czaplewski1 and Sarah Rowland-Jones

MRC Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK
1 British Biotech Pharmaceuticals Ltd, Oxford OX4 5LY, UK

Correspondence to: V. Appay


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
RANTES (regulated upon activation, normal T cell expressed and secreted) is released by cytotoxic T lymphocytes (CTL), and is a potent chemoattractant factor for monocytes and T cells, also known for its ability to suppress HIV infection. At micromolar concentration, RANTES is able to activate leukocytes, and, paradoxically, to enhance HIV infection in vitro. These latter properties are dependent on its ability to self-aggregate. In order to understand further the mechanism of RANTES-induced activation, the effects of both aggregated and disaggregated RANTES on antigen-specific CD8+ clones were studied in comparison with the effects of specific antigens and in the presence of specific inhibitors of RANTES-mediated activation. We observed large amounts of RANTES aggregated on the cell surface, which led to cell activation, including up-regulation of cell surface markers, and secretion of IFN-{gamma} and macrophage inflammatory protein (MIP)-1ß. Specific inhibitors of RANTES-induced activation, such as soluble glycosaminoglycans, MIP-1{alpha} and MIP-1ß, acted by preventing the binding of RANTES on the cell surface. These studies suggest that RANTES acted more like a mitogen than an antigen-independent activator.

Keywords: activation, CD8+ T cells, chemokines, glycosaminoglycans, inhibitors, macrophage inflammatory protein


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The chemokines are soluble factors, well known for their involvement in chemotaxis (1), and are increasingly recognized to have a much broader range of functions, e.g. chemokines like IL-8, macrophage inflammatory protein (MIP)-1{alpha}, MCP-1 and GRO-ß have been shown to be inhibitors of hematopoietic progenitor cell proliferation (2). RANTES (regulated upon activation, normal T cell expressed and secreted) is a CC chemokine which preferentially has chemoattractant properties for monocytes and memory T cells (3). It is a pro-inflammatory chemokine and is found at the sites of many inflammatory disorders (reviewed in 4). It is also intimately linked with the function of cytotoxic T lymphocytes (CTL), which are important in the control of many intracellular pathogens (5). RANTES is present in CTL granules, where it is complexed to glycosaminoglycans (GAG), and is secreted during degranulation (6,7). It has been suggested that CTL responses are enhanced by the binding of RANTES to the CCR3 receptor (8).

RANTES has attracted particular attention following the observation that one of its receptors, CCR5, is a co-receptor for HIV entry into CD4+ cells (9,10). Together with the other chemokines which bind to CCR5, RANTES is able to suppress HIV-1 infection in vitro (11). The potential for the use of RANTES in antiviral therapy for HIV-1 infection has led to more detailed studies of its biology. Recent studies have shown that RANTES at micromolar concentrations can activate T cells, apparently acting as an antigen-independent T cell activator: this activity is independent of binding to CCR5 and other chemokine receptors (1214). RANTES triggers two signalling pathways in T cells. At nanomolar concentrations, G-protein-coupled receptor (GPCR)-mediated signal is generated, leading to cell migration. At higher concentrations (micromolar and above), a protein tyrosine kinase (PTK)-mediated signal is triggered, leading to cell activation. RANTES-induced activation appears not to be confined to T cells, but dual signalling pathways may also be induced in monocytes and neutrophils (14). In addition, high concentrations of RANTES have been shown to enhance HIV infection in vitro, independently of its binding to chemokine receptors (15,16). The enhancement of HIV infection mediated by RANTES is partially due to its activating effect on cells (17). These observations are important to consider before forms of RANTES are used therapeutically.

