TCR-mediated activation of allergen-specific CD45RO+ memory T lymphocytes results in down-regulation of cell-surface CXCR4 expression and a strongly reduced capacity to migrate in response to stromal cell-derived factor-1

Claire Abbal, Patrick Jourdan, Toshiyuki Hori1, Jean Bousquet, Hans Yssel and Jérôme Pène

INSERM U454, Hôpital Arnaud de Villeneuve, 375 Avenue Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France
1 Institute for Virus Research, Kyoto University, Kyoto 606, Japan

Correspondence to: H. Yssel


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The selective migration of functional Th lymphocyte subsets with different cytokine production profiles into inflamed tissue is likely to depend on the state of activation of the cells, as well as on the differential expression of various adhesion molecules and chemokine receptors. In this study, we have analyzed the effect of allergen-specific activation on the expression of the chemokine receptor CXCR4 on T lymphocytes. We show that stimulation of peripheral blood mononuclear cells from atopic patients with the allergen Der p results in down-regulation of CXCR4 surface expression on Der p-activated CD25+CD45RO+ antigen-specific memory cells which was caused by a decrease in CXCR4 gene transcription and did not seem to be mediated by endogenous cytokines, such as IFN-{gamma}. In contrast, however, CXCR4 surface expression was enhanced on naive CD25CD45RO and resting CD25CD45RO+ memory T cells, as a result of the presence of endogenous IL-4, most likely produced by Der p-activated memory T cells. Antigen-specific CD25+CD45RO+ T lymphocytes, purified 7 days after stimulation with Der p, had a strongly reduced capacity to migrate in response to stimulation with stromal cell-derived factor (SDF)-1, the ligand for CXCR4. Together, these results suggest that differential expression of CXCR4 on activated and resting T cells is due to the counteracting effects of TCR-mediated down-regulation and IL-4-mediated up-regulation of this chemokine receptor respectively, and furthermore indicate that antigen-activated memory T cells are unlikely to migrate into inflamed tissue in response to SDF-1.

Keywords: allergy, CD45RO, chemokine receptors, CXCR4, IL-4, stromal cell-derived factor, T cell activation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Th subsets that differ in cytokine production and effector function play an important role in normal and pathological immune responses. Th2 cells, characterized by the production of IL-4, IL-5 and IL-13, but little or no IFN-{gamma}, are involved in allergic responses, and furthermore inhibit acute and chronic inflammatory reactions, which are often a consequence of protective immunity mediated by IFN-{gamma}-producing Th1 cells. Following antigenic challenge, Th subsets are recruited into local tissue, during a migration process mediated by selectins and integrins (reviewed in 1), as well as by locally produced chemokines and chemokine receptors (reviewed in 2). The selective recruitment of Th1 and Th2 cells into local tissues suggests that the differential expression of adhesion molecules and chemokine receptors endows these subsets of Th cells with different migratory capabilities. Indeed, it has recently been demonstrated that murine, as well as human, Th1 and Th2 cells respond to different CXCR and CC chemokines, due to the selective expression of CCR3, CCR4 and CXCR5 on Th2 cells (35), and CCR5 and CXCR3 on Th1 cells (5,6). In addition, CCR8 has been reported to be expressed on Th2 cells, whereas the CCR8 ligand, I-309, is a potent chemoattractant for Th2-polarized cells.

In contrast, the expression of CXCR4, a chemokine receptor member with a unique ligand, stromal-derived factor (SDF)-1 (reviewed in 2), does not seem to be specific for a particular Th subset. Both mouse Th1 and Th2 cells are able to migrate, albeit with different efficacy, in response to stimulation with SDF-1 (4). Furthermore, CXCR4 transcripts can be detected in polarized human Th1, as well as Th2 cells (5). However, since IL-4 is able to specifically induce CXCR4 expression on T cells, CXCR4 expression on T cells seems to be promoted under Th2 polarizing conditions (7).

In view of the prominent role of IL-4 in the pathology of allergic diseases, it is not clear whether the inducing effect of this cytokine of CXCR4 expression endows allergen-specific T cell with the property to home into sites of allergic inflammation. It has been reported that interaction of CXCR4 with SDF-1 results in down-regulation of the receptor–ligand complex (810). Similarly, cell-surface expression of CXCR4 is quickly down-regulated and the receptor internalized on T cells following stimulation with phorbol ester (9,10). However, little information is available about the regulation of expression of CXCR4 following interaction of the TCR with specific antigen or allergen which is the ultimate signal leading to recruitment of T cells into target organs. Therefore, to address the modulation of CXCR4 expression under more physiological relevant experimental conditions, we have analyzed the cell-surface expression of this receptor on peripheral blood T lymphocytes from atopic patients, following a single stimulation with specific allergen in vitro.

Here, we report that the cell-surface expression of CXCR4 is down-regulated on allergen-specific, activated, CD45RO+ memory T lymphocytes, as a result of decreased gene transcription and these cells do no longer migrate in vitro in response to stimulation with SDF-1. In contrast, the presence of endogenous IL-4, most likely produced by the latter cells, is sufficient to induce an up-regulation of CXCR4 on naive, CD45RO, and resting CD45RO+ memory T lymphocytes. Together, these results indicate that CXCR4 does not seem to play a role in the migration of activated allergen-specific T lymphocytes into sites of allergic inflammation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients
Eight patients, ranging in age from 20 to 45 years and allergic to house dust mites, were selected for this study. All patients had perennial symptoms of asthma and rhinitis, a positive skin prick test to a standardized Dermatophagoides pteronyssinus (Der p) allergen extract, and high levels of Der p-specific IgE (Phadebas Cap system; Pharmacia, Uppsala, Sweden). Six non-allergic subjects in the same age group were used as negative control. The expression of CXCR4 during the pollen season was studied on a group of 11 patients, allergic to aero allergens, and 11 healthy controls. None of the patients had received any form of specific immunotherapy or therapy with corticosteroids, at least 3 months prior to the study.

