The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics

In vitro polarized human Th2 cells preferentially express the chemokine receptors CCR3, CCR4, and CCR8 and migrate to their ligands: eotaxin, monocyte-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC), and I-309. We have studied the expression of chemokines and chemokine receptors in the airway mucosa of atopic asthmatics. Immunofluorescent analysis of endobronchial biopsies from six asthmatics, taken 24 hours after allergen challenge, demonstrates that virtually all T cells express IL-4 and CCR4. CCR8 is coexpressed with CCR4 on 28% of the T cells, while CCR3 is expressed on eosinophils but not on T cells. Expression of the CCR4-specific ligands MDC and TARC is strongly upregulated on airway epithelial cells upon allergen challenge, suggesting an involvement of this receptor/ligand axis in the regulation of lymphocyte recruitment into the asthmatic bronchi. In contrast to asthma, T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis produce IFN-γ and express high levels of CXCR3, while lacking CCR4 and CCR8 expression. These data support the role of CCR4, of its ligands MDC and TARC, and of CCR8 in the pathogenesis of allergen-induced late asthmatic responses and suggest that these molecules could be considered as targets for therapeutic intervention.

Trafficking of activated T cells into inflammatory sites is a tightly controlled process directed by multiple molecules, in particular adhesion molecules and chemokines (1, 2). Chemokines are small, secreted polypeptides that regulate lymphocyte trafficking by signaling through G protein–coupled seven transmembrane receptors. A flurry of recent in vitro studies has demonstrated that Th1 and Th2 cells express distinct sets of chemokine receptors that might regulate the recruitment and localization of these cells to inflammatory sites (3, 4). CXCR3 and CCR5 have been associated with the Th1 cytokine profile (5, 6), whereas CCR3, CCR4, and CCR8 have been associated with the Th2 phenotype (7–10). Selective recruitment of subsets of CD4⁺ effector T cells into sites of inflammation may contribute to the development of different pathological conditions.

T lymphocytes are involved in initiating and maintaining airway inflammation and obstruction in asthma (11). Activated CD4⁺ T cells producing IL-4, IL-5, and IL-13 have been identified in bronchoalveolar lavage and bronchial biopsies of both atopic and nonatopic asthmatic patients (12, 13), and they increase during late asthmatic responses (14–16). Despite the clear evidence for T cell involvement in asthma, the mechanisms of Th2 cell recruitment within the airways remain poorly defined. Recent studies have shown that the CCR3 ligand eotaxin is produced at high levels in human asthma and localizes to the airway epithelium and to inflammatory cells (17). Unlike other eosinophil-attracting chemokines, eotaxin binds to a single receptor, CCR3, that is highly expressed on eosinophils (18, 19). Because CCR3 is also expressed on basophils (20) and on in vitro polarized Th2 cells (7), it has been suggested that the sharing of CCR3 may allow these different cell types to colocalize at sites of eotaxin production. In addition to eotaxin, other chemokines have been shown to preferentially attract Th2 cells in vitro. These include monocyte-derived chemokine and I-309, binding to CCR4 and CCR8, respectively. Although these results suggest that these chemokines/chemokine receptor axes may be involved in the recruitment of Th2 lymphocytes that could lead to increased airway damage and altered physiology, very limited information is available on the expression and function of these specific receptors during the asthmatic inflammatory response.

In this study we demonstrate that the majority (>90%) of T cells infiltrating the bronchial biopsies of allergen-challenged asthmatics produce IL-4 and express CCR4. CCR8 was expressed on approximately 28% of infiltrating CCR4⁺ IL-4⁺ T cells. The two CCR4 ligands, monocyte-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), were expressed by airway epithelial cells and their expression was strongly upregulated after allergen challenge. A comparative analysis of T cells infiltrating the bronchi of patients with nonatopic inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD) and pulmonary sarcoidosis revealed that this chemokine/chemokine receptor pattern was specific for atopic asthma. Unlike the T cells infiltrating the asthmatic samples, T cells in both COPD and sarcoidosis produce IFN-γ and express CXCR3, while lacking CCR4 and CCR8 expression. We conclude that CCR4 and, to a lesser extent, CCR8 mark the majority of T cells infiltrating the airway of asthmatic patients after allergen challenge.