A particular characteristic of RANTES is its ability to self-aggregate, forming multimers at high concentration. Czaplewski et al. have been able to produce non-aggregating variants of RANTES, by means of single amino acid substitutions (Glu26 in Ser or Glu66 in Ser), which retain full activity through RANTES-specific chemokine receptors (15). These variants have appeared to have little or no effect on leukocyte activation, leading to the conclusion that the aggregation of RANTES is necessary for its activating effects (14). Non-aggregating RANTES also fails to enhance HIV infection at high concentration (15,17). MIP-1{alpha} and MIP-1ß, which share considerable homology with RANTES, do not activate leukocytes, but surprisingly are able to inhibit RANTES-induced activation (14). Disaggregated RANTES also acts as an inhibitor and the presence of erythrocytes inhibits RANTES-induced activation (14). In addition to binding specific chemokine receptors, RANTES is also able to bind to cell surface GAG which promote RANTES oligomerization (18): this mechanism is involved in RANTES-mediated enhancement of HIV infection (17).

Aggregated and disaggregated RANTES are therefore tools which separate the specific binding of chemokine receptors from the non-specific effects on activation. These reagents can be used to improve our understanding of the role of RANTES in the immune response to infection and the potential problems for its use in therapy. Previous work used peripheral blood mononuclear cells (PBMC) and cell lines without defined antigen specificity. In this report, we have compared the effect of RANTES, both aggregated and disaggregated, with the effect of specific antigens on the activation of CTL clones of known specificity, using staining for extracellular and intracellular markers, and CTL cytotoxicity assays. We find that RANTES is aggregated on the cell surface, but cell-surface RANTES is diminished by inhibitors of activation. We show for the first time that, although RANTES activates T cells, its effects are distinct from those of specific antigens. These observations provide new insights into the mechanism of RANTES-induced activation.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
The chemokines and disaggregated variants were made at British Biotech Ltd. (Oxford, UK) (15). Anti-CD8 (PerCP), CD69 [FITC or phycoerythrin (PE)], CD25 (FITC), HLA-DR (FITC), CD11c (PE), CD11b (PE) and CD11a (FITC) mAb were purchased from Becton Dickinson (Mountain View, CA), and anti-{alpha}ß-TCR (FITC) from Serotec (Oxford, UK). Monoclonal anti-RANTES antibodies (FITC or PE) were purchased from Serotec or PharMingen (San Diego, CA) respectively. Monoclonal anti-MIP-1ß (FITC) or anti-IFN-{gamma} (FITC) antibodies were purchased from R & D Systems (Abingdon, UK). Anti-CD3 antibodies, OKT3, were purchased from Ortho (Raritan, NJ). Peptides were synthesized by Fmoc chemistry and corresponded to the following defined CTL epitopes: `A2-FLU': GILGFVTVL, the influenza matrix protein 58–66 epitope (18); `A2-gag': SLYNTVATL, the immunodominant epitope from the HIV gag p17 protein (19); `A2-tyrosinase': YMDGTGMSQV, the N370D variant of the 368–377 epitope, generated by antigen processing (20); `A2-HCV': LLFNILGGWV, the 1807–1816 epitope of the NS4B protein (21,22); and `B7-CMV': TPRVTGGGAM, the lower matrix protein pp65 417–426 epitope. The Jurkat cell line was obtained from the European Collection of Animal Cells. They were cultured in RPMI 1640, 2 nM glutamine and 10 % (v/v) FCS at 37°C.

Generation of antigen-specific CD8+ clones
Tetrameric HLA–peptide complexes (`tetramers') were synthesized as previously described, (19,23,24) and human CTL were cloned from human peripheral blood lymphocytes (PBL), with or without peptide stimulation, by tetramer-directed sorting (24,25). Briefly, cells were stained with PE-labelled tetramer for 15 min at 37°C before addition of TriColor–anti-CD8 (Caltag, Burlingame, CA) for 15 min on ice, followed by extensive washes and analysis on a FACS Vantage (Becton Dickinson) using CellQuest software. Small lymphocytes were gated by forward and side scatter profile before cloning according to tetramer/CD8 double-staining. To exclude cells showing high non-specific fluorescence at multiple wavelengths, cells exhibiting high signal at 420 nm wavelength (FL1) were gated out. Single cells were cloned directly into U-bottom 96-well plates using CloneCyt software. Each well had previously been coated with anti-CD3 (OKT3; Ortho) and anti-CD28 (Becton Dickinson), both at 100 ng/well in sterile PBS for at least 60 min at 37°C, before flicking off the antibody solution and adding 100,000 irradiated allogenic B cells in 100 µl CTL medium. Cloning plates were incubated at 37°C in 5% CO2 for 10–14 days without any manipulation before inspection. Wells with substantial growth were then expanded in CTL medium, by splitting into U-bottom 96-well plates, then 24-well plates. Selected clones were re-stimulated when proliferation reached a plateau, usually at ~28 days after cloning, by adding ~2x106 irradiated allogeneic PBL in CTL medium with phytohemagglutinin (PHA) 5 µg/ml.