Cells and culture conditions
Peripheral blood leukocytes were isolated by centrifugation on Ficoll-Hypaque and 106 cells were cultured at 37°C and 5% CO2, in the presence or absence of standardized Der p extract (final concentration 160 ng Der p1 and 250 ng Der p2; a generous gift from Dr O. Cromwell, Allergopharma, Reinbeck, Germany) in 24-well tissue culture plates in a final volume of 1 ml of culture medium. To neutralize the effect of endogenous IL-4 in cultures of PBMC, the neutralizing anti-IL-4 mAb MP4-25D2 (a kind gift of Dr J. Abrams, DNAX Research Institute, Palo Alto, CA) was added in parallel experiments, at a final concentration of 10 µg/ml. The neutralizing anti-IL-5 mAb 39D10 (Dr J. Abrams) was used as an isotype-matched control. All cell cultures and experiments were carried out in Yssel's medium (11) (Irvine Scientific, Santa Ana, CA).

Proliferation assays
Five days following stimulation of peripheral blood mononuclear cells (PBMC) with Der p, 100 µl of cell suspension was transferred into 96-well round-bottom culture plates (Nunc, Roskilde, Denmark) and 1 µCi (37 kBq) of [3H]thymidine (Amersham France, Les Ulis, France) was added in a final volume of 10 µl. After 8 h of culture, the cells were harvested onto glass fiber sheets using an automated cell harvester (Tomtec, Orange, CT) and radioactivity was measured using a scintillation counter (Wallac, Turku, Finland). Results are expressed as mean ± SD of triplicate cultures.

Analysis of cytokine production by ELISA
Culture supernatants were harvested 7 days after culture of PBMC with Der p and aliquots were stored at –20°C prior to cytokine measurement. Production of IL-4, IL-5 and IFN-{gamma} was measured by specific ELISA, as described previously (12). For ELISA standards, purified recombinant (r) human IL-4, IL-5 and IFN-{gamma} were purchased from R & D Systems (Abingdon, UK). Production of IL-10 and IL-13 was measured using a commercial ELISA (Diaclone, Besancion, France), according to the manufacturer's instructions. The limits of sensitivity of the assays were: 3 pg/ml for IL-4, IL-10 and IL-13, 4 pg/ml for IL-5 and 5 pg/ml for IFN-{gamma}.

Immunofluorescence, flow cytometry and FACS sorting
All immunofluoresence and flow cytometry procedures were carried out using the method of Lanier and Recktenwald (13), using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest software (Becton Dickinson). PBMC were analyzed by flow cytometry immediately following isolation and following culture of the cells in the presence or absence of Der p, as indicated in the legends to the figures. In addition, cell-surface expression of CXCR4 was analyzed on subsets of resting and activated T cells, as defined by differences in forward scatter and side scatter properties, as well as by their expression of CD4, CD8, CD25 and CD45RO. The following mAb were used in flow cytometry: the non-conjugated CXCR4-specific mAb IVR-7 (14) and anti-IL-4 receptor (IL-4R) {alpha} chain mAb MAB230 (R & D Systems); FITC- or phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD25 and isotype-matched control mAb, purchased from Dako (Gladstrup, Denmark). For indirect staining, non-conjugated mAb and isotype-matched controls were used in combination with a FITC-conjugated goat anti-mouse antibody (Caltag, Burlingame, CA). To prevent non-specific binding, cells were incubated with 10 µg/ml of mouse IgG (PharMingen) for 15 min at 4°C, prior to staining with mAb. For FACS sorting, Der p-stimulated PBMC from an atopic donor were collected after 7 days of culture, stained with anti-CD25 mAb, and CD25+ and CD25 sub-populations respectively were purified using a FACS Vantage flow cytometer (Becton Dickinson). Purity of both sub-populations upon re-analysis after sorting was >99%.

RNase protection assay
Total RNA was extracted from T cells using RNAzol B (Tel-Test, Friendswood, TX) and the multiprobe template set hCR-6, containing, among others, the DNA template for CXCR4, as well as GAPDH as a housekeeping gene (PharMingen) was used to detect CXCR4 transcripts by the RNase protection assay according to the manufacturer's standard protocol. Specific transcripts were measured on 106/ml T cell clones that had been stimulated with the immobilized anti-CD3 mAb SPV-T3b (Immunotech, Marseilles, France) and the anti-CD28 mAb B-T3 (kind gifts of Dr John Wijdenes, Diaclone) in the presence of 2x106 autologous Epstein–Barr virus (EBV)-transformed B cells. The T cell clones and EBV-transformed B cells were generated and cultured as described previously (15).

Chemotaxis assay
Migration of T cells from three atopic patients in response to stimulation with SDF-1 was analyzed in ChemoTx-96-well disposable chambers with a filter sample site of 3.2 mm diameter and 5 µm pore size filters (Neuroprobe, Gaithersburg, MD), using the method described by Bacon and Schall (16). Briefly, 29 µl of Yssel's medium/1% human serum containing 0, 25, 50 or 250 ng/ml of human rSDF-1ß (R & D Systems) was added to the lower wells. Then 20 µl of FACS-purified CD25+ and CD25 T cells from Der p-stimulated PBMC resuspended at 2x106/ml were transferred directly in triplicate on the filter sample sites. After 1 h of incubation in 5% CO2 at 37°C, cells that had migrated through the filter were collected in the lower chamber, re-suspended in culture medium and counted using a hemocytometer. Results are expressed as the ratio of (number of cells migrated in SDF-1-containing medium – number of cells migrated in medium)/total number of cells used in the assayx100%.

