INTRODUCTION

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Monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), and "regulated on activation, normal T-cell expressed and secreted" (RANTES) have been identified as being released by alveolar macrophages in sarcoidosis (1). Studies of cytokines and immune regulation in sarcoidosis also suggest that T lymphocytes in affected organs are of the T helper cell type 1 (Th1)-like phenotype, producing interferon- and interleukin-2 (4). There is increasing evidence that chemokines participate in the differentiation of lymphocytes toward the Th1 or Th2 phenotype (5, 6). Transient upregulation of chemokine receptors on leukocytes allows for the selective amplification of either a cell-mediated Th1-type immune response or an allergic Th2-type response.

C-C chemokine receptor 2 (CCR2) is a receptor for MCP-1 and several other close structural homologues of MCP-1 (MCP-2, -3, -4, and -5). CCR2 also acts as coreceptor for human immunodeficiency virus-1 (HIV-1) infection (7). A point mutation in the CCR2 gene that leads to a single, conservative amino acid change, which substitutes isoleucine for valine at position 64 (CCR2-64I), in the first transmembrane domain of CCR2 was found to correlate with significantly delayed progression to acquired immunodeficiency syndrome (AIDS), especially in African populations (8, 9). Accordingly, CCR2-64I is one of the genetic variations that could influence the development of sarcoidosis. We studied the CCR2 polymorphism to see whether this polymorphism is associated with susceptibility to sarcoidosis using a case-control analysis in a Japanese population.

	METHODS

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Study Subjects

A case-control association analysis was carried out for 100 subjects with sarcoidosis and 122 healthy control subjects. Unrelated Japanese subjects with sarcoidosis were recruited from the pulmonary clinic at the First Department of Medicine, Hokkaido University Hospital. Subjects with sarcoidosis were included on the basis of a biopsy showing noncaseating granulomas, and no evidence of mycobacterial or fungal infection or a history of exposure to agents known to cause granulomatous disease. Healthy control subjects, who had requested annual physical examinations, were included if they had no history of sarcoidosis or any other allergic diseases.

Characteristics of the subjects with sarcoidosis and healthy control subjects are presented in Table 1. One hundred subjects with sarcoidosis had been diagnosed for 66.3 ± 51.6 mo (mean ± SD, range 1 to 278 mo) at the time of the study. Eighty-four subjects showed abnormal chest X-ray findings (Stage I, 45; Stage II, 30; and Stage III, 9) at the time of diagnosis, and 13 subjects have been treated with oral corticosteroid.

All subjects in the study gave informed consent for enrollment in the study and for blood drawing.

Genotyping of the CCR2-64I Polymorphism

Genotype was determined by polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) analysis described previously (10). Briefly, genomic DNA was amplified by PCR using primers CCR2-F, 5'-TTG TGG GCA ACA TGa TGG-3' and CCR2-R, 5'-CTG TGA ATA ATT TGC ACA TTG C-3'. The CCR2-F has a cytosine substituted for by an adenine (in lower case). The PCR conditions were as follows: 1 × PCR buffer II (PE Applied Biosystems, Norwalk, CT), 3.0 mM MgCl₂, 100 mM of each deoxyribonucleoside triphosphate (dNTP), 0.6 mM each primer, and 1 U of AmpliTaq Gold DNA polymerase (PE Applied Biosystems). After initial denaturation at 95° C for 10 min, the reactions were cycled 38 times through a temperature profile of 95° C for 30 s, 58° C for 30 s, and 72° C for 30 s. The final extension was performed at 72° C for 10 min. After amplification, PCR reactions were digested with 3 U of BsaBI (New England Biolaboratories Inc., Beverly, MA) at 60° C for 3 h, electrophoresed through 4% NuSieve (3:1) gel (FMC BioProducts, Rockland, ME), and visualized by ethidium bromide staining. CCR2 wild-type and 64I alleles produced 183-bp and 165-bp fragments, respectively.

Statistical Analysis

Pearson chi-square analysis with 2-sided p values was applied to compare the distribution of CCR2-64I between subjects with sarcoidosis and healthy control subjects. A logistic regression model was used to calculate the odds ratio and its 95% confidence interval adjusted for sex with the program Prophet 5.0 (BBN Systems and Technologies, Arlington, VA).

