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Selective Expression of the Eotaxin Receptor CCR3 by Human T Helper 2 Cells

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Science  26 Sep 1997:
Vol. 277, Issue 5334, pp. 2005-2007
DOI: 10.1126/science.277.5334.2005

Abstract

There is growing evidence that T helper cell subsets (TH1 and TH2) can be differentially recruited to promote different types of inflammatory reactions. Murine TH1 but not TH2 cells are recruited through P- and E-selectin into inflamed tissues, where they induce delayed-type hypersensitivity reactions. The human eotaxin-receptor CCR3, originally described on eosinophils and basophils, was also found to be expressed by TH2 cells. An antibody to CCR3 was used to isolate T cells from peripheral blood that give rise to TH2-polarized cell lines and to identify TH2 cells derived from naı̈ve T cells in vitro. Eotaxin stimulated increases in intracellular calcium and chemotaxis of CCR3+ T cells. The attraction of TH2 cells by eotaxin could represent a key mechanism in allergic reactions, because it promotes the allergen-driven production of interleukin-4 and interleukin-5 necessary to activate basophils and eosinophils.

The regulation of leukocyte migration is a complex process involving the participation of adhesion molecules such as selectins and integrins (1) as well as chemokines and chemokine receptors (2). The combined action of adhesion molecules and chemokines is thought to provide an address code for leukocyte migration to different sites (3).

Effector T lymphocytes are heterogeneous in their functional capabilities (4). TH1 cells produce interferon-γ (IFN-γ) and activate mononuclear phagocytes, thus protecting against intracellular microbes. In contrast, TH2 cells produce interleukin-4 (IL-4) and IL-5 and are involved in responses dominated by immunoglobulin E (IgE), eosinophils, and basophils. Mouse TH1 cells express the ligand for P- and E-selectin, which promotes their migration into inflamed tissues (5). TH2 cells do not enter the same sites as TH1, suggesting a distinct migratory capacity. Because tissues undergoing allergic reactions contain TH2 cells together with eosinophils and basophils, it is conceivable that common factors may be responsible for the recruitment of these three cell types.

The CC-chemokine eotaxin, produced by epithelial and phagocytic cells, is a potent and selective chemoattractant for eosinophils and basophils (6, 7). Eotaxin binds with high affinity and fidelity to a specific receptor, CCR3, which is selectively expressed on eosinophils and basophils (8).

We observed that an antibody to CCR3 (7B11) (9) stained a small proportion of T lymphocytes in the human adult peripheral blood of certain donors but not in cord blood (10). About 1% of peripheral blood T cells express CCR3 (Fig. 1). CCR3+ and CCR3 lymphocytes were sorted, expanded as polyclonal lines by stimulation with phytohemagglutinin (PHA) and IL-2, and tested for expression of CCR3 and cytokine production (11). Lines derived from CCR3+-sorted cells had an increased proportion of CCR3+ cells (19%) and produced IL-4 and IL-5. In contrast, lines derived from unsorted or CCR3-sorted cells comprised only very few CCR3+ cells (<1%) and produced little IL-4 and IL-5. Although IL-4 and IL-5 production correlated with the expression of CCR3, the production of IFN-γ did not, because all the lines produced it in comparable amounts.

Figure 1

CCR3 expression identified type 2 T cells in adult peripheral blood. (A) CCR3 expression on PBMC and (B and E) CCR3 expression on polyclonal T cell lines obtained from CCR3 (R1) and CCR3+ (R2) cells are shown. Data are displayed on a three-decade logarithmic scale. Dotted line represents the staining with isotype-matched control antibody. (C and F) Production of IL-4, IL-5, and IFN-γ by the same T cell lines as measured by ELISA and (D and G) IFN-γ and IL-4 production at the single-cell level measured by intracellular staining (four-decade logarithmic scale) are shown. (H) IFN-γ and IL-4 production by CCR3+ cells sorted from the CCR3-enriched cell line is shown. The CCR3 staining of this population is shown as a thick line in (E). The cells expressing high levels of CCR3 in peripheral blood were identified as basophils by sorting and Giemsa staining.

To identify type 2 T cells unambiguously, cytokine production analysis was performed on single cells (Fig. 1, D and G) (12). The lines derived from CCR3+-sorted cells comprised a high proportion (17%) of typical type 2 cells producing large amounts of IL-4 and no IFN-γ, whereas the lines derived from unsorted or CCR3-sorted cells comprised only small proportions of cells mostly producing intermediate amounts of IL-4 (<2%). A second round of sorting from partially enriched cell lines led to a further enrichment of type 2 cells (>50%) and depletion of type 1 cells (Fig.1H) (13). The expression of CD4 or CD8 was assessed together with cytokine production in polyclonal cell lines by three-color immunofluorescence (12). Cells producing only IL-4 were found in both CD4+ and CD8+ subsets and were enriched in CCR3+ and depleted in CCR3 lines, as compared to unsorted cells (14). We conclude that CCR3 expression identifies a small population of peripheral blood T cells that gives rise to type 2 cell lines in vitro and that most likely have acquired this phenotype in vivo.