Flow cytometry
Cells were incubated in FCS-free RPMI 1640 with chemokines, specific peptide-pulsed T2 cell line or matching Epstein–Barr virus (EBV)-transformed cell line, OKT3 (coated on the wells for 2 h at 37°C), or untreated (addition of PBS as control) for 6 h at 37°C. The cells were harvested, washed in PBS and incubated with appropriate staining antibodies for 15 min at room temperature. Cells were washed and analysed by flow cytometry on a Becton Dickinson FACScalibur using CellQuest software (Becton Dickinson). Erythrocytes were purified from donor blood and re-suspended in RPMI 1640 medium at 109 cells/ml with CTL at 106 cells/ml to reach a similar white/red blood cell ratio as found in whole blood. After incubation with chemokines or peptide-pulsed targets, erythrocytes were lysed using FACS lysis buffer (Becton Dickinson), and CTL were stained and analysed as previously.

Immunofluorescence microscopy
Jurkat cells were incubated at 37°C for 15 min in the presence of RANTES, disaggregated RANTES, MIP-1{alpha}, MIP-1ß or PBS. Cells were fixed in 5% formaldehyde and stained using anti-RANTES antibodies for 15 min at room temperature. Microphotographs were taken using a fluorescence microscope (Nikon) at x40.

Intracellular staining
CTL were incubated in FCS-free RPMI 1640 with chemokines, specific peptide-pulsed T2 cell line or matching EBV-transformed cell line, OKT3 (coated onto wells for 2 h at 37°C), or untreated (addition of PBS as control) for 6 h at 37°C. During the second hour of incubation, Brefeldin A (Sigma), at a final concentration of 10 µg/ml, was added. Cells were washed and permeabilized in FACS permeabilization buffer (Becton Dickinson) for 10 min. After washing, staining was performed for 15 min at room temperature using appropriate anti-cytokine and surface antibodies, and cells were analysed as previously.

Cytotoxicity assay
The T2 cell line or EBV-transformed autologous B cell lines were used as target cells in standard 51Cr-release CTL assays. 51Cr labelling was performed for 1 h, following which cells were extensively washed in RPMI medium. Target cells (5x103) were aliquoted into microtitre plates, where they were pulsed with various concentrations of specific peptides. Controls included target cells incubated with medium or 10% Triton only. Antigen-specific clones were added to the targets at 3:1 E:T ratio, in duplicate. Assays plates were incubated 6 h before harvest. Specific 51Cr release was calculated from the following equation: [(experimental release – spontaneous release)/(maximum release – spontaneous release)]x100%.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of RANTES on cell surface marker expression by CTL clones
To analyse the effect of RANTES in comparison with specific antigen, CTL clones recognizing a variety of different antigens, i.e. HIV (A2 gag), influenza (FLU) (A2 matrix), cytomegalovirus (CMV) (B7 pp65) and hepatitis C virus (A2HCV) were generated by tetramer FACS selection from donors' PBMC and were cultured for several weeks to obtain resting populations. The cells were treated with PBS as a negative control, RANTES (5 µM), disaggregated RANTES (5 µM), or mixed with specific peptide-pulsed antigen-presenting cells (APC). The extent of cellular activation was assessed using several extracellular markers. CD69 is considered as an early activation marker. CD25 (IL-2 receptor) and HLA-DR (MHC class II molecule) can also be used as activation markers. CD11b and CD11c are adhesion molecules induced following cell activation. All these markers were up-regulated in the presence of RANTES and the levels of expressions were very similar to those obtained following peptide stimulation, suggesting identical degrees of activation (Fig. 1Go). However, although the antigen stimulation led to a down-regulation of the TCR, only a modest effect was observed using RANTES. As expected, disaggregated RANTES did not affect the expression of any of these markers, confirming that RANTES-induced activation is aggregation dependent. CD11a, another adhesion molecule, was not altered in these studies (data not shown). No significant differences were observed between clones specific for different antigens.



