Several factors modulate the infectability of hematopoietic cells by HIV-1 [1–3]. Among the most important are expression of CD4 and virus co-receptors, which mediate virus binding to the cell surface and subsequently facilitate viral particle internalization [2–4]. HIV-1 enters cells after binding to the CD4 protein and one of several chemokine receptors. X4 viruses (lymphotropic) utilize the CXCR4 chemokine receptor and R5 viruses (macrophage tropic) the CCR5 chemokine receptor, as co-receptors for entry [2–4]. The primary targets of HIV-1 infection are lympho/hematopoietic cells (T lymphocytes, macrophages, megakaryocytes and dendritic cells) which express CD4 and one or both of these chemokine co-receptors [5,6].
Evidence is accumulating that in addition to lympho/hematopoietic cells other cell types such as endothelial cells, astrocytes and cardiomyocytes, may also be susceptible to HIV-1 infection, and may be an important source of the virus in chronic infections [7,8]. As these cells do not typically express HIV-related co-receptors on their surfaces, other mechanisms must play a role in their infectability. One possibility is that co-receptors are transferred to these cells by small circular membrane fragments, called membrane derived microparticles (MP), that are shed from the surface of eukaryocytic cells. It has recently been shown that MP derived from monocytes may transfer CCR5 between cells .
An important source of MP are platelets and megakaryocytes. While platelets release MP during their activation, megakaryocytes produce substantial amounts of MP during the physiology of platelet generation . It is also well documented that these cells may be infected by HIV-1 [6,11–14]. We previously reported that megakaryocytes and platelets express abundant quantities of CXCR4 [15,16], and demonstrated that CXCR4 is also highly expressed on MP derived from these cells . We also reported that MP derived from platelets transfer platelet-expressed receptors to other cell types . In this manuscript we demonstrate the ability of platelet and megakaryocytic microparticles (PMP and MegaMP, respectively) to transfer CXCR4 to CXCR4-null targets, and thereby, render these cells susceptible to X4-HIV-1 infection.
Materials and methods
Isolation of human CD34 cells and platelets
Light-density bone marrow mononuclear cells (MNC) were obtained, with informed consent, from healthy donors and then depleted of adherent cells and T lymphocytes (A−T−MNC) and enriched for CD34 cells by immunoaffinity selection with MiniMACS paramagnetic beads (Miltenyi Biotec, Auburn, California, USA) as described previously . The purity of isolated bone marrow CD34 cells was > 97% as determined by flow cytometry. Human peripheral blood platelets were isolated from healthy individuals as described previously . Briefly, blood obtained by venipuncture was anticoagulated with acid citrate dextrose and prostaglandin E1 was added to a final concentration of 1 μM. Platelet-rich plasma was prepared by centrifugation of the whole blood for 20 min at 150 g at room temperature. Platelets were pelleted by centrifugation of platelet-rich plasma at room temperature for 20 min at 800 g. The pellets were resuspended and washed twice in a washing buffer as described before being resuspended in a Hepes buffer, pH 7.5.
Isolation of human erythroblasts
Bone marrow CD34 cells were expanded in a serum-free media as described previously [15,18,19]. Briefly, CD34 A−T−MNC were resuspended in Iscove's DMEM (Gibco BRL, Grand Island, New York, USA) (1 × 104/ml) supplemented with 25% artificial serum containing 1% delipidated, deionized, and charcoal-treated bovine serum albumin, 270 μg/ml iron saturated transferrin, 20 μg/ml insulin and 2 mmol/l l-glutamine (all from Sigma, St. Louis, Missouri, USA). BFU-E growth was stimulated with recombinant human erythropoietin (2 U/ml) and rH kit ligand (10 ng/ml). Cytokines were obtained from R & D Systems (Minneapolis, Minnesota, USA). Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO2. Under these conditions, after 14 days approximately 100% of the expanded cells were glycophorin A (GPA) positive and CD33 and CD41 (αIIbβ3) negative. In our experiments we used erythroid cells expanded for 8–11 days as described above.