To identify the factors that induce CCR3 expression in T cells, we cultured cord blood naı̈ve T cells in conditions that lead to either type 1 or type 2 polarization. Type 2 cell lines polarized in the presence of IL-4 plus anti-IL-12 (Fig.2, A through D) (15) comprise up to 50% of CCR3+ cells. When CCR3+ and CCR3 cells were sorted and immediately analyzed for cytokine production, the CCR3+ cells were almost completely of the type 2 phenotype, whereas the CCR3 cells showed a more heterogeneous pattern with few IFN-γ–producing cells. Conversely, type 1 cell lines polarized in the presence of IL-12 plus anti–IL-4 (Fig. 2, E through H) contained very few CCR3+cells. Interestingly, the rare CCR3+ cells gave rise to clones that produced IL-4 and low or no IFN-γ. In contrast, all clones obtained from CCR3 cells produced large amounts of IFN-γ but no IL-4. Thus, CCR3 expression is induced in T cells under type 2–polarizing conditions and correlates with the capacity to produce IL-4 even in the rare cells that develop this phenotype under type 1–polarizing conditions.

Figure 2

CCR3 expression was induced under type 2 polarizing conditions and correlates with IL-4 production. Cytokine production pattern (four-decade logarithmic scale) and CCR3 expression (three-decade logarithmic scale) are shown for cell lines obtained from cord blood T cells under type 2– (A and B) or type 1– (E and F) polarizing conditions. From the type 2–polarized cell line, CCR3(R1) and CCR3+ (R2) cells were sorted and immediately analyzed for cytokine production (C and D). From the type 1–polarized cell line, CCR3 (R1) and the rare CCR3+ (R2) cells were sorted, cloned by limiting dilution, and the clones analyzed for cytokine production by ELISA (G and H). Dashed lines represent the end of the assay. Similar results were obtained with six different cord blood samples.

Because both IFN-α (16) and transforming growth factor–β (TGF-β) (17) can interfere with TH2 development, we tested whether they may also interfere with acquisition of CCR3 expression (Fig.3). When added to the TH2-polarizing condition, IFN-α dominantly induced generation of TH1 cells, whereas TGF-β inhibited development of both IL-4– and IFN-γ–producing cells. In both situations, there was an almost complete inhibition of CCR3 expression, indicating that the expression of this receptor is linked to the program of TH2 differentiation.

Figure 3

IFN-α and TGF-β inhibit polarization to TH2 and acquisition of CCR3. Cytokine production pattern and CCR3 expression are shown for cord blood T cell lines cultured with IL-4 plus anti–IL-12 either alone (Aand D), or together with IFN-α (B andE) or TGF-β (C andF).

To investigate whether the CCR3 expressed on T cells is functional, we measured the increase in intracellular Ca2+ in response to eotaxin (Fig. 4) (18). A CCR3+ but not a CCR3 T cell clone responded with a characteristic rapid rise in intracellular Ca2+ to stimulation with appropriate concentrations of eotaxin. CCR3+ T cell clones were also tested for their chemotactic response to eotaxin by use of a standard chemotaxis assay. CCR3+ but not CCR3 T cells responded to eotaxin with a similar efficiency as to other T cell chemoattractants, such as monocyte chemotactic protein–1 (MCP-1) and IFN-inducible protein–10 (IP-10) (19).

Figure 4

Induction of intracellular Ca2+ increase in CCR3+ T cells by eotaxin. Intracellular Ca2+was recorded following stimulation with 10 nM eotaxin in a CCR3+ (A) or a CCR3(B) T cell clone. Both clones responded similarly when challenged with 10 nM RANTES (25).

We show that CCR3 is acquired as a consequence of type 2 polarization both in vivo and in vitro, and is a stable marker of type 2 T cells, which allows them to respond to eotaxin. Together with the report that murine TH1 cells selectively express ligands for P- and E-selectin (5), our results on human TH2 cells support the concept that the T cell differentiation program goes beyond the cytokine production pattern and involves migratory capacity as well (20).

The expression of the eotaxin receptor on eosinophils, basophils, and TH2 cells shows that these three cells can respond similarily to eotaxin as well as other agonistic chemokines (21). Eotaxin expression is up-regulated in tissues known to be sites of allergic reactions such as the airways (7) and is important for the attraction of eosinophils leading to lung eosinophilia (6). However, it is also known that the generation and maintenance of an allergic reaction requires antigen-specific TH2 cells as a source of IL-4 and IL-5 that serve as growth and stimulation factors for basophils and eosinophils (22). The presence of CCR3 on TH2 cells and on T cells that colocalize with eosinophils in diseased tissues (23) reveals a possible pathogenetic mechanism for T cell recruitment in the airways (24) and provides a target for therapeutic intervention. Thus, the distribution of eotaxin and of its receptor could coordinately and simultaneously attract to particular sites the three cell types that cooperate to generate an inflammatory allergic response.

  • * To whom correspondence should be addressed. E-mail: sallusto{at}bii.ch

  • Present address: Millennium Biotherapeutics Inc., Cambridge, MA 02142, USA.

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