	RESULTS

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Population Study

The frequency of CCR2-64I in healthy control subjects (26.2%) was similar to that reported previously in an Asian population (10). The allelic frequency of CCR2-64I was 12.5% in subjects with sarcoidosis. A decreased frequency of the CCR2-64I allele in sarcoidosis was evident, giving a crude odds ratio of 0.372 (95% confidence interval [CI] 0.208 to 0.666, p = 0.00072). The odds ratio adjusted for sex was also calculated given that females are believed to have a higher risk for sarcoidosis (11), and there was a significant difference in sex between subjects with sarcoidosis and healthy control subjects (Tables 1 and 2). No significant deviation from the Hardy-Weinberg equilibrium was observed in healthy control subjects.

	DISCUSSION

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The expression of sarcoidosis is considered to be the result of the effects of a number of susceptibility genes with inputs from multiple environmental factors. A number of chemokines, including MCP-1, MCP-3, MCP-4, MIP-1, and RANTES, are expressed in the lung of sarcoidosis, and these chemokines could play pivotal roles in the pathogenesis of the inflammatory disease because of their chemotactic activity for T cells and monocytes (1).

In sarcoidosis, it appears likely that genetically predisposed hosts are exposed to unknown antigens that trigger an exaggerated cellular immune response and the formation of granulomas (11). In the search for genes that may confer susceptibility to sarcoidosis, several studies have identified associations with human leukocyte antigen (HLA) regions (12, 13). Examination of other loci, such as those encoding polymorphism in the genes for angiotensin-converting enzyme (14) and tumor necrosis factors (15) also indicates that these genes may be equally important in defining the risk of sarcoidosis.

Our study revealed that the presence of CCR2-64I conferred a lower risk for sarcoidosis. The adjusted odds ratio was 0.369 (95% CI 0.203 to 0.673), suggesting that CCR2 polymorphism was an independent risk factor for the development of sarcoidosis. Interestingly, CCR2/ mice have significant defects in delayed-type hypersensitivity responses, production of Th1-type cytokines (16), and monocyte extravasation (17), which indicates that the presence of CCR2-64I may lead to attenuation of the exaggerated Th1-type immune response to unknown antigens in sarcoidosis.

There are a few examples in which a valine-to-isoleucine substitution can markedly alter the bioactivity of proteins (18). CCR2-64I is, however, considered a conservative change and a priori would not be expected to substantially alter the properties of the protein. No differences were found in MCP-1-induced calcium responses in peripheral blood mononuclear cells (PBMCs) according to the CCR2 genotype (19), and the detailed mechanism by which the point mutation in the CCR2 gene mediates protection against sarcoidosis is currently unknown. Other ligands (MCP-2, -3, -4, and -5) for CCR2 have been identified (1, 3), and functional differences in signaling of these ligands through CCR2 might help to explain in vivo effects of the CCR2-64I. Alternatively, this protection may be through intracellular interactions of variant CCR2 proteins with other chemokine receptors or through linkage disequilibrium with unidentified variants within the CCR gene cluster on chromosome 3p21 (20), such as the recently described polymorphism in the CCR5 gene (21). Actually, a recent study indicated that MCP-1 signaling mediated by CCR2 resulted in heterologous desensitization of both CCR5 and CXCR4 (19).

More than 10% of Caucasians are carriers of a 32-bp deletion in the CCR5 gene, which has been shown to protect from macrophage-tropic HIV infection (22), whereas this deletion is completely absent in African populations (23). Conversely, CCR2-64I is more prevalent and the protective effect of this allele on HIV-1 disease progression is much more significant in African populations (8, 9). These observations suggest basic molecular differences in C-C chemokine biology according to race. Surprisingly, sarcoidosis affects African Americans more commonly and more severely than Caucasians (24) and a higher prevalence of family history of sarcoidosis in African Americans has also been described (25). Given the high prevalence of sarcoidosis in Africans, it is possible that CCR2-64I has different genetic effects according to race. Complex interactions with other genetic and environmental factors that are unique to each race may affect the functional consequences of CCR2 polymorphism (26).

In conclusion, this study identified a significant relationship between CCR2 polymorphism and the risk of sarcoidosis. This opens new prospects for research into the mechanisms underlying sarcoidosis and further studies are needed to define the role of CCR2-64I in the disease.