Role of the Chemokine Receptors CXCR3 and CCR4 in Human Pulmonary Fibrosis

    Allergy and Immunology Unit, Rehabilitative Pneumology Department, and Radiology Unit, Scientific Institute of Pavia
    Pneumology Department, Scientific Institute of Gussago, Fondazione "Salvatore Maugeri"
    Clinic of Respiratory Diseases, University of Pavia, Policlinico San Matteo, IRCCS, Pavia, Italy

    ABSTRACT

    Rationale: The chemokine receptors CXCR3 and CCR4 have recently been described as playing a pivotal role in the mouse model of bleomycin-induced fibrosis.

    Objectives: To evaluate the role of these receptors in human idiopathic pulmonary fibrosis (IPF).

    Methods: We studied 57 patients: 18 with IPF, 17 with non-IPF (nIPF), 12 with sarcoidosis, and 10 healthy control subjects.

    Measurements: We evaluated the expression of CXCR3 and CCR4 in blood and bronchoalveolar lavage (BAL) T lymphocytes by flow cytometry and the chemokine CXCL10, CXCL11 and CCL17 BAL concentration by singular immunoassay.

    Main Results: Patients with IPF had a significantly lower CXCR3 and a higher CCR4 expression on BAL CD4 T cells compared with the other groups. Among patients with IPF, those treated with corticosteroids exhibited higher CXCR3 and lower CCR4 expression compared with untreated patients. CXCR3 expression correlated with BAL lymphocytes and CCR4 with BAL neutrophils and eosinophils. CXCL10 levels correlated with the expression of CXCR3 on BAL CD4 cells. CXCL11 was undetectable in almost all patients, whereas CCL17 was primarily detectable in patients with IPF. The percentage of BAL CCR4CD4 cells negatively correlated with DLCO. The changes in the total lung capacity, VC, and of the alveolar–arterial PO2 gradient in patients with IPF and those with nIPF 6 to 12 mo after the first evaluation were associated with CD4CXCR3 percentage on BAL cells.

    Conclusions: We found an imbalance in CXCR3/CCR4 expression on BAL CD4 lymphocytes and reduced CXCL10 BAL levels in patients with IPF, suggesting a pivotal role of these molecules in IPF.

    Key Words: chemokines  CXCL10  interstitial lung diseases

    Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrosing lung disease characterized by the remodeling of the lung parenchyma and collagen deposition (1), with an increasing prevalence in Western countries (2).

    The pathogenesis of the disease still remains unknown, despite many efforts toward understanding the mechanisms leading to its development. The role of inflammation has currently been revised, from major to minor component of the pathogenesis of the disease, favoring studies on matrix production and matrix deposition (3). In this view, the inflammatory process might only be the result of the microenvironment that has been generated in the alveolar spaces of lungs in patients with IPF (4). The presence of a predominantly T-helper type 2 (Th2) background in the lung appears to favor the development of the disease both through the induction of profibrotic events and through the inhibition of Th1 cytokine production with antifibrotic properties (5, 6). The Th1/Th2 balance can be evaluated primarily by the detection of the specific cytokines or through the evaluation of chemokine receptors on T cells: CXCR3 or CCR5 for Th1 and CCR3, CCR4, and CCR8 for Th2 cells (7). The evaluation of these chemokine receptors on alveolar lymphocytes obtained from bronchoalveolar lavage (BAL) or pulmonary biopsies allow the characterization of the immune response in other airway disorders, such as asthma (8, 9) and chronic obstructive pulmonary disease (COPD) (10) or interstitial lung diseases, including sarcoidosis (11), eosinophilic pneumonia (12), and Sjgren syndrome (13).