PMP were prepared from human platelets as described previously . Prior to use, platelets were activated by thrombin (0.1 U/ml) and collagen (4 μg/ml) for 20 min at 37°C with agitation. After activation, platelets were centrifuged twice at 2000 g for 15 min at 4°C and the PMP-enriched supernatants were collected. Supernatants were again centrifuged at 28 000 g for 1 h at 4°C. The pellets were washed and resuspended in Hepes buffer, pH 7.4. PMP were examined by flow cytometry analysis using phycoerythrin (PE)-conjugated anti-human antibodies against αIIbβ3 (Coulter-Immunotech, Marseille, France), P-selectin (CD62) (Becton Dickinson, San Jose, California, USA), CXCR4, PAR1 (Becton Dickinson Pharmingen, San Diego, California, USA), CD154 (Coulter-Immunotech) and PF4 (Peprotech Inc., Rocky Hill, New Jersey, USA). PMP were fixed in 1% paraformaldehyde prior to analysis using the FACScan (Becton Dickinson).
Bone marrow CD34 cells were cloned in serum-free methylcellulose cultures as described previously [15, 18,20]. Briefly, CD34 A−T−MNC (1 × 104/ml) cultured for 1 h with or without PMP were suspended in Iscove's DMEM (Gibco BRL) supplemented with 25% artificial serum containing 1% delipidated, deionized and charcoal-treated bovine serum albumin, 270 μg/ml iron-saturated transferrin, insulin (20 μg/ml) and 2 mM l-glutamine (Sigma) and cultured in 1% methylcellulose and CFU-Meg growth factor with the recombinant human growth factor thrombopoietin (50 ng/ml; R & D Systems). Cultures were incubated at 37°C in a humidified atmosphere supplemented with 5% CO2. MegaMP were isolated from the supernatants of these cells at day 11–14 by centrifugation as described above for PMP.
HEL, TF-1 and UT-7 cells used in these studies were maintained in RPMI medium (Gibco BRL) supplemented with 10% bovine calf serum (Hyclone, Logan, Utah, USA). U87/CD4 cells were maintained in DMEM (Gibco BRL) supplemented with 10% bovine calf serum and geniticin (Gibco BRL) at a concentration of 0.2 mg/ml. MAGI/CD4 cells were maintained in DMEM also with geniticin and hygromycin B (Roche BMB, Indianapolis, Indiana, USA) at a concentration of 0.1 mg/ml. U87/CD4 and MAGI/CD4 cell lines were obtained from F. Gonzales-Scarano (Department of Neurology, University of Pennsylvania, Philadelphia, Pennsylvania, USA).
FACS analysis of chemokine receptor expression
The expression of CD4, CCR5 and CXCR4 on PMP, MegaMP, and cell lines was evaluated by FACS as described previously . CD4 antigen was detected with PE–anti-CD4 monoclonal (MAb) clones RPA-T4 (BD Pharmingen, San Diego, California, USA). The MAb used for the analysis of chemokine receptor expression were a gift from R & D Systems: clone # 45549.111 (for CCR5) and clone # 12.G5 (for CXCR4). To detect CCR5 we also used PE–anti-CCR5 MAb clone # 2D7 (BD PharMingen). Briefly, the cells were stained in Ca2+- and Mg2+-free physiologic buffered saline (Invitrogen, Carlsbad, California, USA) supplemented with 5% bovine calf serum (Hyclone). Primary MAb were detected with secondary PE-conjugated goat anti-mouse MAb (1 : 100; Sigma). After the final wash, cells were fixed in 1% paraformaldehyde prior to FACS analysis. Analysis was performed using the FACscan (Becton Dickinson). PMP and CFU-Meg were stained for 30 min with appropriate MAb and analyzed without washing.