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Fig. 1. RANTES and antigen stimulation lead to similar up-regulations of extracellular activation markers, except for the TCR. A2-gag-specific CD8+ clones were incubated overnight in FCS-free medium in the presence of PBS, RANTES (5 µM), disaggregated RANTES E66S (5 µM) or specific peptide-pulsed APC. Cell surface expression of CD69, CD25, HLA DR, CD11b, CD11c and TCR was evaluated by flow cytometry. Data are expressed as mean fluorescence intensity.

 
RANTES is aggregated on the cell surface
The mechanism by which RANTES is able to activate T cells is dependent on its ability to aggregate and is distinct from its usual activity through GPCR (14). To study the behavior of RANTES on the surface of T cells, Jurkat cells, which are activated by RANTES in a similar way to primary T cells, were treated with RANTES or disaggregated RANTES and were stained for RANTES using specific antibodies (Fig. 2AGo), which could recognize both aggregated and disaggregated RANTES (as demonstrated by Western Blot, data not shown). A striking observation was that high levels of aggregated RANTES could be seen around the cell surface. Disaggregated RANTES could also be detected on the cell surface, but at a much lower level. The binding of RANTES aggregated on the cell surface could also be observed by flow cytometry, on Jurkat cells (data not shown) or on antigen-specific CD8+ clones (Fig. 2BGo), as RANTES staining of cells treated with RANTES or disaggregated RANTES led to different fluorescence signals. The intensity of fluorescence correlated with the concentration of RANTES used to treat the cells (data not shown) and therefore with the amount of RANTES bound to the cells. This observation was subsequently used to study the effect of inhibitors of RANTES-induced activation.



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Fig. 2. RANTES is aggregated on the cell surface. (A) Jurkat cells were incubated in FCS-free medium for 15 min with either PBS, RANTES (5 µM) or disaggregated RANTES E66S (5 µM), washed, fixed and stained with anti-RANTES antibodies coupled with FITC. Photomicrographs were taken using a fluorescence microscope at x40. One of two representative experiments is shown. (B) Antigen-specific CD8+ clones were incubated in FCS-free medium with either PBS, RANTES (5 µM) or disaggregated RANTES E66S (5 µM), washed, fixed and stained with anti-RANTES antibodies. The cells were analysed by flow cytometry.

 
Presence of soluble GAG and FCS prevents the binding of RANTES to the cell surface
Some reports have demonstrated the importance of the GAG in mediating the attachment and oligomerization of RANTES on the cell surface (17,26,27). Therefore experiments were performed to study CTL activation by RANTES in the presence of soluble GAG, using increasing concentrations of heparan sulphate. As shown in Fig. 3AGo, heparan sulphate inhibited RANTES-induced activation in a concentration-dependent manner, proportional to the decrease in the amount of RANTES bound to the cell surface, but did not have any effect on antigen-induced activation (data not shown). We also found that the presence of FCS during the assay could inhibit cell activation by RANTES. Antigen-specific CD8+ T cells were incubated with RANTES and varying concentrations of FCS, and assessed for their degree of activation and for the amount of bound RANTES, as described previously (Fig. 3BGo). There was an inverse relationship between the proportion of FCS used and the degree of RANTES-induced T cell activation, which correlated directly with the amount of RANTES bound to the cells. In contrast, activation of the CTL in response to antigen was not affected by the presence of FCS (data not shown). These observations suggest that some constituents of FCS are able to prevent the attachment of RANTES to the cell surface. It is worth noting that the levels of inhibition of CD69 expression and RANTES binding mediated by both heparan sulphate and FCS appear quite similar, suggesting that GAG may be the principal inhibiting factor in FCS. It is also interesting that high levels of RANTES bound to the cell surface are required to activate the clones.