Statistical analysis
Statistical analysis was performed by means of non-parametric tests. The Mann–Whitney U-test was used for inter-group analysis, and the Wilcoxon signed-rank test and Spearman correlation test were used for intra-group analysis of the results.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Der p-induced stimulation modulates CXCR4 expression on T cells from atopic donors
Freshly isolated PBMC from atopic and non-atopic donors generally express low levels of CXCR4 on their cell surface which are enhanced following in vitro culture of T cells in medium alone (Fig. 1Go). To determine the effect of allergen-specific activation on T cell-surface expression of CXCR4, PBMC from atopic and non-atopic donors were cultured in the presence or absence of Der p and CXCR4 expression on CD3+, CD4+ or CD8+ T cells was measured by flow cytometry. At day 7, CXCR4 expression was up-regulated on ~70% of the T cells from atopic donors following allergen-specific stimulation, as compared to its expression on these cells cultured in medium alone, whereas 30% of the cells expressed strongly reduced CXCR4 levels on their cell surface (Fig. 1EGo). This bimodal effect following allergen-specific stimulation was observed on T cells from all eight atopic donors, and was maximal between day 5 and 7 of culture (Fig. 1A–EGo). In contrast, stimulation of PBMC from non-atopic donors with Der p did not significantly affect surface expression of CXCR4 on T cells (Fig. 1FGo and Table 1Go).



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Fig. 1. Effect of Der p-specific stimulation on CXCR4 surface expression on T cells from atopic and non-atopic donors. PBMC from an atopic (A–E) and a non-atopic donor (F) were cultured in the absence (plain histograms) or presence (bold histograms) of 10 µg/ml of Der p, and the cell-surface expression of CXCR4 on T cells was analyzed by flow cytometry at days 1, 4, 5, 6 and 7 of culture. The x- and y-axes represent fluorescence (four-decade log scale) and relative cell number respectively. Histograms from cells stained with control isotype-matched mAb are shown in dotted lines.

 

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Table 1. Effect of Der p on CXCR4 expression by T cells of atopic and non-atopic donorsa
 
Increased levels of CXCR4 cell-surface expression were detected on all CD8+ cells, but only on a fraction of CD4+ cells (Table 1Go). Although the frequency of CXCR4+ T cells present in either subset was about equal, CD4+ T cells expressed significantly higher levels of CXCR4, as measured by the intensity of antibody staining. In contrast, Der p-induced down-regulation of CXCR4 expression was exclusively observed on CD4+ T cells (Table 1Go).

PBMC from atopic donors proliferated strongly following Der p-specific stimulation (Table 2Go). Although Der p also induced proliferative responses in PBMC from non-atopic donors, these responses were significantly lower (P < 0.007, Mann–Whitney U-test). Moreover, a positive correlation was observed between the up-regulation of CXCR4 expression and the magnitude of Der p-induced activation (r = 0.8 and P < 0.0012, Spearman correlation test), determined on PBMC from atopic and non-atopic subjects.


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Table 2. Induction of T cell proliferation by Der pa
 
CXCR4 surface expression is down-regulated on Der p-activated CD4+CD25+ T cells, but is up-regulated on CD4+ naive and resting memory T cells
Flow cytometric analysis of PBMC 7 days after stimulation with Der p showed two populations of lymphocytes based on forward scatter and side scatter characteristics (Fig. 2AGo). A minor population of cells (36 ± 5% of total; n = 8), which was characterized by increased size and granularity (designated G1), was detected among Der p-stimulated PBMC from atopic donors (Fig. 2AGo), but not from control donors (data not shown). In addition, the emergence of the G1 population was observed for CD4+ T cells, but not for CD8+ T cells (data not shown). A larger population of cells (66 ± 20%) that did not respond with increased cell size following stimulation in the presence of Der p (designated G2) was present in the cultures (Fig. 2AGo). More than 95% of PBMC from atopic patients (Fig. 2BGo) and control donors (data not shown), cultured for 7 days in medium only, belonged to the G2 population of cells.



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Fig. 2. Effect of Der p-specific stimulation on the emergence of activated and memory T cells. PBMC from an atopic donor (A–E) were stimulated with Der p (A) or cultured in medium alone (B) and density plots, representing internal complexity (SSC) as a function of size (FSC), were obtained after 7 days of culture. Indicated percentages represent the relative quantity of activated (G1) and resting (G2) cells respectively. Cell-surface expression of CD25 (x-axis) and CD45RO (y-axis) on T cells, in the total population of stimulated PBMC (C), gated on the G1 region (D) and the G2 region (E), is represented on a four-decade log scale as dot-plots of correlated FITC and PE fluorescence. Quadrant markers were positioned to include >98% of control Ig-stained cells in the lower left quadrant (not shown). Representative data of eight atopic donors.

 
Analysis of the cells within the G1 and G2 populations for the expression of activation and memory cell surface antigens showed that ~35% of the PBMC, cultured for 7 days in medium alone, expressed CD45RO (data not shown) and that their frequency increased to 50% when cultured in the presence of Der p (Fig. 2CGo). More specifically, up to 90% of the Der p-activated T cells expressed CD45RO, as well as CD25+, indicating that the G1 population consists of activated memory T cells (Fig. 2DGo). In contrast, among the CD4+ T cells in the G2 population, only 35% of the cells had a CD45RO+ memory phenotype and >60% of these cells were CD25 (Fig. 2EGo). In addition, none of the CD45RO cells expressed significant levels of CD25 on their surface (Fig. 2C and DGo). These results indicate that the G2 population consists mainly of resting naive and memory T cells. Although a small number of activated memory T cells could be detected within the G2 population, their presence is likely to be due to the arbitrary definition of this population, based on forward and side scatter properties.