Th2 lymphocytes represent the predominant infiltrate in bronchial biopsies of atopic asthmatics after allergen challenge. Bronchial biopsies were obtained from six atopic asthmatics 24 hours after allergen challenge, six atopic asthmatics 24 hours after sham challenge (nonsmokers; one female; mean age 27.6 ± 2.8; mean base-line FEV₁ 98.5 ± 4.5; PC₂₀FEV₁ methacholine 1.3 [0.9] mg/ml), and three healthy controls (one female; mean age 39 ± 6.3). Each of the six asthmatic patients challenged with allergen developed both an EAR and an LAR (nonsmokers; two female; mean age 31.5 ± 2.1; mean base-line FEV₁ 95.4 ± 5.8; PC₂₀FEV₁ methacholine 1.5 [1.1] mg/ml). Bronchoalveolar eosinophils and lymphocytes were significantly increased at 24 hours after allergen challenge (5% [3–5.25%] vs. 1% [0–1.5%]; P < 0.01) and (3% [2–6%] vs. 1.5% [0.88–2%]; P < 0.05).

Immunohistochemical analysis with anti-CD3, anti-EG2, anti-CD14, anti-CD1a, and anti-tryptase antibodies revealed that only the number of CD3⁺ cells per square millimeter was significantly increased in the airway mucosa of sham-challenged asthmatics when compared with the nonasthmatic control group (874 [384–1480] vs. 274 [112–440], respectively; P ≤ 0.005). Upon allergen challenge, the number of subepithelial and intraepithelial CD3⁺ cells per square millimeter increased significantly (874 [384–1480] vs. 4400 [3870–7060], in sham-challenged and in allergen-challenged asthmatics, respectively; P = 0.0022). The number of infiltrating eosinophils was not significantly increased at 24 hours after allergen challenge. At this time, cells staining positive for CD14, CD1a, or tryptase could hardly be detected in any of the tissues analyzed (data not shown). We then analyzed IL-4 and IFN-γ production in infiltrating CD3⁺ cells by double immunofluorescence. A significant proportion of CD3⁺ cells in healthy controls (34%) and in sham-challenged asthmatics (40%) produced IL-4, while virtually all the CD3⁺ cells infiltrating the airway mucosa of allergen challenged asthmatics were IL-4⁺ (99.5%) (Figure 1). By contrast, almost no IFN-γ–producing cells were present in the airway mucosa either after sham challenge (1% among all the CD3⁺ cells) or allergen challenge (0.5% among all the CD3⁺ cells). Few IFN-γ–producing cells were found in the airway mucosa of healthy controls (11% among all CD3⁺ cells). These results indicate a predominant airway infiltration of Th2 cells at 24 hours after sham or allergen challenge.

Figure 1

Cytokine production by T lymphocytes that infiltrate the airway subepithelium of atopic asthmatics. Cryostat sections of a bronchial biopsy from a representative healthy subject (a and d), a representative sham-challenged atopic asthmatic (b and e), and a subject with atopic asthma 24 hours after allergen challenge (c and f). Double immunofluorescent staining with anti-CD3 (green) and anti–IL-4 (red) (a–c) or anti–IFN-γ (red) (d–f) is shown. Arrows point to double-stained cells (yellow). Original magnification: ×40. e, airway epithelium.

CD3⁺ cells in bronchial biopsies of allergen-challenged atopic asthmatics express CCR4 and CCR8 but not CCR3. The selective migration of CD4⁺ cells to inflammation sites is a tightly regulated event depending upon the restricted expression of chemokine receptors on different Th cell subsets. We analyzed, by immunofluorescence, chemokine receptor expression on T cells infiltrating the airway mucosa. Since CCR3 is expressed on in vitro polarized Th2 cells, we first analyzed the tissue distribution of CCR3 expression in allergen-challenged and sham-challenged asthmatics. As shown in Figure 2, CD3⁺ cells infiltrating the bronchi of allergen-challenged asthmatics did not express CCR3. A few cells expressing CCR3 were only detectable in the airway mucosa of allergen-challenged asthmatics, not in sham-challenged asthmatics or in healthy controls. These cells are likely to be eosinophils, as they coexpress the marker EG2.

Figure 2

Expression of CCR3 by EG2⁺ cells and airway epithelial cells but not by T cells infiltrating the airway of atopic asthmatics. Cryostat sections of bronchial biopsies from a representative healthy subject (a), a representative sham-challenged atopic asthmatic (b), and a representative subject with atopic asthma 24 hours after allergen challenge (c and d). (a–c) Double immunofluorescent staining with anti-CD3 (green) and anti-CCR3 (red). (d) Double immunofluorescent staining with anti-EG2 (green) and anti-CCR3 (red). Arrows point to double-stained cells (yellow). Original magnification: ×40. e, airway epithelium.