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Fig. 3. The presence of soluble GAG and FCS prevents the binding of RANTES onto the cell surface. A2-gag-specific clones were incubated in RPMI for 6 h with PBS (control) or RANTES (5 µM) and with different concentrations of heparan sulphate (A) or FCS (B). After washing and fixation, the cells were stained for CD69 or RANTES and analysed by flow cytometry. Data are expressed as mean fluorescence intensity.

 
Cell-surface binding of RANTES is abrogated by the presence of disaggregated RANTES, MIP-1ß, MIP-1{alpha} or erythrocytes
We previously showed that the activation of leukocytes induced by RANTES could be inhibited by high concentrations (in the micromolar range) of MIP-1ß, MIP-1{alpha} or disaggregated RANTES, or the presence of erythrocytes (14). Similar studies were carried out using antigen-specific clones, assessing both expression of CD69, as a marker of activation, and the amount of RANTES bound to the cells. CD69 up-regulation on CTL in response to RANTES was inhibited by these compounds (Fig. 4AGo), although by themselves the inhibitors had no direct effect on CTL nor did they have any effect on the level of antigen-mediated stimulation (data not shown). The decrease in CD69 up-regulation in the presence of these inhibitors correlated with a reduction in RANTES staining on the cell surface. This suggests that the inhibitors act by preventing RANTES from binding to the cell surface, thereby preventing activation of the cells by this mechanism. CTL were therefore incubated with either MIP-1ß or MIP-1{alpha}, at concentrations similar than those used to inhibit RANTES-induced activation, and then stained for surface binding of MIP-1ß or MIP-1{alpha} respectively using flow cytometry. No significant staining was observed for either molecule, although the specific antibodies used were able to recognize the proteins by Western blot (data not shown). In order to test whether erythrocytes could bind RANTES and therefore compete for RANTES binding with the CTL, surface staining for RANTES was carried out on erythrocytes incubated with RANTES. Both native RANTES and, at a much lower level, disaggregated RANTES could bind to erythrocytes (Fig. 4BGo).



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Fig. 4. The presence of disaggregated RANTES, MIP-1ß, MIP-1{alpha} or erythrocytes prevents the binding of RANTES onto the cell surface. (A) FLU-specific clones were incubated in FCS-free medium for 6 h in the presence of PBS (negative control), RANTES only (5 µM, positive control) or RANTES (5 µM) and an inhibitor: RANTES E66S, MIP-1ß, MIP-1{alpha} (at 10 µM) or erythrocytes. After washing steps and lysis of erythrocytes if necessary, CD69 and RANTES stainings were carried out on the clones, and the cells were analysed by flow cytometry. (B) Purified erythrocytes were incubated in FCS-free medium for 15 min in the presence of RANTES or disaggregated RANTES E66S. After washing, the red blood cells were stained for RANTES on their surface and analysed by flow cytometry. Data are expressed as mean fluorescence intensity.

 
Differences in the induction of IFN-{gamma} and MIP-1ß expression induced by RANTES and specific antigens
Another way to study T cell activation is to analyse their secretion of cytokines after stimulation. It has already been shown that RANTES is able to trigger IL-2 and IL-5 release from CD4+ T cells (12). In these studies, we examined the secretion of two soluble factors closely associated with the function of antigen-specific CTL, IFN-{gamma}, an antiviral cytokine produced by the majority of CTL, and MIP-1ß, a pro-inflammatory chemokine which is able to suppress HIV infection in vitro and is a potent inhibitor of RANTES-induced activation. The secretion of IFN-{gamma} and of MIP-1ß was assessed by intracellular staining of antigen-specific clones in the presence of RANTES, disaggregated RANTES, anti-CD3 antibodies (OKT3) or their cognate antigen. RANTES, but not disaggregated RANTES, induced the release of both IFN-{gamma} and MIP-1ß (IFN-{gamma} production was also observed using ELISPOT assays, data not shown) (Fig. 5Go). However, although CTL stimulated by either RANTES, OKT3 or specific antigen displayed similar levels of the activation marker CD69, production of MIP-1ß and, in particular, IFN-{gamma} was lower in response to RANTES than specific antigen. The expression of cytokines induced by RANTES more closely resembled the pattern of stimulation by OKT3 and also PHA (data not shown). It therefore seems likely that RANTES acts more as a mitogen than an antigen-independent activator. Since optimal cytokine release occurs when antigen is presented by an APC, it may therefore require appropriate co-stimulation by the APC in addition to the correct antigen.