Among the population of Der p-stimulated PBMC, ~50% of the CD25, resting T cells expressed up-regulated levels of CXCR4 (Fig. 3BGo), as compared to those on T cells cultured in medium alone or to Der p-stimulated T cells from non-atopic donors (data not shown). However, >80% of the CD25+ T cells that had been specifically activated following culture of PBMC with Der p no longer expressed detectable cell-surface expression levels of CXCR4 (Fig. 3CGo), suggesting that antigen-specific activation resulted in the down-regulation of CXCR4 surface expression. Indeed, among Der p-stimulated PBMC from atopic donors a decrease in cell-surface CXCR4 expression was observed only on CD45RO+CD25+ activated memory T cells (Fig. 4A and BGo). In contrast, CXCR4 expression was slightly, but significantly, enhanced on CD45RO+CD25 resting memory T cells (Fig. 4A and CGo), as well as naive CD45RO T cells (data not shown), as compared to its expression on T cells that had been cultured in medium alone.



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Fig. 3. Down-regulation of cell-surface CXCR4 expression and concomitant up-regulation of CD25 expression on Der p-activated T cells. PBMC of an atopic donor were stimulated with 10 µg/ml Der p and surface expression of CXCR4 (x-axis) and CD25 (y-axis) was analyzed by flow cytometry after 7 days of culture of T cells in the total population of stimulated PBMC (A), gated on the G2 region (B) or the G1 region (C), as defined in Fig. 2Go. Representative data of eight atopic donors.

 


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Fig. 4. CXCR4 expression is down-regulated on activated memory T cells. PBMC of an atopic donor were stimulated for 7 days in the presence (bold histogram) or absence (plain histogram) of 10 µg/ml Der p and the expression of CXCR4 was analyzed on CD45RO+ T cells in the total population of stimulated PBMC (A), gated on the G1 region (B) or the G2 region (C), as defined in Fig. 2Go. Representative data of eight atopic donors.

 
TCR–CD3 complex-mediated activation results in an inhibition of CXCR4 transcription
To investigate whether TCR–CD3 complex-mediated down-regulation of CXCR4 expression was due to a decrease in CXCR4 transcripts in the activated cells, expression of CXCR4 mRNA was analyzed using an RNAse protection assay. However, in view of the bimodal distribution of CXCR4 expression on PBMC T cells following stimulation with Der p, analysis of CXCR4 transcripts on the entire population of T cells would not be informative. In addition, preliminary experiments indicated that it was not possible to directly analyze CXCR4 gene transcription on all Der p-activated T cells at a single time point in the cultures, due to the complex stimulation kinetics. Therefore, Der p-specific T cell clones were derived from one of the atopic donors which were used to analyze CXCR4 mRNA following stimulation with either Der p and autologous EBV-transformed B cells as antigen-presenting cells or with anti-CD3 and anti-CD28 mAb which mimics antigen-specific stimulation, as a control. As is shown in Fig. 5Go, CXCR4 mRNA levels were strongly decreased already following 6 h of stimulation of these cells with either anti-CD3 and anti-CD28 mAb or with Der p and antigen-presenting cells, indicating that TCR–CD3 complex-mediated down-regulation of CXCR4 expression is mediated at the transcriptional level.



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Fig. 5. TCR–CD3 complex-mediated activation results in an inhibition of CXCR4 transcription. Der p-specific T cell clones Mo-98 ({blacksquare}) and Mo-142 ({square}) were stimulated with immobilized anti-CD3 and soluble anti-CD28 mAb for different periods of time and CXCR4 mRNA expression was analyzed by RNAse protection assay. In addition, T cell clone Mo-142 ({blacksquare}) was stimulated with 10 µg/nl Der p and autologous EBV-transformed B cells. Values are expressed as the ratio between specific CXCR4 mRNA and GAPDH mRNA expression by the T cell clones, as well as by the autologous EBV-transformed B cells ({blacksquare}).

 
Induction of CXCR4 surface expression on resting T cells present in Der p-stimulated PBMC is mediated by endogenous IL-4
PBMC from atopic donors stimulated with Der p had a Th2-like cytokine production profile, producing IL-4, IL-5 and IL-13, although levels of IL-4 production were just above the limit of sensitivity of the assay (Fig. 6AGo and Table 3Go). In addition, specific IL-4 transcripts could be detected in PBMC of four out of four atopic donors tested by RNase protection assay (results not shown). In contrast, neither IL-4 protein nor IL-4 transcripts were detected in Der p-stimulated PBMC from non-atopic donors. As shown in Fig. 6Go(B), the presence of a neutralizing anti-IL-4 mAb during stimulation of PBMC from atopic donors with Der p prevented the up-regulation of cell-surface expression of CXCR4. On activated T cells, however, Der p-mediated down-regulation of CXCR4 was not affected by the addition of the anti-IL-4 mAb to the cultures (Fig. 6CGo). Together, these results indicate that the induction of CXCR4 on resting T cells is indirect and is, at least in part, mediated by the endogenous production of IL-4 by Der p-activated CD4+ memory T cells.



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Fig. 6. The induction of CXCR4 on resting cells is mediated by endogenous IL-4. PBMC from healthy donors (C; n = 6) and atopic donors (A; n = 8) were incubated in the absence (M) or presence (Dp) of 10 µg/ml Der p and the production of IL-4 was measured in the culture supernatants after 7 days of culture by cytokine-specific ELISA (A). PBMC of an atopic donor were incubated in medium (grey histogram) or with 10 µg/ml Der p in the absence (bold histogram) or presence (plain histogram) of 10 µg/ml of the neutralizing anti-IL-4 mAb 25D2 and the expression of CXCR4 was analyzed on T cells gated on the G2 region (B) or the G1 region (C), as defined in Fig. 2Go. Representative data of three atopic donors.