The tissue distribution of CCR4 expression in challenged asthmatics versus nonchallenged asthmatics and nonasthmatic controls is shown in Figure 3. Challenged asthmatics showed a significantly higher number of infiltrating CCR4⁺ Th2 cells per square millimeter compared with sham-challenged asthmatics (3410 [2790–4480] vs. 390 [261–606], respectively; P = 0.0022). CCR4 expression was also found on airway epithelial cells in all sections examined both in asthmatics and in controls (see Figures 2, 4, and 6).

Figure 3

(a–c) Expression of CCR4 by T cells infiltrating the airway of atopic asthmatics. Cryostat sections of bronchial biopsies from a representative healthy subject (a), a representative sham-challenged atopic asthmatic (b), and a representative subject with atopic asthma 24 hours after allergen challenge (c). Double immunofluorescent staining with anti-CD3 (green) and anti-CCR4 (red) is shown. Arrows point to double-stained cells (yellow). Original magnification: ×40. e, airway epithelium. (d) Individual counts of CCR4-expressing T lymphocytes in the airway subepithelium of atopic asthmatic patients 24 hours after allergen challenge (n = 6) compared with sham-challenged asthmatic subjects (n = 6). The horizontal bars indicate the mean value for each group (419 ± 77 SE, 3560 ± 349 SE in sham-challenged vs. allergen-challenged). The results are expressed as number of cells per square millimeter of subepithelium. Differences between groups were analyzed using the Mann-Whitney test.

Figure 4

(a–c) Expression of CCR8 by T cells infiltrating the airway of atopic asthmatics. Cryostat sections of bronchial biopsies from a representative healthy subject (a), a representative sham-challenged atopic asthmatic (b), and a representative subject with atopic asthma 24 hours after allergen challenge (c). Double immunofluorescent staining with anti-CD3 (green) and anti-CCR8 (red) is shown. Arrows point to double-stained cells (yellow). Original magnification: ×40. e, airway epithelium. (d) Individual counts of CCR8-expressing T lymphocytes in the airway subepithelium of atopic asthmatic patients 24 hours after allergen challenge (n = 6) compared with sham-challenged asthmatic subjects (n = 6). The horizontal bars indicate the mean value for each group (269 ± 25 SE and 984 ± 84 SE in sham-challenged vs. allergen-challenged). The results are expressed as number of cells per square millimeter of subepithelium. Differences between groups were analyzed using the Mann-Whitney test.

Figure 6

Upregulation of MDC and TARC expression on airway epithelial cells of allergen-challenged atopic asthmatics. Cryostat sections of bronchial biopsies from a representative healthy subject (a and d), a representative sham-challenged atopic asthmatic (b and e), and a representative subject with allergic asthma 24 hours after allergen challenge (c and f). Immunofluorescent staining with anti-MDC (a–c) and anti-TARC (d–f) is shown. Original magnification: ×40. e, airway epithelium.

T cells expressing CCR8 were also increased in bronchial biopsies from challenged asthmatics as compared with sham-challenged asthmatics (1070 [729–1160] vs. 251 [229–326], respectively; P = 0.0022) and were undetectable in healthy controls (Figure 4). Double immunofluorescence staining with anti-CCR4 and anti-CCR8 antibodies revealed that CCR8⁺ Th2 cells represent a small subset (28%) of the CCR4⁺ Th2 population (Figure 5). Our data indicate that 24 hours after allergen challenge, the central airways of atopic asthmatics are infiltrated by Th2 cells that express CCR4, by a smaller population of Th2 cells coexpressing CCR4 and CCR8, and by a few eosinophils expressing CCR3. The maximal decrease in FEV₁ during LAR positively correlated with the number of CCR8⁺ cells (r² = 0.7361; P = 0.0288) but not of CCR4⁺ cells (r² = 0.2732; P = 0.2874) infiltrating the airway mucosa.

Figure 5

Coexpression of CCR4 and CCR8 by infiltrating T cells (a and b) and airway epithelial cells (a) of atopic asthmatics. Double immunofluorescent staining with anti-CCR8 (green) and anti-CCR4 (red) on a bronchial biopsy from a representative subject with atopic asthma 24 hours after allergen challenge. Arrows indicate double positive T lymphocytes (yellow). Original magnification: ×40. e, airway epithelium.

MDC and TARC are upregulated in airway epithelium after allergen challenge. On the same biopsies we also examined the expression of MDC and TARC. Both MDC and TARC expression was exclusively detected as a cytoplasmic staining in airway epithelial cells (Figure 6). While we did not observe any difference in the expression of MDC and TARC between healthy controls and sham-challenged asthmatics, their expression on airway epithelial cells was strongly upregulated upon allergen challenge.