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Fig. 5. RANTES and antigen stimulation lead to different patterns of IFN-{gamma} and MIP-1ß secretion. B7 CMV-specific CD8+ clones were incubated for 6 h in FCS-free medium in the presence of PBS, RANTES (5 µM), disaggregated RANTES E66S (5 µM), anti-CD3 antibodies (OKT3) or specific peptide-pulsed APC. Secretion of IFN-{gamma} and MIP-1ß was measured by flow cytometry after intracellular staining. The mean fluorescence mean is given for each condition.

 
Effect of RANTES on the cytotoxic activity of CTL
It was recently reported that, at low concentrations, RANTES could mediate enhancement of the cytotoxicity of some HIV-specific CTL cultured under `autologous conditions' (8,28). Since in our experiments RANTES, at high concentrations, was able to activate cells and induce cytokine release, we went on to study the effect of RANTES on the cytotoxic activity of antigen-specific CD8+ clones. Peptide titration CTL assays were carried out in the presence of low (50 nM) or high (5 µM) concentrations of RANTES or disaggregated RANTES (Fig. 6Go). No enhancement by RANTES of either specific or non-specific CTL killing was observed at any of the concentrations tested. At high concentrations, a modest decrease of specific killing was observed: this may be a consequence of RANTES-induced activation of the clones, leading to increased susceptibility to apoptosis in FCS-free medium, as suggested by an increase in annexin V expression (an early marker of apoptosis) on the surface of peripheral blood T cells and Jurkat cells treated with RANTES (data not shown).



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Fig. 6. Effect of RANTES on the cytotoxic activity of antigen-specific CD8+ clones. A2 tyrosinase-specific clones were incubated in FCS-free medium in the presence of 51Cr-loaded T2 cells (at an E:T ratio of 3:1), at different concentrations of specific peptide, or PBS ({blacksquare}), RANTES (50 nM, {blacktriangleup}; 5 µM, •), disaggregated RANTES E66S (50 nM, {triangleup}; 5 µM, {circ}) or anti-RANTES antibodies (5 µg/ml, x). 51Cr-release was measured after 6 h of incubation. Data are expressed in percentage of lysis. Similar data were obtained using an A2 HCV-specific clone and an A2-gag-specific clone

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In 1995 it was reported that at high concentrations RANTES could induce T cell signalling via a PTK-mediated pathway which led to T cell activation (12), suggesting that RANTES was able to act as an antigen-independent activator of T cells. In a recent report, we demonstrated that such activation was dependent on the self-aggregation of RANTES and was also seen in other leukocyte populations, such as monocytes and neutrophils (14). We further showed that this activity could be inhibited by disaggregated RANTES or by more physiological inhibitors such as other members of the CC chemokine family, MIP-1{alpha} and MIP-1ß, and erythrocytes. The potential importance of RANTES-induced activation is illustrated by the observation that HIV infection of macrophages is actually enhanced by high concentrations of RANTES: this activity is dependent on RANTES aggregation and is not apparently mediated through GPCR binding and signalling (17). GAG, present on the surface of cells and viruses, are known to bind RANTES and to promote its oligomerization (26): these proteins were found to be involved in the enhancement of HIV infection mediated by RANTES (17). Studies of the biology of RANTES at high concentration raise questions about the relevance of the phenomenon at more physiological concentrations of RANTES in inflammatory disorders and viral infection. However, the local levels of RANTES achieved at sites of inflammation may be substantially higher than those measured in blood. Peak levels of RANTES preparations administered therapeutically could also be higher than normal blood levels, so these observations are potentially important.