 

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Table 3. Induction of cytokine production by Der pa
 
Endogenous IL-4 induces IL-4R surface expression on resting T cells present in Der p-stimulated PBMC
To determine whether the inducing effect of endogenously produced IL-4 on CXCR4 expression on resting cells correlated with the presence of a functional IL-4R, PBMC from atopic patients were stimulated with Der p and kinetics of induction of IL-4R expression on resting, CD25, and activated, CD25+, T cells was analyzed by flow cytometry. Less than 5% of freshly isolated peripheral blood T cells expressed IL-4R on their surface and only at low levels. This expression did not increase following culture of the cells in medium alone. A substantial number of T cells expressed IL-4R on their surface after 48 h of culture with Der p, with maximal expression levels at day 4 of culture which subsequently declined (Fig. 7AGo). Furthermore, no cell-surface expression of IL-4R could be detected on activated CD25+ T cells in cultures of PBMC stimulated with Der p (Fig. 7BGo). In contrast, ~25% of the CD25 T cells expressed detectable levels of IL-4R which were, however, absent when the cells had been stimulated in the presence of a neutralizing anti-IL-4 mAb, whereas the addition of an isotype-matched anti-IL-5 mAb did not have any effect (data not shown). Together, these results suggest that endogenous IL-4, produced in cultures of Der p-stimulated PBMC, up-regulates the expression of its own receptor on resting T cells, leading to a subsequent increase in surface CXCR4 expression on these cells.



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Fig. 7. Der p-induced up-regulation of IL-4R expression on non-activated T cells is mediated by endogenous IL-4. PBMC of an atopic donor were cultured in the absence (plain histogram) or presence (bold histogram) of 10 µg/ml Der p and surface expression of IL-4R was analyzed on the total population of PBMC T cells at different days after stimulation (A). In addition, PBMC were stimulated in the presence or absence of 10 µg/ml of the neutralizing anti-IL-4 mAb 25D2, and surface expression of both IL-4R (x-axis) and CD25 (y-axis) was analyzed after 5 days of culture on the total population of stimulated PBMC (B). Representative data of three donors.

 
Der p-induced modulation of CXCR4 surface expression results in differences in chemotactic properties of resting and activated T cells respectively
CXCR4 has a unique ligand, SDF-1, which specifically induces the migration of CXCR4-expressing cells. To determine whether Der p-induced modulation of CXCR4 expression was reflected in functional changes of the responding cells, CD25+ and CD25 T cells from the PBMC from atopic donors that had been stimulated with Der p for 7 days were purified by FACS sorting and the chemotactic responses of both populations, following stimulation with rSDF-1ß, were studied. A dose-dependent migration of CD25 T cells was induced by rSDF-1ß that reached a net migration index of 20–30% at concentrations of 25–50 ng/ml of chemokine. The migration of CD25+ T cells in response to SDF-1ß, however, was far less efficient and did not exceed 4–5% at the same concentrations. At higher doses of rSDF-1ß, the difference in migratory capacity between both groups of cells was less important, but net migration of CD25 cells remained almost 4-fold higher than that of CD25+ cells. These results indicate that allergen-mediated down-regulation of CXCR4 on activated T cells results in a reduced capacity of these cells to migrate in response to stimulation with SDF-1ß, as compared to that of non-responding T cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
There is increasing evidence for differential expression of various chemokine receptors on subsets of Th1 and Th2 cells, which might explain their selective migration pattern into inflamed tissue. Although a few studies have addressed the effect of polyclonal activators such as phytohemagglutinin and phorbol esters on the expression of CXCR4 on T cells (10,17,18), as well as interaction with its natural ligand (8), little is known about the effects of TCR-mediated stimulation on the expression of this chemokine receptor. The results presented here demonstrate that a single stimulation of PBMC from atopic donors with Der p results in a bimodal effect on T cell-surface-expressed CXCR4, which is characterized by a down-regulation of the receptor on activated memory T cells, and an enhanced surface expression on resting memory and naive T cells, as a result of endogenous IL-4 production, most likely by specifically activated memory cells.

Der p-mediated effects on CXCR4 expression were maximal between day 5 and 7 of culture, and were, in all cases, accompanied by a strong proliferative response, as well as by the production of Th2 cytokines. In contrast, stimulation of PBMC from non-atopic donors with Der p did not result in significant proliferation and no changes in CXCR4 expression were observed, underscoring the specificity of the Der p-mediated effects. In addition, although the frequency of allergen-specific T cells in peripheral blood is very low (19), the results obtained in vitro were supported by the observation that the frequency of CXCR4-expressing cells among PBMC from 11 atopic donors, but not from 11 healthy controls, declined during the pollen season as a result of natural exposure to aero allergens (results not shown).

Consistent with reports in the literature (reviewed in 2), freshly isolated peripheral blood T cells express low, but significant, levels of CXCR4. However, the spontaneous expression of CXCR4 is rapidly enhanced on T cells cultured in medium only and remains elevated for as long as the cells can be maintained in culture. The up-regulation of CXCR4 surface expression is observed on CD45RA+ and CD45RO+ T cells, cultured in the presence of (autologous) human or fetal calf serum, as well as in serum-free medium, is dependent on the presence of external Ca2+ and is not due to the presence of steroid hormones in the culture medium (data not shown). This increased cell-surface expression was recently proposed to be due to the relocation of intracellular CXCR4 (20), similar to what has been reported for short-term cultured Langerhans cells and macrophages (21). However, the addition of cycloheximide to the cultures prevented the up-regulation of surface expression of CXCR4 (P. Jourdan and H. Yssel, unpublished data), suggesting that the enhanced expression of CXCR4 is in part due to de novo synthesis of CXCR4 protein as well. The in vivo relevance of this observation is however not clear and it cannot be excluded that the spontaneous up-regulation of CXCR4 on peripheral blood T cells represents a culture-induced artefact which masks the up-regulation of cell-surface expression of this receptor induced by immunologic stimuli.

