A haplotype of the human CXCR1 gene protective against rapid disease progression in HIV-1+ patients

Abstract

Chemokines and their receptors are key factors in the onset and progression of AIDS. Among them, accumulating evidence strongly indicates the involvement of IL-8 and its receptors, CXCR1 and CXCR2, in AIDS-related conditions. Through extensive investigation of genetic variations of the human CXCR1-CXCR2 locus, we identified a haplotype of the CXCR1 gene (CXCR1-Ha) carrying two nonsynonymous single nucleotide polymorphisms, CXCR1_300 (Met to Arg) in the N terminus extracellular domain and CXCR1_142 (Arg to Cys) in the C terminus intracellular domain. Transfection experiments with CXCR1 cDNAs corresponding to the CXCR1-Ha and the alternative CXCR1-HA haplotype showed reduced expression of CD4 and CXCR4 in CXCR1-Ha cells in human osteosarcoma cells as well as in Jurkat and CEM human T lymphocytes. Furthermore, the efficiency of X4-tropic HIV-1(NL4-3) infection was significantly lower in CXCR1-Ha cells than in CXCR1-HA cells. The results were further confirmed by a series of experiments using six HIV-1 clinical isolates from AIDS patients. A genetic association study was performed by using an HIV-1(+) patient cohort consisting of two subpopulations of AIDS with extreme phenotypes of rapid and slow progression of the disease. The frequency of the CXCR1-Ha allele is markedly less frequent in patients with rapid disease onset than those with slow progression (P = 0.0003). These results provide strong evidence of a protective role of the CXCR1-Ha allele on disease progression in AIDS, probably acting through modulation of CD4 and CXCR4 expression.

Fig. 1.

Physical map of the human CXCR1 and CXCR2 genes. Coding and untranslated regions are indicated by black and gray bars, respectively. The location of polymorphisms with frequencies of >1% is indicated. The asterisk corresponds to polymorphisms that are in strong LD. A horizontal line below each gene shows the regions that have been sequenced.

Fig. 2.

Expression of HIV receptor/coreceptor on HOS cells (A–C) or Jurkat cells (D–F). (A) Flow cytometric analysis of HIV receptor CD4 and coreceptor CXCR4. (B and C) Western blot analysis (B) and RT-PCR analysis (C) of HIV receptor CD4, and coreceptors CXCR4 and CCR5. (D) Flow cytometric analysis of HIV receptor CD4. (E and F) Western blot analysis (E) and RT-PCR analysis (F) of HIV receptor CD4. A representative result from three independent experiments is shown.

Fig. 3.

CXCR1 expression and cellular responses to its cognate ligand IL-8 by using HOS cells. (A) Flow cytometric analysis of CXCR1 expression. (B) Intracellular Ca²⁺ mobilization. (C) Receptor internalization. (D) Chemotactic activity. (E) Confocal fluorescence microscopic images showing the subcellular distribution of CD4 (left column, green) and CXCR1-HA or CXCR1-Ha (middle column, red) on each transfected HOS cells. Overlaid green and red images show the colocalization between CD4 and CXCR1 (right column, yellow).

Fig. 4.

The efficiency of X4-tropic HIV infection in HOS cells transfectants by using HIV-1_NL4–3 strain (A), S.I. clinical isolates (B), and N.S.I. clinical isolates (C). S.I. represents an isolate with CXCR4 predominance, and N.S.I. represents and isolate with CCR5 predominance. Red, blue, and black lines show the means from three three-replicate assays for CXCR1-Ha (red lines), CXCR1-HA (blue lines) and control cells transfected with empty vector (black lines). Vertical bars indicate the range of the results obtained for each set of measurements. The amount of p24 protein in culture medium is indicated as ng/ml. Differences between CXCR1-Ha and CXCR1-HA for day 8 are significant in all instances as evaluated by a t test: NL, P = 0.002; S.I.-1, P = 0.001; S.I.-2, P = 0.01; S.I.3, P = 0.008; N.S.I.-1, P = 0.003; N.S.I.-2, P = 0.006; N.S.I.-3, P = 0.03.