    Recent data shed new light on the mechanisms of IPF, showing a nonredundant role for CXCR3 in limiting the development of fibrosis in the bleomycin mouse model of the disease (14) and a pivotal role of CXCL11 (IFN-inducible T-cell -chemoattractant, I-TAC) (15), and CXCL10 (IFN-–induced protein, IP-10) (16), the agonist chemokines of the CXCR3 receptor. On the other hand, Belperio and colleagues (17) demonstrated that the chemokines CCL17 (thymus- and activation-regulated chemokine ) and CCL22 (macrophage-derived chemokine ), the agonists of the CCR4 receptor, are also involved in the development of pulmonary fibrosis in bleomycin-treated mice. Considering the emerging role of the chemokine receptors in the mechanisms of pulmonary fibrosis, this study aimed to evaluate CXCR3 and CCR4 on BAL and peripheral blood T lymphocytes of patients with IPF and patients with other interstitial lung diseases.

    Some of the results of this study have been previously reported in the form of an abstract (18).

    METHODS

    A more detailed description of the methods is available in the online supplement.

    Patients

    We enrolled 57 patients: 18 with IPF, 17 with diffuse parenchymal lung diseases other than IPF (nIPF), 12 with sarcoidosis, and 10 control subjects with normal radiologic/functional data. Patients' characteristics are shown in Table 1. Eight of 18 patients received a diagnosis of IPF without lung biopsies according to American Thoracic Society (ATS)/European Respiratory Society (ERS) diagnostic criteria (19). Among the patients with nIPF, five were affected by progressive systemic sclerosis with pulmonary involvement (20), six by pulmonary asbestosis (21), three by cryptogenic organizing pneumonia, and three by nonspecific interstitial pneumonia (19). Sarcoidosis diagnosis was made according to the ATS/ERS/World Association of Sarcoidosis and Other Granulomatous Disorders joint statement (22). Five patients had stage I, six had stage II, and one had stage III disease. A histologic diagnosis was obtained in 10 patients with IPF, four patients with nIPF, and seven patients with sarcoidosis. Patients with immunosuppressive drugs other than corticosteroids, and with neoplastic or systemic infectious diseases at the diagnosis, were excluded. Patients gave their informed consent to extra blood drawing during routine venipuncture. The study conformed to the Declaration of Helsinki and was approved by the internal review board of our institute (Fondazione Salvatore Maugeri).

    Lung Function Tests

    All patients underwent spirometry, evaluation of single-breath carbon monoxide diffusing capacity (DLCO), PO2, PCO2, and alveolar–arterial PO2 gradient (a–aPO2). The values of VC, total lung capacity (TLC), FEV1, and DLCO were expressed as percentage of the predicted value (23, 24). A 6-min walk test (6MWT) was performed (25); the walk distance (6MWD) and the oxygen desaturation (desatO2) were evaluated. The clinical and functional progression of the disease was reevaluated in 28 patients (15 with IPF and 13 with nIPF) 6 to 12 mo after the BAL. The difference of VC (VC), TLC (TLC), DLCO (DLCO), a–aPo2 (a–aPo2), 6MWD (6MWD), and desatO2 (desat O2) between the two time points was expressed as percentage of the baseline value.

    High-Resolution Computed Tomography

    High-resolution computed tomography (HRCT) was performed with standard technical parameters, and the Kazerooni score for fibrosis pattern (26) was blindly assigned by a radiologist.

    BAL

    BAL was performed and processed following the ERS guidelines (27). An aliquot of the BAL was microbiologically analyzed in 47 patients.

    Flow Cytometric Evaluation of CXCR3 and CCR4 Expression

    Ethylenediaminetetraacetic acid anticoagulated peripheral blood and BAL cells were incubated with CD4 or CD8 fluorescein isothiocyanate and CXCR3 phycoerythrin or CCR4 phycoerythrin monoclonal antibodies (PharMingen and Becton Dickinson, San Jose, CA). Cells were acquired with a flow cytometer (FacScan; Becton Dickinson), and data were analyzed with the CellQuest software (Becton Dickinson).

    Chemokine Determination

    Levels of CXCL10, CXCL11, and CCL17 were evaluated in undi- luted BAL samples with commercial immunoassays (R&D Systems, Minneapolis, MN) following manufacturer's instructions.

    Statistical Analysis

    Data were expressed as mean ± SD and analyzed using the Mann-Whitney U test. The comparison between blood and BAL expression of CXCR3 and CCR4 in each patient was performed with Wilcoxon matched pairs test. Correlations were assessed with Spearman's rank test; p values less than 0.05 were considered as significant.