The down-regulation of CXCR4 on allergen-activated T cells contrast with that of Carroll et al. who have reported an increased expression of CXCR4 mRNA following activation of peripheral blood T cells with cross-linked anti-CD3 and anti-CD28 mAb (18). Although no surface expression CXCR4 was measured in the latter study, activated T cells were susceptible to infection with a T-tropic strain of HIV, suggesting the induction of enhanced levels of CXCR4 surface expression following activation. The discrepancies between our data and those described in the study of Carroll et al. might be explained by the above-mentioned observation that in vitro culture of T lymphocytes in medium alone results in an up-regulation of CXCR4 expression. Moreover, chemokine receptors other than CXCR4 may act as co-receptors for certain T-tropic strains (22), thereby enabling entry of the virus in the absence of CXCR4. Furthermore, our results support those of Bermejo et al. who reported that CXCR4 is down-regulated on T lymphocytes following activation with phytohemagglutinin or anti-CD3 mAb (20). Although interaction of CXCR4 with SDF-1 results in a down-regulation of CXCR4 due to internalization of the receptor–ligand complex (810), this mechanism is unlikely to be operational in TCR-mediated down-regulation of CXCR4. Indeed, it is demonstrated here that the decrease of CXCR4 cell-surface expression is due to an inhibition of CXCR4 mRNA, suggesting a direct TCR-mediated effect at the level of transcription. In view of the fast kinetics of decrease in CXCR4 mRNA levels, as well as the failure of IFN-{gamma}, reported to down-regulate CXCR4 expression (23), to affect the expression of CXCR4 in our culture system (results not shown), this TCR-mediated effect does not seem to involve the action of endogenous cytokines.

The enhancement of CXCR4 on CD45RO and resting CD25CD45RO+CD4+ T cells, although masked to great extend by the high levels of CXCR4 surface expression induced by culture in medium alone, was likely to be due to the presence of endogenous IL-4, since its increase was almost completely blocked by the addition of a neutralizing anti-IL-4 mAb to the cultures. None of the other cytokines, produced in this culture system, among others IL-2, IL-5 and IL-10, were found to have an inducing effect on CXCR4 expression as demonstrated by the addition of neutralizing mAb or relevant recombinant cytokines to the culture system (results not shown). Moreover, although IL-13 has largely overlapping biological functions with IL-4, the absence of a functional IL-13 receptor on T cells (24) excludes a direct role for endogenously produced IL-13 in the induction of the CXCR4.

In addition to Th2 cells, basophils are a potential source for IL-4, following stimulation of their high-affinity Fc{varepsilon}RI with allergen (25,26). However, IL-4 production by basophils is mostly the result of rapid release of pre-synthesized IL-4, whereas IL-4 transcripts are only transiently expressed (26) and kinetics of IL-4 by basophils is very rapid (27). The presence of specific IL-4 mRNA, as measured using the RNase protection assay, in Der p-activated PBMC, even after 7 days of activation (data not shown), suggests that activated memory T cells are one of the sources of endogenous IL-4 in the in vitro culture system, used in this study. However, a role for basophil-derived IL-4 in the induction of CXCR4 cell-surface expression on resting CD45RO and CD45RO+ T cells cannot be totally excluded.

The inducing effects of endogenous IL-4 on the expression of CXCR4 on resting cells indicate that a functional IL-4R is present on their cell surface and our results are in line with previously published reports showing that IL-4R mRNA, as well as cell-surface expression of IL-4R is up-regulated on T cells, cultured in the presence of exogenous IL-4 (28,29), a process which is inhibited by the addition of an anti-IL-4R mAb. Our data, including those on the kinetics of IL-4R and CXCR4 expression, suggest that allergen-activated T cells do not express detectable levels of cell-surface IL-4R. However, the presence of endogenous IL-4, produced by allergen-specific T cells and likely by basophils as well, early during activation, results in the enhancement of IL-4R expression on resting cells which in its turn, following interaction with IL-4, will result in the induction/enhancement of CXCR4 expression. The differential expression of CXCR4 on resting and specifically activated, CD25-expressing T lymphocytes has functional consequences for both populations of cells. Indeed, allergen-activated T lymphocytes were found to have a reduced capacity to migrate in response to stimulation with SDF-1 in vitro. Importantly, while most chemokines are released following activation of the producer cells, SDF-1 is ubiquitous and constitutively expressed (30,31), and it has been proposed that the interaction of CXCR4 and its ligand play a role in the homing of naive cells to lymph nodes (32,33). If activation is required for allergen-specific T cells to enter tissue, such as lung wall or atopic skin, paradoxically, allergen-specific T cells which have been activated in secondary lymphoid nodes will have lost CXCR4 surface expression, leaving the cells unable to respond to the migration-inducing capacity of SDF-1. These results either preclude a role for SDF-1 in the homing of activated allergen-specific T cells into inflamed tissue or suggests that resting memory T cells might encounter specific allergen in sites of inflammation, resulting in a subsequent loss of CXCR4 expression and confinement of the cells at the site of inflammation. In this respect, it is worth noting that cultured human dermal endothelial cells are able to both process and present allergen, such as Der p, in vitro (34), suggesting that endothelium might be able to specifically activate memory T cells. Indeed, the loss of CXCR4 by allergen-activated T lymphocytes could sequester them into secondary lymphoid tissues, allowing these cells to respond selectively to other cytokines, specifically produced during inflammation, such as RANTES and MIP-1{alpha}. Although it has been reported that the receptor for the latter chemokines, CCR5, is down-regulated following polyclonal activation of cloned T cell lines, our preliminary results, using the culture system described in this study, indicate that at least 50% of allergen-activated T cells do express CCR5 at their cell surface (C. Abbal, unpublished data) and therefore might still be able to migrate in response to its specific ligand.

