Activation of HIV-1 Coreceptor (CXCR4) Mediates Myelosuppression*

Chemokines are cytokines that activate and induce the migration of leukocytes. Stroma-derived factor-1 (SDF-1) is a novel chemokine that blocks the entry of T-tropic HIV-1 mediated by fusin/CXCR4/LESTR (leukocyte-derived seven-transmembrane domain receptor). In this work we demonstrate that SDF-1 triggers increases in intracellular calcium and inhibits the proliferation of myeloid progenitor cell line 32D. By contrast, SDF-1 neither triggers a calcium response nor affects the proliferation of the myeloid progenitor cell line 32D-GR that is deficient in CXCR4. Responsiveness to SDF-1 was rescued by transfection of 32D-GR cells with a cDNA encoding the human CXCR4. The data indicate that SDF-1 induces myelosuppression by activation of CXCR4. The constitutive production of SDF-1 by bone marrow stromal cells argues for a major role of SDF-1 on the regulation of myelopoiesis.

Chemokines are peptides of 70 -100 amino acids secreted by many cell types in response to injury and infection. Two major subfamilies of chemokines are distinguished according to the position of the first two cysteines, the CXC or CC. Chemokines activate and induce migration of leukocytes in a cell-specific fashion (1). SDF-1 1 is a CXC chemokine secreted constitutively from several cell types. Two isoforms of SDF-1 have been identified, ␣ and ␤, which are generated by differential splicing (2). SDF-1 was initially characterized as a pre-B-cell stimulatory factor (3). Recently, SDF-1 has been identified as a highly efficient chemotactic factor for T-cells, monocytes (4), and CD34 ϩ human progenitor cells (5). The constitutive expression of SDF-1 by many tissues has suggested that SDF-1 plays a key role in homing T-cells and monocytes under basal conditions (4). Targeted disruption of the SDF-1 gene in mice was shown to be lethal with severe abnormalities in B-cell lymphopoiesis, bone marrow myelopoiesis, and development of the cardiac ventricular septum (6). The signaling of SDF-1 is mediated by CXCR4/fusin/LESTR (leukocyte-derived seven-transmembrane domain receptor), a G protein-coupled receptor expressed preferentially in leukocytes (4,(7)(8)(9). Recent studies have demonstrated that CXCR4 is the coreceptor for the entry of T-tropic HIV-1 (10) and HIV-2 (11) into CD 4 ϩ and CD 4 Ϫ cells, respectively. Of importance is the observation that SDF-1 blocked the entry of T-tropic HIV-1 strains (7,8). Since SDF-1 is constitutively expressed by bone marrow cells we investigated whether SDF-1 also regulates myelopoiesis. Our data indicate that SDF-1 induces myelosuppression of bone marrow-derived cell line 32D by activation of CXCR4.

EXPERIMENTAL PROCEDURES
Cell Cultures and Transfections-The murine IL-3-dependent 32D and 32D-GR cell lines were kindly provided by Dr. J. Greenberger, University of Pittsburgh Medical School, Pittsburgh, PA. These cell lines were maintained in RPMI 1640 plus 15% heat-inactivated fetal bovine serum and 15% conditioned medium from the murine myelomonocytic cell line WEHI-3B as a source of crude IL-3 (12). Cells were cultured at 37°C in a 5% CO 2 atmosphere and maintained at a cell density of 0.5 ϫ 10 6 cells/ml. 32D-GR cells (10 7 cells/ml) were transfected by electroporation with the human CXCR4 cDNA subcloned into the unique EcoRI site of the expression vector pMEXneo (13). Transfected cells were selected by growing the cells in the presence of 500 g/ml G418. Colonies were scored on days 7 and 14 of culture.
Expression and Purification of SDF-1-To prepare the murine SDF-1 (mSDF-1) proteins, the cDNA fragment encoding mature mSDF-1␣ or mSDF-1␤ was subcloned into the Escherichia coli expression vector pAL781 (14) behind the translation initiation codon ATG. The protein expression was carried out in the E. coli strain GI934 as described (15). Inclusion bodies were harvested, washed sequentially with buffers containing 1 M NaCl and 0.5% Triton X-100, solubilized in 6 M guanidine HCl, and then dialyzed against a pH 6.5 buffer containing 15 mM sodium acetate, 15 mM sodium phosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM p-aminobenzamidine. The refolded mSDF proteins were then further purified from the clarified dialysates by ion-exchange chromatography.
Agar Colony Assays-Cultures of 32D or 32D-GR cells were carried out as described by Metcalf (16). In brief, 300 cells were seeded in 35-mm Petri dishes containing 1 ml of Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated fetal bovine serum, 0.3% agar, and IL-3 or 10% of conditioned medium from the cell line WEHI-3B. SDF-1 resuspended in phosphate-buffered saline or an equal volume of phosphate-buffered saline was added to the empty culture dish prior to the addition of the cell suspension in agar medium. After 7 days of incubation clusters of 50 or more cells were scored as colonies.
Northern Blot Analysis-Total RNA (10 g) was fractionated on denaturating formaldehyde gels and transferred onto nylon membranes. RNA was hybridized with 32 P-labeled cDNA encoding human CXCR4. Blots were washed twice in 0.25 ϫ SSC, 0.1% SDS at 55°C for 30 min and once in 0.1 ϫ SSC, 0.1% SDS at 65°C for 15 min. Blots were exposed to x-ray film at Ϫ80°C for 24 h.