    RESULTS

    BAL Cells

    Patients with IPF had a higher amount of neutrophils and eosinophils in BAL fluid compared with patients with nIPF or those with sarcoidosis and control subjects (Table 2). Among the patients with sarcoidosis, six had a high-activity alveolitis (BAL lymphocytes: 30.4 ± 4.4%) and six presented a low-activity alveolitis (BAL lymphocytes: 7.3 ± 4.8%).

    Expression of CXCR3 and CCR4 on T Lymphocytes

    Patients with IPF had a significantly lower expression of CXCR3 and a higher expression of CCR4 on BAL CD4 cells compared with patients with nIPF, patients with sarcoidosis, and control subjects (for CD4 CXCR3, p = 0.0004, vs. nIPF, p = 0.00004, vs. patients with sarcoidosis, p = 0.0003, vs. control subjects; for CD4CCR4, p = 0.0004, vs. nIPF, p = 0.0002, vs. patients with sarcoidosis, p = 0.03, vs. control subjects; Figure 1). As a consequence of the different chemokine receptor expression on CD4 cells among the groups considered, the CXCR3/CCR4 ratio on CD4 cells was lower in patients with IPF compared with patients with nIPF, with patients with sarcoidosis, and with control subjects (CD4CXCR3/CCR4: 1.42 ± 1.23, 3.24 ± 1.96, 4.89 ± 2.54, and 2.14 ± 0.41; p = 0.00008, p = 0.00005, and p = 0.003, respectively). In contrast, the expression of CXCR3 on CD4 peripheral blood of patients with IPF was higher than in patients with nIPF (Figure 1). The expression of CXCR3 and CCR4 on BAL and peripheral blood CD8 lymphocytes was comparable among the groups considered (Figure 1).

    Patients with a positive BAL culture (n = 7) showed no difference in the expression of CXCR3 and CCR4 on BAL and peripheral blood T cells compared with patients with a negative BAL culture (n = 40). No association between CXCR3, CCR4 expression, and the age of the patients was found.

    To better characterize CXCR3- and CCR4-positive cells, we evaluated, in a small number of patients (n = 6: 2 with IPF, 2 with nIPF, and 2 with sarcoidosis), the expression of activation markers both in peripheral blood and in BAL. Peripheral blood and BAL CD4CXCR3- and CD4CCR4-positive cells were all CD45RO positive (> 85%) and CD95 positive (> 80%), and in BAL they were also CD69 positive (> 65%), with different expression of CD25 and HLA-DR (data not shown).

    Effect of Corticosteroid Therapy on the Expression of CXCR3 and CCR4 in Patients with IPF

    When patients were divided according to therapy, we found in corticosteroid-treated patients with IPF a significant increase in CXCR3 and a tendency toward a decrease of CCR4 expression on BAL CD4 lymphocytes (p = 0.006 and p = 0.08, respectively; Figure 2). The same trend was detected on BAL CD8CXCR3 lymphocytes but without any statistical significance, suggesting that steroid therapy tended to recover the normal expression of these receptors. No significant difference in CXCR3 and CCR4 expression was found on peripheral blood T cells between corticosteroid-treated and untreated patients. Considering subjects without corticosteroid treatment, the expression of CXCR3 on CD4 BAL cells correlated with the amount of BAL lymphocytes (r = 0.42, p = 0.006) and CCR4 with the amount of neutrophils and eosinophils in the BAL (r = 0.57, p = 0.0001, and r = 0.40, p = 0.008, respectively).

    Comparison between BAL and Peripheral Blood Expression of CXCR3 and CCR4 on CD4 T Cells

    The expression of CXCR3 was higher in BAL than on peripheral blood CD4 T lymphocytes with the exception of patients with IPF who had comparable expression of CXCR3 in peripheral blood and BAL. Patients with IPF with higher BAL CXCR3 expression compared with blood expression were the corticosteroid-treated patients (Figures 3A–3D). CCR4 expression was higher on BAL CD4 lymphocytes in patients with IPF and in healthy control subjects compared with blood expression (Figures 3E–3H).