Taken together, the data presented here demonstrate that the differential expression of CXCR4 on resting and activated T cells depends on the activation state of the cells, and is the result, at least in part, of the counteracting effects of specific, TCR-mediated, down-regulation and IL-4-mediated up-regulation of CXCR4. Finally, in spite of the prominent role of IL-4 in allergic disease, our results suggest that CXCR4 is not likely to be involved in the trafficking of allergen-specific cells to the site of allergic inflammation, although the precise role of this chemokine receptor in atopic disease remains yet to be established.



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Fig. 8. Der p-induced modulation of CXCR4 results in differences in chemotactic properties of resting and activated T cells respectively. CD25+ (•) and CD25 ({circ}) T cells, purified by FACS sorting from the PBMC of an atopic donor after 7 days of culture in the presence of 10 µg/ml Der p were stimulated with various concentrations of human rSDF-1ß, in an in vitro migration assay, as described in Methods. Results represent mean ± SD of triplicate determinations using three different atopic donors.

 

    Acknowledgments
 
The authors would like to thank Dr Kevin Bacon (Neurokine, San Diego, CA) for helpful advice on the chemotaxis assay and Christophe Duperray (INSERM U291, Montpellier, France) for FACS sorting. The technical assistance of Vera Boulay is greatly appreciated.


    Abbreviations
 
Der pDermatophagoides pteronyssinus
EBVEpstein–Barr virus
IL-4RIL-4 receptor
PBMCperipheral blood mononuclear cell
PEphycoerythrin
rrecombinant
SDFstromal cell-derived factor

