aDepartment of Morphology and Embryology, Human Anatomy Section, University of Ferrara, 44100 Ferrara, Italy; and bInstitute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, MD 21201-1192, USA.
Sponsorship: This research was supported by `AIDS project' of the Italian Ministry of Health, and University of Ferrara local funds.
Received: 16 November 1999; accepted: 2 December 1999.
As well as its primary role in regulating the homing and retention of leukocytes in hematopoietic and lymphoid tissues, stromal-derived factor 1-alpha (SDF-1α), the high affinity ligand of CXCR4, can mediate an apoptotic stimulus in different cell types, including cells of hematopoietic origin [1–3]. In particular, ligation of CXCR4 by SDF-1α or by HIV-1 gp120 can result in the induction of CD8+ T cell apoptosis . Because surface CXCR4 is highly expressed on various cell types, including CD4+ T cells , which represent the main target of HIV-1 infection, we have investigated the effect of SDF-1α on the survival of primary CD4+ T cells.
For this purpose, peripheral blood mononuclear cells were isolated by Ficoll–Hypaque density–gradient centrifugation (Pharmacia, Uppsala, Sweden) of heparinized leukocyte units obtained from healthy adult donors. Highly enriched populations of primary resting CD4+ T cells (> 85% pure) were obtained by immunomagnetic negative selection , followed by overnight adhesion to plastic to eliminate residual monocytes.
Although only a fraction (20–30%) of freshly isolated CD4+ T cells expressed detectable levels of surface CXCR4, virtually all (> 95%) cells showed a bright expression of surface CXCR4 after 24 h of culture. Therefore, to evaluate the effect of SDF-1α on the survival of primary resting CD4+ T cells, these cells were seeded in culture for 24 h to allow the spontaneous upregulation of surface CXCR4 before adding SDF-1α (10 ng/ml; PeproTech, London, UK). Cells were maintained viable and in a resting state by keeping them in RPMI plus 10% fetal bovine serum in the absence of exogenous IL-2 or mitogenic agonists. Under these culture conditions, background levels (< 4%) of apoptosis were noticed up to day 5 of culture. No significant increase of cell death was observed in the presence of SDF-1α (Fig. 1a).
In the next experiments, primary CD4+ T cells were stimulated with anti-CD3 mAb (0.5 μg/ml) plus IL-2 (40 U/ml) for 66 h, washed and then cultured with IL-2 (40 U/ml) plus SDF-1α for additional 5 days. Compared to resting CD4+ T cells, mitogenic stimulation resulted in an abrupt downregulation of surface CXCR4 expression, as evaluated both in terms of number of positive cells and in terms of mean fluorescence intensity, within 48 h of culture (data not shown). The addition of SDF-1α immediately after mitogenic stimulation did not show any significant effect on CD4+ T cell survival (Fig. 1b).
To evaluate whether the lack of apoptosis induction in mitogenically stimulated CD4+ T cells was caused by an intrinsic resistance of primary cells to SDF-1α-mediated apoptosis or the downregulation of surface CXCR4 expression, additional experiments were performed on cells stimulated with anti-CD3 mAb plus IL-2 and then cultured with IL-2 alone for 12 days before adding SDF-1α. Under these culture conditions, CXCR4 was progressively re-expressed on the surface of CD4+ T cells (Fig. 1c). Remarkably, when SDF-1α was added after 12 days of culture with IL-2 alone, a progressive and significant (P < 0.01) increase in apoptosis was noticed in cells prestimulated with anti-CD3 mAb plus IL-2 (Fig. 1d).
CXCR4, which plays a prominent role as a coreceptor for the highly cytopathic T cell line-adapted isolates of HIV-1 , belongs to a family of G-protein-coupled receptors that are involved in the regulation of numerous biological processes, and is selective for a single chemokine, SDF-1. In this study we demonstrated that SDF-1α induces a slow and progressive increase of apoptosis in activated primary CD4+ T cells. Of note, this effect was observed at a relatively low concentration (10 ng/ml) of SDF-1α. The ability of SDF-1α to induce apoptosis in these cells was clearly related to the surface expression of CXCR4. However, primary resting CD4+ T cells were completely refractory to the apoptosis-inducing activity of SDF-1α, in spite of a high density of surface CXCR4. Surface CXCR4 thus cannot transduce an apoptotic signal unless CD4+ T cells are previously induced to proliferate.
As HIV-1 disease is characterized by an initial phase of immune activation , the ability of SDF-1α to induce apoptosis in activated CD4+ T cells may represent an additional mechanism of CD4+ T cell depletion in HIV-1-infected patients.
Maria Luisa Colamussia
1. Herbein G, Mahlknecht U, Batliwalla F. et al. Apoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4. Nature 1998, 395: 189–194.
2. Hesselgesser J, Taub D, Baskar P. et al. Neuronal apoptosis induced by HIV-1 gp120 and the chemokine SDF-1α is mediated by the chemokine receptor CXCR4. Curr Biol 1998, 8: 595–598.
3. Sanchez X, Cousins-Hodges B, Aguillar T, Gosselink P, Lu Z, Navarro J. Activation of HIV-1 coreceptor (CXCR4) mediates myelosuppression. J Biol Chem 1997, 272: 27529–27531.
4. Secchiero P, Zella D, Capitani S, Gallo RC, Zauli G. Immobilized HIV-1 Tat protein upregulates the expression of surface CXCR4 in resting CD4+ T cells. J Immunol 1999, 162: 2427–2431.
5. Moore JP, Trkola A, Dragic T. Co-receptors for HIV-1 entry. Curr Opin Immunol 1997, 9: 551–562.
6. Fauci AS. Multifactorial nature of human immunodeficiency virus disease: implications for therapy. Science 1993, 262: 1011–1018.
© 2000 Lippincott Williams & Wilkins, Inc.