Human immunodeficiency virus gp120-induced apoptosis of human neuroblastoma cells in the absence of CXCR4 internalization

Abstract

The chemokine receptor CXCR4 functions as human immunodeficiency virus (HIV)-1 coreceptor and is involved in acquired immunodeficiency virus (AIDS) neuropathogenesis. CXCR4 is expressed by most cell types in the brain, including microglia, astrocytes, and neurons. Studies have shown that the HIV envelope protein gp120 binds to neuronal CXCR4 and activates signal transduction pathways leading to apoptosis. However, the natural CXCR4 ligand (CXCL12) has been referred to induce both neuronal survival and death. Here the authors used flow cytometry to determine whether gp120 and CXCL12 differ in their ability to induce CXCR4 internalization in the human neuroblastoma cells SH-SY5Y, which constitutively express CXCR4. As expected, increasing concentration of CXCL12 reduced surface expression of CXCR4 in a time-and concentration-dependent manner. Conversely, gp120IIIB (monomeric or oligomeric, in presence or absence of soluble CD4) did not change CXCR4 membrane levels. Similar results were obtained in a murine lymphocyte cell line (300-19) stably expressing human CXCR4. Nevertheless, gp120IIIB was still able to activate intracellular signaling and proapoptotic pathways, via CXCR4. These results show that gp120IIIB toxicity and signaling do not require CXCR4 internalization in SH-SY5Y cells, and suggest that the viral protein may alter normal CXCR4 trafficking thus, interfering with activation of prosurvival pathways.

Figure 1

HIV gp120 induces cell death in SH-SY5Y cells. SH-SY5Y neuroblastoma cells were serum starved for 48 h and treated with monomeric or oligomeric gp120_IIIB (200 pM), monomeric or oligomeric gp120_IIIB/CD4 complexes (200 pM), or CXCL12 (20 nM) for the following 24 h at 37°C. Two groups of samples were pretreated for 15 min at 37° C with 100 ng/ml of AMD3100 and subsequently treated with vehicle or monomeric gp120_IIIB (200 pM) for 24 h at 37°C. At the end of the treatment, cells were fixed, stained for cleaved caspase 3 (red in A), and counted. The graph shows percentage of dead cells (mean ± SEM) from three independent experiments (B). Six microscopic fields per coverslip were counted and three coverslips per treatment were used for each experiment. (*** P < 0.001 gp120s versus control; ^ P < 0.001, gp120_IIIB versus AMD3100 + gp120_IIIB).

Figure 2

HIV gp120 is unable to induce CXCR4 internalization in SH-SY5Y cells. SH-SY5Y neuroblastoma cells were incubated with different concentrations of CXCL12 and gp120_IIIB (20 pM to 20 nM). The relative cell surface expression of CXCR4 was determined by flow cytometry after staining with PE-conjugated monoclonal anti-human CXCR4 antibody. (A) Representative traces from experiments in which cells were incubated with the two ligands for 60 min. (B) The average of three independent experiments reported as the mean fluorescence intensity of CXCL12-treated (red filled triangle) and gp120_IIIB-treated (open triangles) groups, compared to untreated cells (100%, blue dot). Some of the error bars of gp120_IIIB treated cells are not clearly visible in the graph and thus reported here as well: at 0.02 nM, 99.37% ± 1.26%; at 0.2 nM, 98.63% ± 1.23%; at 2 nM, 97.8% 1.72%; at 20 nM, 98.67% ± 1.30%. (C) Data from time-course experiments (from 30 min to 12-h) after treatment with 200 pM gp120_IIIB (open triange) or 20 nM CXCL12 (filled red triangles). The graph represents the averages of three independent experiments ± SEM. (treatments versus control, ** P < 0.01; *** P < 0.001).

Figure 3

-HIV gp120_IIIB/CD4 complex does not affect CXCR4 expression. (A) CXCR4 cell surface expression on SH-SY5Y cells after incubation for 60 min at 37°C without (control) or with 20 nM of the indicated ligands. Bars represent the averages of two to four independent experiments ± SEM. (B) 300-19/CXCR4 cells were incubated for 60 min at 37°C with increasing concentrations of gp120_IIIB (open triangles) or CXCL12 (red filled triangles). The percentage of receptor expression from two independent experiments as compared to control cells is shown (mean ± SEM) (treatments versus control, * P < 0.05; ** P < 0.01; *** P < 0.001). (C) Representative traces from a FACS analysis experiment in 300-19/CXCR4 cells (60 min at 37°C). A total of three independent experiments with both monomeric and oligomeric gp120_IIIB as well as the CXCL12 control were performed; untreated cells (blue trace), 20 nM monomeric-gp120_IIIB (orange trace), 20 nM oligomeric-gp120_IIIB(green trace), 20 nM CXCL12 (red trace). The gray trace represents the antibody isotype control.

Figure 4

HIV gp120_IIIBsignals through CXCR4 on SH-SY5Y cells. Pretreatment of SH-SY5Y cells with the specific CXCR4 antagonist AMD3100 (100 ng/ml) abolishes CXCL12- (2 nM, 5 min) and gp120_IIIB- (200 pM, 5 min) induced phosphorylation of ERK (A). (B) The densitometry analysis of the bands (mean ± SEM) of three independent experiments (CXCL12 + AMD versus CXCL12,P* < 0.05; gp120_IIIB + AMD versus gp120_IIIB, *** P < 0.001).