Expression and functional activity of CXCR-4 and CCR-5 chemokine receptors in human thymocytes

Abstract

In this paper we addressed the expression of the HIV co-receptors CXCR-4 and CCR-5 in human thymocytes by phenotypic, molecular and functional approaches. Cytofluorimetric analysis disclosed that CXCR-4 was constitutively expressed by freshly isolated thymocytes (~10 000 molecules/cell in about 30% of thymocytes); the receptor was endowed with functional activity, as it mediated polarization, migration and intracellular Ca2+ increase in response to its ligand, SDF-1. On the contrary, CCR-5 expression in freshly isolated thymocytes was significantly lower (<4000 molecules/cell in less than 5% of the cells), and no functional response to CCR-5 agonists could be documented. Northern blot analysis of freshly isolated thymocytes showed high CXCR-4 mRNA levels, whereas the message for CCR-5 was barely detectable. On the other hand, a modest increase in the expression of CCR-5 was associated with in vitro thymocyte stimulation, and CCR-5 density at the cell surface attained CXCR-4 figures in most cases. None the less, no functional response to CCR-5 agonists could be documented in in vitro stimulated thymocytes. In vitro infection of thymocytes by CAT-expressing recombinant HIV bearing the envelope glycoproteins from different isolates showed that T-tropic strains, which use CXCR-4 as a co-receptor, were more efficient in infecting thymocytes than M-tropic strains, which preferentially use CCR-5. Altogether, these data indicate that expression of the major co-receptors involved in infection by M-tropic HIV strains is very poor in human thymocytes, and would suggest that thymocyte infection by M-tropic HIV strains may be a rare event in vivo.

Fig. 1

Percentage expression (a) and surface density (b) of CXCR-4 and CCR-5 co-receptors in different thymocyte subsets. (a) The results represent the relative percentage of CD4⁺CD8^–, CD4⁺CD8⁺ and CD4^–CD8⁺ cells also expressing CXCR-4 (hatched columns) or CCR-5 (open columns). Mean values of six consecutive experiments were reported; the bars indicate the s.d. (b) CXCR-4 (hatched columns), CCR-5 (open columns) and CD4 (closed columns) antibody-binding sites (ABS)/cell were calculated in CD4⁺CD8^–, CD4⁺CD8⁺ and CD4^–CD8⁺ thymocytes. Mean values of six consecutive experiments were reported; the bars indicate the s.d. *P = 0·02 compared to CD4⁺CD8^– thymocytes; **P = 0·02 compared to CD4⁺CD8⁺ thymocytes; ***P < 0·01 compared to CD4⁺CD8^– thymocytes.

Fig. 2

Cytofluorographic analysis of CD3 and CXCR-4/CCR-5 expression in CD4^–CD8^– (double-negative) thymocytes. Unfractionated thymocytes were depleted in CD4⁺ and CD8⁺ cells, and the resulting double-negative population was analysed for CD3 and co-receptor expression: (a) anti-CD3 and anti-CXCR-4; (b) anti-CD3 and anti-CCR-5 by two-colour immunofluorescence. One representative experiment of three consecutive experiments is shown; the numbers indicate the percentage of cells included in each section of the plot.

Fig. 2

Cytofluorographic analysis of CD3 and CXCR-4/CCR-5 expression in CD4^–CD8^– (double-negative) thymocytes. Unfractionated thymocytes were depleted in CD4⁺ and CD8⁺ cells, and the resulting double-negative population was analysed for CD3 and co-receptor expression: (a) anti-CD3 and anti-CXCR-4; (b) anti-CD3 and anti-CCR-5 by two-colour immunofluorescence. One representative experiment of three consecutive experiments is shown; the numbers indicate the percentage of cells included in each section of the plot.

Fig. 3

Northern blot analysis of CXCR-4 and CCR-5 mRNA expression in freshly isolated thymocytes. Three thymocyte preparations from different subjects (lanes 1, 2 and 3) were analysed. The CXCR-4 and CCR-5 messages revealed in a representative freshly isolated PBL sample is also shown. Autoradiographs for CCR-5 were obtained after 5-day exposure.

Fig. 4

Response of freshly isolated thymocytes to chemokines in chemotactic (a) and polarization (b) assays. (a) The chemotactic assay was carried out in Transwell system with 5 μm pore filter; optimal concentrations of SDF-1 and MIP-1β were added in the lower filter. Unfractionated freshly isolated thymocytes were added in the upper filter and incubated for 2 h. Transmigrated cells were collected from the lower well and counted. (b) Thymocytes were exposed to SDF-1 and MIP-1β for 15 min, and then fixed. Polarized cells were scored by phase contrast microscopy and identified as cells that changed from a spherical shape to a shape characterized by head–tail polarity. In both assays, zymosan-activated serum was used as a reference chemoattractant. Mean values of four consecutive experiments were reported; the bars indicate the s.d., and the asterisks indicate significative responses (P < 0·001), compared to controls (medium alone).

Fig. 5

Effect of chemokines on intracellular calcium mobilization. Freshly isolated thymocytes were loaded with Fura-2AM, as detailed in Materials and methods, and exposed to MIP-1β (100 ng/ml) or SDF-1 (1000 ng/ml). The flux profile is representative of three consecutive experiments.

Fig. 6

CAT activity in human thymocytes infected with recombinant HIV variants. Left panels: freshly isolated or 4-day cultured thymocytes were infected with recombinant viruses containing envelope glycoproteins of the T-tropic HXBc2 (▪), of the M-tropic ADA (), and of the M-tropic Ba-L (□) isolates. The CAT assay was performed as detailed in Materials and methods; data from one of three representative experiments are presented. Right panels: non-infected thymocytes were analysed in parallel for percentage expression and surface density (ABS/cell) of CD4 (Δ), CXCR-4 (○) and CCR-5 (□).