In these studies we have compared the effect of RANTES with that of specific antigens on the activation of antigen-specific CD8+ clones. As previously described for leukocytes and cell lines, the activation of CTL by RANTES appears to be dependent on its aggregation. Although at first sight the effect of RANTES on activation markers appears very similar to that of specific peptides, RANTES fails to trigger the TCR down-regulation induced by cognate antigen. Since CD3 expression is necessary for RANTES-induced activation of Jurkat cells (29) it is possible that RANTES, through its aggregation on the cell-surface, is able to cross-link CD3 molecules, leading to signalling through the PTK pathway and hence to T cell activation. The down-regulation of the TCR by antigens but not by RANTES would be in favour of a non-specific cross-linking of the TCR complex or CD3 molecules by RANTES. It is worth noting that although CD3 seems to be necessary for the activation of T cells, other surface molecules can be cross-linked and lead to activation in other cell types.

Surface staining of cells treated with RANTES shows that it binds to and aggregates on the cell surface. Cellular activation appears to be directly proportional to the amount of RANTES bound to the cell surface and inhibitors of RANTES-induced activation, such as FCS, appear to act by preventing the binding of RANTES to the cell surface. The fact that heparan sulphate and FCS inhibit completely RANTES-induced CD69 up-regulation, while causing only a small decrease of RANTES binding, suggests that there is a tight threshold of surface bound RANTES, below which no activation can be achieved, despite the presence of significant amounts of RANTES on the cell surface. GAG mediate the oligomerization of RANTES on the cell surface (26) and soluble GAG can inhibit the RANTES-mediated enhancement of HIV infection (17). GAG such as heparan sulphate may therefore be the key elements which mediate interactions between aggregated RANTES and its target cells. Treatment of the cells with heparinases led to lower binding of RANTES to the cell surface (data not shown). It is likely that soluble GAG are the active ingredients in FCS which block RANTES-induced activation. These data suggest that RANTES binds to GAG on the cell surface, which increase its local concentration and promote its self-aggregation, leading to the cross-linking of signalling molecules such as CD3 molecules on T cells and ultimately to cell activation. RANTES may be able to engage proteins located in lipid rafts, thereby inducing their aggregation and triggering cell signalling (30). High levels of aggregated RANTES seem to be required on the cell surface to trigger activation, explaining why disaggregated RANTES, although binding to the cell surface, cannot trigger any signal.

Disaggregated variants of RANTES, obtained by single amino acid substitutions at one of two positions (15), are much less efficient at triggering signalling than the native form (14). It is clearly important for the interpretation of our results that the ability of these compounds to bind to GAG is not affected by the amino acid substitutions which disrupt self-aggregation. Burns et al. demonstrated that lysine and arginine residues within the C-terminal region of RANTES mediate binding to GAG (31). Thus it is possible that the substitution of residue Glu66 (in the C-terminal region) might have an effect on GAG binding, but the substitution at Glu26 would not be predicted to affect binding. Moreover, these two variants have previously been used to demonstrate that only aggregated forms of RANTES are able to induce signalling (14). The disaggregated form of RANTES E66S has been shown in this study to be able to bind to the cell surface by both flow cytometry and microscopic analysis. It is very unlikely that this is due to chemokine receptor binding alone, as no staining was observed using MIP-1ß or MIP-1{alpha}. Assuming that disaggregated RANTES can indeed bind to GAG, we would predict that disaggregated RANTES would inhibit RANTES-induced activation by competing for binding sites on GAG, thereby preventing the binding of native RANTES and its subsequent aggregation on the cell surface, since wild-type RANTES is unable to aggregate with either of the non-aggregating variants.