    Notes
 
Transmitting editor: K.-i. Arai

Received 15 February 1999, accepted 21 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Butcher, E. C. and Picker, L. J. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  2. Baggiolini, M., Dewald, B. and Moser, B. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15:675.[ISI][Medline]
  3. Sallusto, F., Mackay, C. R. and Lanzavecchia, A. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005.[Abstract/Free Full Text]
  4. Siveke, J. T. and Haman, A. 1998. T helper 1 and T helper 2 cells respond differentially to chemokines. J. Immunol. 160:550.[Abstract/Free Full Text]
  5. Bonecchi, R., Bianchi, G., Bordignon, P. P., d'Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P. A., Mantovani, A. and Sinigaglia, F. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129.[Abstract/Free Full Text]
  6. Loetscher, P., Uguccioni, M., Bordoli, L., Baggiolini, M., Moser, B., Chizzolini, C. and Dayer, J.-M. 1998. CCR5 is characteristic of Th1 lymphocytes. Nature 391:344.[ISI][Medline]
  7. Jourdan, P., Abbal, C., Noraz, N., Hori, T., Uchiyama, T., Bousquet, J., Taylor, T., Pène, J. and Yssel, H. 1998. Interleukin-4 induces functional cell-surface expression of CXCR4 on human T cells. J. Immunol. 160:4153.[Abstract/Free Full Text]
  8. Amara, A., Legall, S., Schwartz, O., Salamero, J., Montes, M., Loetscher, P., Baggiolini, M., Virelizier, J. L. and Arenzana-Seisdedos, F. 1997. HIV coreceptor down-regulation as antiviral principle: SDF-1 {alpha}-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med. 186:139.[Abstract/Free Full Text]
  9. Förster, R., Kremmer, E., Schubel, A., Breitfeld, D., Kleinschmidt, A., Nerl, C., Bernhardt, G. and Lipp, M. 1998. Intracellular and surface expression of the HIV-1-coreceptor CXCR4/fusin on various leukocyte subsets: rapid internalization and recycling upon activation. J. Immunol. 160:1522.[Abstract/Free Full Text]
  10. Signoret, N., Oldridge, J., Pelchen-Matthews, A., Klasse, P. J., Tran, T., Brass, L. F., Rosenkilde, M. M., Schwartz, T. W., Holmes, W., Dallas, W., Luther, M. A., Wells, T. N. C., Hoxie, J. A. and Marsh, M. 1997. Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4. J. Cell Biol. 139:651.[Abstract/Free Full Text]
  11. Yssel, H., de Vries, J. E., Koken, M., Van Blitterswijk, W. and Spits, H. 1984. Serum-free medium for the generation and propagation of functional human cytotoxic and helper T cell clones. J. Immunol. Methods 72:219.[ISI][Medline]
  12. Pène, J., Rousset, F., Brière, F., Chrétien, I., Bonnefoy, J. Y., Spits, H., Yokota, T., Arai, N., Arai, K.-I., Banchereau, J. and De Vries, J. E. 1988. IgE production by normal human lymphocytes is induced by interleukin (IL)-4 and suppressed by interferon (IFN)-{gamma} and -{alpha}, and prostaglandin E2. Proc. Natl Acad. Sci. USA 85:6880.[Abstract]
  13. Lanier, L. L. and Recktenwald, D. J. 1991. Multicolor immunofluorescence and flow cytometry. Methods: A Companion to Methods in Enzymology 2:192.
  14. Hori, T., Sakaida, H., Sato, A., Nakayama, T., Shida, H., Yoshie, O. and Uchiyama, T. 1997. Detection and delineation of CXCR-4 (fusin) as an entry and fusion co-factor for T-tropic HIV-1 by three different monoclonal antibodies. J. Immunol. 160:180.[Abstract/Free Full Text]
  15. Spits, H., Yssel, H., Terhorst, C. and de Vries, J. E. 1982. Establishment of human T lymphocyte clones highly cytotoxic for an EBV transformed B cell line in serum-free medium: isolation of clones that differ in phenotype and specificity. J. Immunol. 128:95.[Free Full Text]
  16. Bacon, K. B. and Schall, T. J. 1997. Chemokine-induced lymphocyte migration: analysis by fliter-based bioassays. In Herzenberg, L. A., Weir, D. M., Herzenberg, L. A. and Blackwell, C., ed., Weir's Handbook of Experimental Immunology, p. 71. Blackwell Science, Malden, MA.
  17. Bleul, C. C., Wu, L. J., Hoxie, J. A., Springer, T. A. and Mackay, C. R. 1997. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc. Natl Acad. Sci. USA 94:1925.[Abstract/Free Full Text]
  18. Carroll, R. G., Riley, J. L., Levine, B. L., Feng, Y., Kaushal, S., Ritchey, D. W., Bernstein, W., Weislow, O. S., Brown, C. R., Berger, E. A., June, C. H. and St Louis, D. C. 1997. Differential regulation of HIV-1 fusion cofactor expression by CD28 costimulation of CD4+ T cells. Science 276:273.[Abstract/Free Full Text]
  19. Sager, N., Feldmann, A., Schilling, G., Kreitsch, P. and Neumann, C. 1992. House dust-mite-specific T cell in the skin of subjects with atopic dermatitis: frequency and lymphokine profile in the allergen patch test. Eur. J. Dermatol. 89:801.
  20. Bermejo, M., Martin-Serrano, J., Oberlin, E., Pedraza, M. A., Serrano, A., Santiago, B., Caruz, A., Loetscher, P., Baggiolini, M., Arenzana-Seisdedos, F. and Alcami, J. 1998. Activation of blood T lymphocytes down-regulates CXCR4 expression and interferes with propagation of X4 HIV strains. Eur. J. Immunol. 28:3192.[ISI][Medline]
  21. Zaitseva, M., Blauvelt, A., Lee, S., Lapham, C. K., Klaus-Kovtun, V., Mostovski, H., Manischewitz, J. and Golding, H. 1997. Expression and function of CCR5 and CXCR4 on human Langerhans cells and macrophages: implications for HIV primary infection. Nature Med. 3:1369.[ISI][Medline]
  22. Bergen, E. A., Doms, R. W., Fenyö, E.-M., Korber, B. T. M., Littman, D. R., Moore, J. P., Sattentau, Q. J., Schuitemaker, H., Sodroski, J. and Weiss, R. A. 1998. A new classification for HIV-1. Nature 391:240.[ISI][Medline]
  23. Galli ,G., Annunziato, F., Mavilia, C., Romagnani, P., Cosmi, L., Manetti, R., Pupilli, C., Maggi, E. and Romagnani, S. 1998. Enhanced HIV expression during Th2-oriented responses explained by the opposite regulatory effect of IL-4 and IFN-{gamma} of fusin/CXCR4. Eur. J. Immunol. 28:3280.[ISI][Medline]
  24. De Waal Malefyt, R., Abrams, J. S., Zurawski, S. M., Lecron, J.-C., Mohan-Peterson, S, Sanjanwala, B., Bennett, B. F., Silver, J., de Vries, J. E. and Yssel, H. 1995. Differential regulation of IL-4 and IL-13 expression by human T cell clones and Epstein–Barr virus-transformed B cells. Int. Immunol. 7:1405.[Abstract]
  25. Brunner, T., Heusser, C. H. and Dahinden, C. A. 1993. Human peripheral blood basophils primed by interleukin-3 (IL-3) produce IL-4 in response to immunoglobulin E receptor stimulation. J. Exp. Med. 177:605.[Abstract]
  26. MacGlashan, D. J., White, J. M., Huang, S. K., Ono, S. J., Schroeder, J. T. and Lichtenstein, L. M. 1994. Secretion of IL-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils. J. Immunol. 152:3006[Abstract/Free Full Text]
  27. McHugh, S., Deighton, J. Rifkin, I. and Ewan, P. 1996. Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture. Eur. J. Immunol. 26:1260.[ISI][Medline]
  28. Armitage, R. J., Beckmann, M. P., Idzerda, R. L., Alpert, A. and Fanslow, W. C. 1990. Regulation of interleukin-4 receptors on human T cells. Int. Immunol. 2:1039.[ISI][Medline]
  29. Renz, H., Domenico, J. and Gelfland, E. W. 1991. IL-4-dependent up-regulation of IL-4 receptor expression in murine T and B cells. J. Immunol. 146:3049.[Abstract/Free Full Text]
  30. Tashiro, K., Tada, H., Heilker, R., Shirozu, M., Nakano, T. and Honjo, T. 1993. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261:600.[ISI][Medline]
  31. Nagasawa, T., Kikutani, H. and Kishimoto, T. 1994. Molecular cloning and structure of a pre-B-cell growth-stimulating factor. Proc. Natl Acad. Sci. USA 91:2305.[Abstract]
  32. Bleul, C. C., Fuhlbrigge, R., Casasnovas, J. M., Aiuti, A. and Springer, T. A. 1996. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J. Exp. Med. 184:1101.[Abstract]
  33. Aiuti, A., Webb, I. J., Bleul, C. and Springer, T. A. 1997. The chemokine SDF-1 is a potent chemoattractant for human CD34+ hemopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitor cells to peripheral blood. J. Exp. Med. 185:111.[Abstract/Free Full Text]
  34. Vora, M., Yssel, H., de Vries, J. E. and Karasek, M. 1994. Antigen presentation by human dermal microvascular endothelial cells: immunoregulatory effect of interleukin 10. J. Immunol. 152:5731.