We found that, in contrast to our initial hypothesis, CCR5 Δ32 carriers had significantly higher lung CT scores as compared with wt/wt individuals. Stratification on CCL5 G-403A genotype which was not related to disease phenotypes alone, did not modify the associations of CCR5 with CT score. Interestingly, we found the CX3CR1 1249 allele was associated with lower 1999 pneumoconiosis prevalence, the effect being evident in miners with high dust exposure. Further, CX3CR1 1249 and CCR5 Δ32 carriers had lower CT score in 1990, slower progression in score between 1990 and 1994, and the proportion of miners with pneumoconiosis in 1999 in each group was consistent with these results.
This study is the first to consider simultaneously CCR5 and CCL5, CCR2 and CCL2, and CX3CR1 polymorphisms in an inflammatory lung disease. A strength of the study was the functional relevance of each SNP under study. Observed genotype and allele frequencies were similar to those reported previously in other Caucasians populations [5–7, 16, 17, 37, 38], with a higher frequency of subjects homozygous for the CX3CR1 M280 allele observed in our study. Other strengths of the study design include the availability of a quantitative validated phenotype measured twice 4 years apart and objective measurements of coal dust exposure, the main cause of pneumoconiosis. A limitation of the study was the relatively small sample size, which precludes detailed analyses to address simultaneously all genetic factors. Although Bonferroni correction for the independent comparisons on the main outcome (CT score in 1990) did not remove the statistical significance of the association with CCR5 Δ32, and the internal coherence of the results with other outcomes support the findings, the limited sample size imposes caution in the interpretation of the results. Further, no family-based data were available, and replication in other studies is warranted. The association of CCR5 Δ32, alone and after stratification on CCL5 G–403A, with more fibrosis measured by CT score, agrees with the association previously found with sarcoidosis . In contrast, Spagnolo et al.  recently found no association of CCR5 haplotypes (including the CCR5 Δ32 insertion/deletion) with susceptibility to sarcoidosis, and CCR5 Δ32 has been associated with protection against asthma . Distinct biological pathways during disease pathogenesis, and particularly the fibrotic process may partly account for the difference. As a loss of CCR5 is associated with macrophage dysfunction, we hypothetized that CCR5 Δ32 carriers could have impaired their cleaning capacity, leading to an increase in lung fibrosis. Further, association of CCR5 Δ32 SNP with fibrosis remains after stratification on CCL5 –403 genotype. The lack of clear association between the CCL5 –403 SNP and pneumoconiosis phenotypes and prevalence in our study was not surprising. Only one subject was homozygous for CCR5 Δ32, and heterozygosity in CCR5 Δ32 results in only a 50% decrease of CCR5 molecule expression on the cell suface . Therefore, we were unable to completely evaluate the association of the CCL5 –403 SNP with pneumoconiosis according to the CCR5 Δ32 polymorphism.
We also found no association of CCR2 V64I and CCL2 A–2578G SNPs with pneumoconiosis phenotypes and prevalence, in contrast to results previously reported for sarcoidosis and asthma [8,17]. Recently, Valentonyte et al.  found no association between CCR2 gene polymorphisms, including the V64I SNP, and the risk of sarcoidosis in 1203 patients and their relatives. However, they found positive linkage results in the 3p21 chromosomal region, suggesting a susceptibility gene in this location. In mice, contrasting effects of Ccr5 and Ccr2 deficiency on pulmonary inflammatory response to influenza A virus have been reported , and targeted deletion of Ccr3 (Ccr3−/−) were found to have enhanced hyper-reactivity in an airway inflammation model . Further, whereas the hypothesis that inactivation of CCR2 or CCR5 would ameliorate rheumatoid arthritis, it was shown in murine models that Ccr5 null mice phenotype was similar to wild type and that collagen-induced arthritis phenotype of Ccr2 null mice mimicked that of human disease . Pneumoconiosis is another collagen-related disease, and all these results suggest that the complex network of the chemokine system needs to be evaluated in detail, as recently shown in the study of Ferreira et al. .
Within the CCR cluster, we previously identified two common SNPs in the open reading frame of the CX3CR1 gene (V249I and T280M)  that associated with reduced risk of cardiovascular diseases [35,46,47]. Similarly, the present study found that the CX3CR1 I249 allele was associated with reduced pneumoconiosis prevalence. Our study is the first to evaluate the role of polymorphisms in CX3CR1 in an inflammatory lung disease. Fractalkine, the ligand of CX3CR1, is constitutively expressed in pulmonary endothelial and epithelial cells . We hypothesize that fractalkine may be the primary signal allowing capture of CX3CR1-expressing inflammatory cells, and that CX3CR1 I249 variant, associated with enhanced adhesiveness , may decrease the extravasation of monocytes, leading to attenuation of airway inflammation. Association of CX3CR1 SNPs with reduced pneumoconiosis prevalence was more evident in miners with high exposure, i.e. in those having the higher recruitment of inflammatory cells in their airways. We hypothesize that the CCR5 axis may promote the migration of monocytes through the lung, and that both pairs of chemokines and their receptors may act sequentially or simultaneously to allow robust migration and fine positioning of cells expressing both chemokine receptors.
In summary, our findings suggest that chemokine receptors CCR5 and CX3CR1 may be involved in the development of pneumoconiosis, an inflammatory and fibrotic lung disorder. Further, association of the CX3CR1 I249 allele with CT score and pneumoconiosis prevalence was more evident in miners with high cumulative exposure or in CCR5 Δ32 carriers, suggesting that interactions of chemokine receptor polymorphism with coal dust exposure (gene X environment) and between polymorphisms (gene X gene) may control disease susceptibility and progression. Our results also suggest the importance of considering simultaneously genetic variations in several chemokine receptors and balance with their ligands, and of combining gene with environmental parameters to better understand the aetiology of inflammatory diseases.
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