Airway CD3⁺ cells of COPD and pulmonary sarcoidosis express CXCR3 but not CCR4. To evaluate the disease specificity of the CCR4/CCR8 expression pattern, we analyzed chemokine receptor expression in bronchial biopsies from patients with COPD and pulmonary sarcoidosis. COPD is a nonatopic inflammatory process of the airway mucosa and lung parenchyma resulting in a chronic airflow limitation (31). Double immunofluorescent analysis with anti–IFN-γ/anti-CD3, anti–IL-4/anti-CD3, anti-CCR4/anti-CD3, anti-CCR8/anti-CD3 (data not shown), and anti–IFN-γ/anti-CXCR3 or anti–IL-4/anti-CCR4 (Figure 7) antibodies was performed on bronchial biopsies from six allergen-challenged asthmatic patients and six patients with COPD, and on transbronchial biopsies from two patients with active pulmonary sarcoidosis. In contrast to asthma patients (Figure 7, a and d), T cells infiltrating the airways of COPD patients produce IFN-γ and express the CXCR3 chemokine receptor, while lacking expression of IL-4, CCR4 (Figure 7, b and e), and CCR8 (data not shown). IL-4, CCR4, and CCR8 expressions were also not detected on lung T cells from pulmonary sarcoidosis (Figure 7c and data not shown), a disease characterized by a typical cell-mediated Th1-type inflammatory response (28, 32–34). The Th1 nature of T cells in pulmonary sarcoidosis was confirmed by IFN-γ expression. Furthermore, Th1 cells surrounding the granuloma express CXCR3 (Figure 7f).

Figure 7

Coexpression of CCR4/IL-4 and CXCR3/IFN-γ by lymphocytes infiltrating the airway in allergic asthma, COPD, and pulmonary sarcoidosis. Cryostat sections of bronchial biopsies from a representative subject with allergic asthma 24 hours after allergen challenge (a and d), and from a representative subject with COPD (b and e). Cryostat sections of transbronchial biopsy from a representative subject with pulmonary sarcoidosis (c and f). Double immunofluorescent staining with anti-CCR4 (red) and anti–IL-4 (green) (a–c), and with anti-CXCR3 (green) and anti–IFN-γ (red) (d–f), is shown. Arrows point to double positive T lymphocytes. Original magnification: ×40. e, bronchial epithelium.

In this study we found that the T cells infiltrating the airway mucosa of atopic asthmatics have a Th2 phenotype, that they express the chemokine receptors CCR4 and, to a lesser extent, CCR8, and that their number increases after allergen challenge. Interestingly, we found a correlation between the number of CCR8⁺ Th2 cells and the severity of the asthmatic response to allergen challenge. Finally, we observed that MDC and TARC are expressed by airway epithelial cells of healthy controls and stable asthmatics. Furthermore, their expression is strongly upregulated after allergen challenge.

Effector T helper cells are polarized with respect to their chemokine receptor expression as well as their cytokine production, and in vitro polarized Th2 cells preferentially express CCR3, CCR4, and CCR8. Our studies demonstrate that after allergen challenge, virtually all T cells infiltrating the airways produce IL-4 and express CCR4 but not CCR3. The expression of CCR4 was paralleled by strong expression of its ligands MDC and TARC in the airway epithelial cells after allergen challenge, suggesting that the CCR4/ligands axis may be involved in the Th2 cell recruitment. Recent evidence indicates that IL-4 in combination with TNF-α upregulates TARC production by an airway epithelial cell line (35). Thus, it is tempting to speculate that the CCR4 ligands induced by IL-4 in Th2 diseases such as asthma chemoattract CCR4⁺ T cells, which in turn are induced to express more IL-4, establishing a mechanism for amplifying Th2 responses (36). Collectively, these data support the role of the CCR4/ligands axis in the pathogenesis of inflammation and tissue damage in asthma. Studies in a mouse model of antigen-induced airway inflammation have indeed provided evidence for a crucial role of the CCR4/MDC and CCR4/TARC axes in the recruitment of Th2 cells into the lung (37, 38). Repeated allergen challenge resulted in an increased frequency of CCR4-expressing Th2 cells in the inflamed airways, and anti-MDC as well as anti-TARC antibodies suppressed cell influx and airway hyperresponsiveness (38, 39). Surprisingly, CCR4-deficient mice are still capable of developing allergic airway inflammation (40). Since CCR4 and its ligands have been implicated in thymocyte development (41), it is possible that CCR4-deficient T cells from gene-ablated mice upregulate some compensatory mechanisms. Recent data concerning the upregulation of CCR4 in vivo have also been obtained in atopic dermatitis (AD) patients (42). AD is a disease in which the lymphocytes invading the skin are, at least in the initial phase, Th2-type cells. Expression of the CCR4 ligand MDC was found in both dendritic cells and Th2 cells in the skin of AD patients (43). Furthermore, in a mouse model of bacteria-induced fulminant hepatitis, in which Th2 cells are the cause of liver injury after LPS administration, CCR4 was found preferentially expressed in IL-4–producing Th2 cells in vivo. Neutralization of the CCR4 ligand TARC decreased IL-4 production by liver-infiltrating T cells (44).