    Chemokine Evaluation

    The levels of CXCL10, one of the CXCR3 ligands, were lower in patients with IPF compared with those with nIPF and sarcoidosis (p = 0.0002 and p = 0.04, respectively; Figure 4). Patients with IPF treated with corticosteroids tended to have a higher amount of CXCL10 in BAL fluid (56.2 ± 33.7 pg/ml) compared with untreated patients (32.7 ± 44.1 pg/ml, p > 0.05).

    When we divided patients with sarcoidosis on the basis of higher or lower activity alveolitis (BAL lymphocytes > or < 25%), patients with higher BAL lymphocyte had higher CXCL10 levels (p = 0.003; Figure 4).

    Taking into account all the patients without corticosteroid treatment, CXCL10 BAL levels correlated with the expression of CXCR3 on CD4 BAL lymphocytes (r = 0.48, p = 0.019) and were negatively associated with CCR4 expression on CD4 BAL cells (r = –0.52, p = 0.009; Figures 5 and 6). Furthermore, CXCL10 BAL levels correlated with the amount of total BAL lymphocytes (r = 0.56, p = 0.004). CXCL11, another CXCR3 ligand, was detectable in 0 of 11 patients with IPF, in 2 of 12 patients with nIPF (mean, 60.5 ± 0.7 pg/ml), in 0 of 8 patients with sarcoidosis, and in 0 of 6 control subjects. CCL17, one of the CCR4 ligands, was detectable in 6 of 11 patients with IPF (mean, 30.2 ± 14.2 pg/ml) in 2 of 13 patients with nIPF (mean, 22.0 pg/ml ± 2.8 pg/ml), in 0 of 8 patients with sarcoidosis, and in 0 of 6 control subjects.

    Correlation between CXCR3 and CCR4 Expression and Functional and Radiologic Parameters

    Considering that corticosteroid therapy could modulate the expression of CXCR3 and CCR4 on BAL T lymphocytes, we performed the statistical analysis only on untreated subjects. We found that the DLCO expressed both in absolute value and in percentage negatively correlated with the expression of CCR4 on CD4 BAL T lymphocytes (r = –47, p = 0.003, and r = –0.42, p = 0.0081, respectively; % values in Figure 7). None of the other functional parameters evaluated at the time of enrollment (Table 1) correlated with CXCR3 and CCR4 expression.

    The fibrosis score was higher in patients with IPF compared with patients with nIPF (p = 0.003; Table 1), but no association was found between the fibrosis score and the expression of CXCR3 and CCR4 in patients with IPF and nIPF, whereas CXCL10 BAL levels negatively correlated with the fibrosis score (r = –0.59, p = 0.02).

    In patients with IPF the amount of CCR4 expressed on CD4 BAL T lymphocytes, represented by the mean intensity fluorescence, was associated with patients' disease duration (r = 0.71, p = 0.012).

    Correlation between the Disease Progression and CXCR3 and CCR4 Expression

    Pulmonary function of some patients with IPF and nIPF enrolled in the study was reevaluated 6 to 12 mo after BAL (n = 28). Taking into consideration only the patients without baseline corticosteroid treatment (n = 20), the expression of CXCR3 on BAL CD4 T cells correlated with the difference between the TLC at the two time points (TLC) and with the difference between the VC at the two time points (VC; n = 19; Figures 8A and 8B). Furthermore, the expression of CXCR3 on BAL CD4 cells negatively correlated with the a–aPO2 (a–aPO2; n = 17; Figure 8C).

    No association between DLCO, 6MWT, desat O2, and the expression of CXCR3 or CCR4 on CD4 cells was found.