MIP-1ß and MIP-1{alpha} share a substantial degree of homology with RANTES, and their self-aggregation is governed by the same amino acids (15). Surprisingly these chemokines appeared to inhibit RANTES-induced activation by preventing its cell surface binding, even though they do not bind to the cell surface via GAG as RANTES does. Disaggregated MIP-1ß and MIP-1{alpha} were unable to inhibit the effect of RANTES (14). Taken together, these data suggest that MIP-1ß and MIP-1{alpha} could inhibit RANTES-induced activation by trapping RANTES in aggregates formed of hetero-multimers. The implication is that RANTES, MIP-1{alpha} and MIP-1ß can bind to each other. (We were unable to demonstrate this directly, by using immunoprecipitation assays of RANTES mixed with the two other chemokines, since each of the allegedly specific antibodies used to immunoprecipitate the chemokines cross-reacted with the other chemokines.) It is likely that erythrocytes also inhibit RANTES-induced activation by trapping RANTES on their cell surface GAG, as Duffy receptors have been shown to have no involvement in the inhibition mechanism (14).

None of the inhibitors tested had any effect on cellular activation induced by specific antigen, which is consistent with the differences we observed in the manner of activation by RANTES and specific antigen. Although similar levels of activation markers can be obtained with both RANTES and cognate antigen, substantial differences are seen in their profile of IFN-{gamma} and MIP-1ß release. In this regard, the effect of RANTES is similar to that of anti-CD3 antibodies or PHA, which suggests that RANTES may more closely resemble a mitogen than an antigen-independent activator, as it was originally defined (12).

We have also examined the effect of RANTES on the cytotoxic activity of CTL in this report. Hadida et al. described an enhancement of HIV-specific T cell cytotoxicity induced by low concentration of RANTES (8), which more recently was shown to occur only in the small subset of CTL which exert their killing by means of Fas ligand (28). We did not observe any significant effect of RANTES on the cytotoxicity of any of our CTL clones; at high concentration their cytotoxicity was actually decreased, almost certainly a consequence of the activation of the cells by RANTES. Our data do not contradict the observations of Hadida et al., as the CTL clones used in our assays were grown under different conditions and predominantly mediated their killing by means of perforin release (data not shown). However, these data emphasize the differences between the activation mediated by RANTES and by specific antigens.

In conclusion, we have shown that in addition to its well-known chemoattractant properties, RANTES is able to mediate leukocyte activation at high concentrations. The mechanism of this activation is thought to be through RANTES binding to GAG and subsequent self-aggregation on the cell surface, which in turn leads to non-specific cross-linking of signalling molecules and ultimately to cell activation. This activation occurs very rapidly: intracellular calcium mobilization is triggered in a few seconds (12,14) and changes in cellular shape can be observed within 10 min of incubation, indicating a very fast rate of cytoskeletal mobilization (unpublished data). Just a few minutes later, RANTES-treated cells form clusters, suggesting rapid changes in the affinity or the expression of cell surface adhesion molecules (32) (and unpublished data). This effect of RANTES on cells is comparable to that seen with a mitogen.

The high and apparently non-physiological concentration of RANTES required to activate the cells may raise questions concerning the validity of these observations and their physiological relevance. However, we still do not know what conditions T cells may experience locally in vivo—thus high in vivo concentrations of RANTES certainly cannot be ruled out, particularly at sites of inflammation. The best way to address the in vivo relevance of RANTES-induced activation would be the study of inflammatory conditions in transgenic mice constructed to express a non-aggregating form of RANTES.


    Acknowledgments
 
We wish to thank David Jackson, Kevin Bacon and Alexandra Trkola for helpful discussions. This work was supported by the Medical Research Council (UK).


    Abbreviations
 
APC antigen-presenting cell
CMV cytomegalovirus
FLU influenza virus
GAG glycosaminoglycan
GPCR G-protein coupled receptor
HCV hepatitis C virus
MIP macrophage inflammatory protein
PBL peripheral blood lymphocyte
PBMC peripheral blood mononuclear cell
PE phycoerythrin
PHA phytohemagglutinin
PTK protein tyrosine kinase
RANTES regulated upon activation, normal T cell expressed and secreted

    Notes
 
Transmitting editor: P. Beverley

Received 5 January 2000, accepted 2 May 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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