CCR8 was expressed, after allergen challenge, in approximately 28% of the CCR4/IL-4 double positive airway T cells. To the best of our knowledge this is the first report of CCR8 expression on T cells in a human pathology. The functional role of this T cell population is currently unclear; however, it was recently reported that the deletion of CCR8 attenuates eosinophil accumulation, Th2 cytokine production, and airway hyperresponsiveness in a cockroach allergen model of asthma (45). Interestingly, we found that the number of CCR8⁺ Th2 cells infiltrating the airway mucosa in challenged asthmatics correlates with the degree of airflow limitation during LAR. The precise mechanism by which CCR8⁺ Th2 cells are linked to airflow limitation needs to be further investigated both in vitro and in vivo. However, our data suggest that a CCR8 selectively oriented recruitment of Th2 cells into the airways may be involved in the development of airflow limitation after allergen exposure. Surprisingly, we could not find expression of the CCR8-specific ligand I-309 either in the airway mucosa or in epithelial cells. TARC, whose expression is strongly upregulated by airway epithelial cells after allergen challenge, could serve, in vivo, as a ligand for CCR8, although this possibility is debatable (46, 47). An additional possibility is that CCR8⁺ cells in the airway mucosa respond to a yet unidentified ligand.

In the present study of bronchial biopsies from atopic asthmatics we show that CCR3 is not expressed by airway-infiltrating T cells either before or after allergen challenge. These data are in agreement with previous observations reporting in vivo expression of CCR3 on eosinophils and basophils but not on T cells in the skin of patients with systemic sclerosis (48). Interestingly, in a mouse model of airway hyperresponsiveness, expression of CCR3 was transient and was found only during the initial aeroallergen challenge (37). This may be explained by the observation that while in the mouse CCR3 is upregulated by anti-CD3 stimulation, in human T cells TCR-mediated activation results in downregulation of CCR3 (49).

The comparative analysis of chemokine receptor expression in a different airway disease, such as COPD, clearly indicated that CCR4 expression is specific to asthma. COPD is a disease characterized by the progressive development of airflow limitation associated with a chronic inflammatory process that differs from that seen in asthma, with different mediators, inflammatory effects, and response to treatment (31). In contrast to asthma, T cells in the bronchial mucosa of COPD patients predominantly express IFN-γ and CXCR3 but do not express IL-4 and CCR4. The lack of IL-4 expression by infiltrating T cells confirms the nonatopic nature of COPD, while the expression of CXCR3 is in agreement with a number of published in vitro and in vivo studies in which CXCR3 was consistently found associated with Th1-type responses (5, 6, 50). Similarly, in sarcoidosis, a disease characterized by a typical cell-mediated Th1-type inflammatory response (25, 28, 32–34), we describe that lung T cells express IFN-γ and CXCR3 but not IL-4 and CCR4. These results are consistent with a previous report documenting CXCR3 expression on CD4⁺ T cells from the bronchoalveolar lavage of sarcoidosis patients (51).

In conclusion, we have demonstrated that upon allergen challenge, CCR4 and to a lesser extent CCR8 are strongly expressed by T cells in airway mucosa, and their ligands TARC and MDC are upregulated in airway epithelial cells of atopic asthmatics. The coexpression of these chemokines and their receptors indicate their involvement in the regulation of lymphocyte recruitment into the asthmatic lung, suggesting that neutralization of this pathway in vivo may modify T cell migration into the airways and the consequent disease progression.

We thank Maura Francolini for help with the confocal analysis, Lorenzo Corbetta and Franco Ravenna for performing bronchoscopies, and Daniele D’Ambrosio and Luciano Adorini for the critical reading of this manuscript. The work was supported by grants from the Italian Ministry of University and Research (MURST), Associazione per la Ricerca e la Cura dell’Asma, and Consorzio Ferrararicerche.

Paola Panina-Bordignon and Alberto Papi contributed equally to this work.

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