    DISCUSSION

    Our study presents evidence in support of the role of CXCR3 and CCR4 in human pulmonary fibrosis. We found a lower expression of CXCR3 and a higher expression of CCR4 on CD4 BAL T lymphocytes in patients with IPF compared with patients with nIPF or sarcoidosis and healthy control subjects. Patients with IPF treated with corticosteroids presented higher CD4CXCR3 and lower CD4CCR4 expression than untreated patients. Furthermore, the expression of CCR4 on BAL CD4 lymphocytes negatively correlated with DLCO values and the amount of CCR4 molecules in patients with IPF was associated with the disease duration. Patients with lower CD4CXCR3 BAL expression clinically reevaluated 6 to 12 mo after the BAL had a greater reduction of the TLC, a higher decrease in VC values, and an increase in the a–aPO2.

    The current etiopathogenetic hypothesis for IPF is based on repeated insults in the lung with an abnormal fibrotic response characterized by altered mesenchymal phenotype with matrix production and reduced matrix mobilization (28). What determines this abnormal and fibrotic response to injury is still unknown: a genetic predisposition of patients with IPF to mount an abnormal fibrotic response is the most plausible explanation and many genetic studies are focused on clearly defining this predisposition (29). It seems quite evident from published data that a Th2 microenvironment is present in the lungs of patients with IPF (5, 6). Different methodologies, including in situ hybridization on biopsies, soluble cytokine determinations, or mRNA in the BAL, have been chosen to demonstrate this condition (30–33). Although it has been reported that interleukin 4 (IL-4) induces the synthesis of extracellular matrix proteins (34) and is a chemotactic factor for fibroblasts (35), it is still a question of debate whether the Th2 pattern favors progression of disease in humans. CXCR3 and CCR4 receptors are extensively characterized as Th1 and Th2 markers in numerous articles on basic immunology and also in different clinical contexts. CD4CXCR3- and CD4CCR4-positive cells were previously characterized as activated T cells (36) and we confirmed in a small number of patients that in BAL they express CD45RO, CD95, and CD69 activation molecules.

    The decreased expression of CXCR3 and the increased expression in CCR4 on BAL CD4 T lymphocytes we found in the lungs of patients with IPF clearly reflect the imbalance between Th1 and Th2 in IPF. Differences in CXCR3 and CCR4 expression were also found between patients with IPF and those with nIPF, confirming the different fibrotic patterns presented by the two groups. The increase of CD4CXCR3 cells in the blood of patients with IPF compared with patients with nIPF reflects an accumulation of these cells in the blood and confirms the defect in recruiting them in the lung.

    A completely different pattern of chemokine receptor expression was found in patients with sarcoidosis: high CXCR3 and low CCR4 expression on BAL CD4 lymphocytes, confirming for CXCR3 the previous data of Agostini and colleagues (11).

    The decreased CXCR3 expression on BAL CD4 cells we found in patients with IPF agreed with recent published data demonstrating that CXCR3-deficient mice had increased mortality with progressive interstitial fibrosis compared with wild type after bleomycin-induced fibrosis (14). This suggests that, in an animal model, CXCR3 expression could be protective toward the development of fibrosis. Furthermore, Belperio and colleagues (17) demonstrated that the chemokines CCL17 (TARC) and CCL22 (MDC), the agonists of CCR4 receptor, favor the development of pulmonary fibrosis in bleomycin-treated mice. According to these data, we found an increased CCR4 expression, the receptor of CCL17 and CCL22, in BAL CD4 T cells and a higher number of subjects with detectable levels of CCL17 in patients with IPF compared with the other subjects considered in our study. This confirms that an imbalance between chemokine/chemokine receptors might be a predisposing factor for the development of the disease.

    We also found that patients with IPF treated with corticosteroids had higher CXCR3 and lower CCR4 expression on CD4 BAL lymphocytes, but no effect of corticosteroid treatment was detected on peripheral blood CD4 and CD8 T cells, suggesting an effect of this drug on tissue activated T cells. Kurashima and coworkers (37) found a significant effect of steroid therapy in decreasing CCR4 expression on blood CD4 memory T cells but only in patients with asthma and not in healthy control subjects. The effect of corticosteroid treatment could be directly on the expression of CXCR3/CCR4 or the consequence of the down-modulation of the mRNA for IL-4 and IL-5 and the increase of mRNA for IFN- in BAL lymphocytes (37). Jiang and colleagues demonstrated that adoptive transfer of CXCR3+ leukocytes partially reverses the fibrotic response in CXCR3 knock-out mice 24 h after bleomycin injection (14). Despite the fact that corticosteroid treatment seems to recover the imbalance of CXCR3/CCR4 expression in humans (38), it fails to inhibit the progression of the disease (39). It is plausible that CXCR3-positive T lymphocytes could help in preventing the disease (14) but are useless when the fibrotic process is already established in humans.

