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IDDM in animal models is T cell mediated and requires the participation of both CD8+ class I MHC-restricted and CD4+ class II MHC-restricted T cells(1). Studies have shown the important roles of several regulatory and proinflammatory cytokines(2). Chemotactic cytokines, or chemokines, are small signaling proteins that are deeply involved in the physiology of acute and chronic inflammatory processes, as well as in the pathologic dysregulations of these processes, by attraction and stimulation of specific subsets of leukocytes(3). Chemokines are 70-90 amino acids in length and are subdivided into two gene families (CC and CXC chemokines) depending on the relative position of the first two conserved cysteines(4). The CXC chemokines predominantly activate neutrophils, whereas the CC chemokines generally activate monocytes, lymphocytes, basophils, and eosinophils(4). Recent studies have shown that the actions of chemokines are mediated by subfamilies of G-protein-coupled receptors(5). Several human CCR have been cloned and characterized recently(6). The genes for the receptors are dispersed in small clusters of genes that have related function and sequence. The genes for CCR2 and CCR5 have been mapped to human chromosome 3p21(7,8). CCR5, which is expressed in monocytes, macrophages, and primary T cells, binds, among others, to RANTES (regulated on activation normal T cell expressed)(8). RANTES is a chemoattractant for monocytes, memory T cells, and eosinophils and induces the release of histamine by basophils. CCR2 binds to monocyte chemotactic protein-1, which is highly effective on CD4+ and CD8+ T cells(9). IL-2 enhances the CCR2 expression and chemotactic responsiveness of CD4+ T cells(10). A common 32-bp deletion mutation in the CCR5 gene (CCR5Δ32), which causes truncation and loss of CCR5 receptors on lymphoid cell surfaces, has recently been described(11). In the CCR2 receptor, a G-to-A nucleotide substitution has been detected at position 190 that substitutes the amino acid residue valine to isoleucine (CCR2-64I), a conservative change located within the first transmembrane domain of the CCR2 receptor(12). The CCR2 and CCR5 loci are tightly linked (17.5 kb apart) on chromosome 3 and the mutant alleles are in strong, perhaps complete, linkage disequilibrium with each other. This means that CCR5Δ32 invariably occurs on a haplotype that is wild-type CCR2, whereas CCR2-64I occurs on a haplotype that contains wild-type CCR5. The CCR5Δ32 mutation is common only in individuals of Caucasian origin, whereas the CCR2-64I mutation is present in all ethnic groups(12,13).

As part of an ongoing study of genetic polymorphisms in children with IDDM(14), we have determined the frequency of the CCR5Δ32 and CCR2-64I alleles in children with IDDM and in nondiabetic subjects.

METHODS

Subjects. One hundred fifteen children with IDDM [age 1-14 (9.3 ± 4.3) y] were investigated. The mean age at onset of IDDM was 5.2 y (range 0.5-13 y). All patients were Hungarian and were treated in the Heim Pál Pediatric Hospital, Budapest, or in the 3rd Department of Internal Medicine, Semmelweis University of Budapest.

We have also determined the frequency of the two mutations in nondiabetic Hungarian children [n = 280, age 1-14 (8.5 ± 4.5) y]. The nondiabetic children were randomly selected from patients in the Heim Pál Pediatric Hospital.

All human studies were approved by an Institutional Review Board.

Protocols. Total genomic DNA was extracted from white blood cells by the method of Miller(15).

Genotyping of CCR5 was performed by DNA amplification by PCR by use of the CCR5-specific primer pair (F: 5′-CTT CAT TAC ACC TGC AGC TCT CA-3′; R: 5′-CAC AGC CCT GTG CCT CTT CTT CTC A-3′) that flanks the 32-bp deletion, separated in 4% agarose gel, and stained with ethidium bromide. The CCR2-64I mutation was determined with a PCR-RFLP assay using a BsaBI site introduced into the PCR primer next to the C-T transition. Amplification with the primers F: 5′-TTG TGG GCA ACA TG a TGG-3′, which has a cytosine substituted with an adenine (in lower case), and R: 5′- GAG CCC ACA ATG GGA GAG TA-3′ generated a 128-bp product. Digestion with BsaBI yields 110 and 18-bp fragments when an isoleucine is present instead of valine at position 64. The products were separated in 4% agarose gel and stained with ethidium bromide(12).

Statistical methods. Allele frequencies were calculated by allele counting. Hardy-Weinberg equilibrium was tested by using a χ2 goodness-of-fit test. Fischer's exact test was used to test for differences in allele distributions between the two groups.

RESULTS

The results are presented in Table 1. There were no significant differences in the frequency of CCR5Δ32 mutation between IDDM and healthy children. The CCR2-64I allele frequency in children with IDDM was 0.226, which differed significantly from the allele frequency in controls (0.114, p = 0.001). The results overall were in Hardy-Weinberg equilibrium.

Table 1 CCR2-64I and CCR5Δ32 genotypes and allelic frequencies of control children and children with IDDM
Full size table

DISCUSSION

The recently characterized CCR5 has rapidly become the object of interest because it has been found to be the major coreceptor on CD4+ cells for primary M-tropic HIV-1 strains(11). The mutant CCR5 allele, which carries the 32-bp deletion, is not expressed on the cell surface. Until now, no obvious physiologic defect has been found associated with the absence of functional CCR5. In the present study, we have found no differences in the frequency of CCR5Δ32 mutations between IDDM children and nondiabetic individuals, indicating that there is no association of the deletion with IDDM.

CCR2 can serve as a minor coreceptor for some strains of HIV-1 in the initial stages of infection. It has not yet been proven whether the 64I mutation impairs the function of the receptor(12) or is just a neutral polymorphism(16). The CCR2-64I allele frequency was significantly higher in children with IDDM than in the control group. The importance of this mutation in IDDM cannot be explained yet, but, because CCR2 mediates the chemotaxis of CD4+ and CD8+ T cells to areas of inflammation(9) and because these cells play important roles in insulitis, a mutation in the CCR2 gene may contribute to the susceptibility of contracting the disease. In animal models, efficient transfer of diabetes is achieved by spleen CD4+ T cells from diabetic and prediabetic donors(17). CD4+ T cells recognize antigens presented by class II major histocompatibility molecules that fail to be expressed by β cells. Thus CD4+ T cells are likely to act through either the release of soluble mediators (e.g. chemokines) or non-T-cellular effectors (e.g. activated macrophages, natural killer cells). Both macrophages and natural killer cells express CCR2 on their surfaces and migrate in response to monocyte chemotactic protein-1(3). The CCR2-64I mutation may influence both the expression and/or the response of the receptor and may contribute to the alteration of the immune system.

An alternative explanation is also possible: The 64I allele may be a marker of a linked mutation through linkage disequilibrium.

According to these results, the CCR2 gene may be a new candidate for the susceptibility locus of IDDM. Because no IDDM locus has been identified near 3p21 until now, further investigations using larger IDDM populations, animals, or cell cultures are urgently needed.