RESULTS AND DISCUSSION
Since SDF-1 is constitutively expressed by bone marrowderived stromal cells (2) we investigated whether SDF-1 plays a role in myelopoiesis. SDF-1-induced intracellular signals in myeloid precursor cells were monitored by increases in the * This work was supported by National Institutes of Health Grant R01 AI 34031. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  level of intracellular Ca 2ϩ . We found that SDF-1␣ or -␤ increased the intracellular Ca 2ϩ in 32D cells (Fig. 1), a nontumorigenic cell line that exhibits features of normal myeloid progenitor cells (16). In contrast to leukemic cell lines, the proliferation of 32D cells requires the presence of IL-3 (18). The Ca 2ϩ response mediated by SDF-1␤ was concentration-dependent, and a maximal response was achieved with 100 nM SDF-1␤ (Fig. 1). Cells treated with SDF-1␣ or -␤ were weakly respon-sive to subsequent additions of SDF-1␣ or -␤ showing homologous desensitization. Other chemokines including IL-8, MIP-2, neutrophil activating peptide-2, platelet factor-4, melanocyte growth stimulatory activity, monocyte chemotactic protein-1, or ATP did not desensitize the Ca 2ϩ response mediated by SDF-1␤. The SDF-1-induced calcium responses in 32D cells were similar to those previously observed with T-cells and monocytes (7). In addition, SDF-1␤ failed to induce calcium responses in 32D cells pretreated with pertussis toxin (data not shown), demonstrating that the SDF-1␤ receptor is coupled to G i proteins. To evaluate the functional role of SDF-1 on myeloid progenitor cells we tested the effect of SDF-1␤ on the proliferation of 32D cells using agar colony assays (16). Like normal hematopoietic progenitor cells, 32D cells form colonies in semisolid culture that are strictly dependent on the presence of IL-3. As shown in Fig. 2, SDF-1␤ inhibited colony formation of 32D cells, suggesting that SDF-1␤ is a myelosuppressor.
To determine whether the calcium responses and suppression of proliferation of 32D cells are mediated by activation of the HIV-1 coreceptor CXCR4 we first explored the expression of CXCR4 in 32D cells by Northern blot analysis. Blots of RNA extracted from 32D cells, Jurkat T cells, and white blood cells were probed with human CXCR4 cDNA. As shown in Fig. 3, all these cells express the mRNA of CXCR4. Second, we found that 32D-GR cells (19), a cell line derived from 32D cells, neither express the mRNA of CXCR4 (Fig. 3) nor exhibit the typical SDF-1␤-dependent Ca 2ϩ response (Fig. 4). However, similarly to 32D cells, 32D-GR cells exhibit the typical ATP-dependent Ca 2ϩ responses (Fig. 4, A and C), form colonies in semisolid culture, and require IL-3 for proliferation. Both 32D and 32D-GR form a similar number of colonies; however, SDF-1␤ failed to inhibit the proliferation of 32D-GR cells (Fig. 4). Finally, responsiveness to SDF-1 was rescued by transfection of 32D-GR with human CXCR4 cDNA. Figs. 2 and 4 show that SDF-1 triggers a calcium response and inhibits proliferation of 32D-GR cells expressing the human CXCR4. These data indicate that SDF-1␤ induces myelosuppression by activation of CXCR4. Previously, several chemokines including IL-8, platelet factor-4, MIP-1␣, and MIP-1␤ have shown myelosuppressive activity in bone marrow myeloid progenitor cells; however, the heterogeneity and low frequency of precursor cells from bone marrow has precluded the identification of receptor systems that mediate the effect of these chemokines (20 -22). It is unlikely that these chemokines mediate their myelosuppressive activity via CXCR4 since they do not activate CXCR4 or desensitize the calcium responses mediated by SDF-1. Probably, these chemokines bind specific receptors. Indeed, we have recently shown the endogenous expression of the murine homolog of the human IL-8 receptor B in 32D cells. 2 The constitutive expression of SDF-1 by bone marrow stromal cells strongly argues for a major role of the HIV-1 coreceptor CXCR4 on myelopoiesis.