    The correlation between CCR4 expression on CD4 cells and patients' disease duration and the negative correlation of the same receptor with DLCO values suggest that this marker could be related to the degeneration of the alveolar structure. These data point to the progressive Th2 switch in patients with IPF and underline the need to diagnose and treat these subjects early. Furthermore, we found that patients with IPF and nIPF with lower CXCR3 on BAL CD4 cells are the ones who decrease their TLC and their VC and increase their a–aPO2 gradient after a 6- to 12-mo follow-up. Considering recent published data showing that changes in physiologic parameters appear to be correlated with the predicted survival of patients with IPF (40) independently from the histologic lung pattern (41–43), our data suggest a protective role of CXCR3 not only in the onset but also in the progression of the fibrosis process, giving a prognostic value to CXCR3 evaluation.

    The decrease in CXCR3 expression on BAL CD4 T cells compared with patients with other pulmonary disorders could be secondary to the lack of chemokine stimuli in the site of lung injury. We found in patients with IPF decreased levels of CXCL10, one of the chemokines that attracts CXCR3-positive cells, and increased levels of CCL17, one of the ligands of CCR4. However, it is quite evident from the high percentage of CD4CXCR3 cells in the BAL of healthy control subjects, who had low levels of CXCL10, that additional mechanisms are involved in the recruitment of CXCR3-positive cells in the lung. The evaluation on BAL of CXCL11, the other agonist of CXCR3, did not provide any other useful information since it was detectable only in two patients with nIPF.

    A great deal of evidence has recently demonstrated that CXCR3 and CXCL10 are involved in the process of angiogenesis (15–44). Tager and colleagues found that CXCL10-deficient mice exhibited increased pulmonary fibrosis after administration of bleomycin, showing that CXCL10 directly inhibits fibroblast recruitment through a mechanism not regulated by CXCR3, as fibroblasts do not express this receptor (16). On the other hand, recent studies show that CXCR3 exerts an angiostatic effect mediated by an alternative splicing of the CXCR3 gene (CXCR3-B) (45). Dysregulated expression of CXC chemokines and their receptors during inflammatory processes might alter the equilibrium between angiostatic and angiogenic processes. Patients with IPF present neovascularization processes in the lung tissue as originally identified by Turner-Warwick (46). Therefore, it is conceivable that the reduction of CXCL10 levels and of CXCR3 expression on CD4 cells found in the BAL of patients with IPF corresponds to an imbalance of the same chemokine/chemokine receptors also in the vascular compartment which might favor the angiogenic activity.

    In conclusion, we found an imbalance in CXCR3/CCR4 expression on BAL CD4 T lymphocytes and reduced CXCL10 BAL levels in patients with IPF compared with those with nIPF or sarcoidosis and healthy control subjects, confirming in humans the relevant role of these molecules as described in animal studies. Taking into account our data, we can speculate that low levels of CXCL10, low expression of CXCR3, and high expression of CCR4 on CD4 BAL cells are characteristics of IPF and could be interesting for the evaluation during the follow-up of patients with IPF. Furthermore, the evaluation of these receptors could also be useful in selecting patients for the treatment with immune modulator drugs such as IFN-1b.

    Acknowledgments

    The immunoassays for the evaluation of chemokine levels were kindly provided by Boehringer Ingelheim Italia.

    FOOTNOTES

    This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

    Originally Published in Press as DOI: 10.1164/rccm.200502-244OC on October 20, 2005

    Conflict of Interest Statement: P.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.B. has been reimbursed by Boehringer Ingelheim Italia for attending several conferences. D.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.-R.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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