FACS analysis of MP on target cells
To demonstrate the presence of MP (PMP or MegaMP) on the surface of target cells, cells incubated with MP were stained with PE–anti-CXCR4 or PE–anti-CD41 and analyzed by FACS as described . As negative controls we used PE–goat-anti mouse antibodies. In some experiments PMP or MegaMP were stained with PKH26 as described , and the presence of MP on the surface of the target cells was analyzed by FACS.
T-tropic HIV IIIB was a generous gift of F. Gonzalez-Scarano and dual-tropic DH-12 and M-tropic Ba-L virus was obtained from the University of Pennsylvania Center for AIDS Research core facility. The viral stocks used were prepared using normal peripheral blood lymphocytes stimulated before infection with phytohemagglutinin (1 μg/ml) and interleukin-2 (200 U/ml). Ba-L and DH-12 viral stocks were obtained after infection of normal peripheral blood monocytes. Peripheral blood lymphocytes and peripheral blood monocytes to prepare these stocks were isolated from whole blood units obtained from the New York Blood Center (New York, USA). All viral stocks us were cell free and carefully purified from contaminating viral DNA by incubation with DNase (15 U/ml for 30 min at room temperature). To support this, we did not detect any contaminating viral DNA in our stocks using standard PCR screening tests with both gag and long terminal repeat (LTR) primers.
Infection of different cell lines with HIV-1 strains in the presence of MP
The U87/CD4 and MAGI/CD4 glioblastoma, and hematopoietic HEL, TF-1 and UT-7 cell lines were plated at 1 × 105 cells/well, and subsequently infected at day 0 with IIIB (X4 HIV) or DH-12 (dual-tropic) at a concentration of 10 ng/ml following 12 h preincubation with MegaMP or PMP. The multiplicity of infection for the IIIB virus was 0.02. The cells were subsequently incubated for 12 h at 37°C, 5% CO2 after which they were washed and grown in the presence of DMEM with 10% bovine calf serum. Aliquots of media were collected on day 0, 5 and 10 of culture for p24 ELISA assays. Following removal of the media, DNA was isolated from the cells as described below for PCR analysis.
Infection of human erythroblasts with different HIV-1 strains
Human erythroblastic precursors were obtained and grown as described above. After 11 days of culture following primary isolation, the cells were washed and resuspended in Iscove's medium as described previously  supplemented with erythropoietin and kit ligand, and plated in 48-well plates (5 × 105 cells/well). Cultures were infected with two different HIV-1 strains: T-tropic IIIB and M-tropic Ba-L at a final concentration of 20 ng/mL (all HIV-1 stocks were obtained from F. Gonzales-Scarano) following 12 h preincubation with PMP. After 12 h incubation with viruses, the cultures were washed, grown for an additional 48 h in the presence of fresh media, and then p24 ELISA and DNA isolations were conducted.
Detection of HIV infection
For PCR, cells (> 3 × 104/well), were collected from 96-well plates. To isolate DNA, 20 μl proteinase K (Qiagen, Valencia, California, USA) was added to the bottom of each well after removal of media. Physiologic buffered saline (200 μl) was mixed with the cells, and 200 μl lysis buffer were added. This mixture was heat inactivated for 2 h at 60°C and subsequently boiled for 15 min. DNA was purified using the blood and body fluid protocol of the Qiamp DNA Blood Mini Kit (Qiagen). From a final resuspension volume of 50 μl, 5 μl of each purified DNA sample was used as a template for PCR in the following reaction mixture: 1 × PCR buffer, 1.5 mM MgCl2, 15 mM each dNTP, 1 × Q reagent, 6 μM LTR-plus primer, 6 μM LTR-minus primer, 1 μL PlatinumTag Polymerase (5 U/μl, Invitrogen). In some experiments, primers amplifying within the gag region were used at the same concentrations . The PCR were denatured for 1 min at 94°C and then subjected to the following amplification cycle: denaturation at 94°C (30 sec), annealing at 55°C (60 sec), and extension at 72°C (90 sec) for 35 cycles, followed by a 10 min 72°C extension. The 430-base pair products were visualized on 1.2% agarose gels (Invitrogen). All PCR reagents were obtained from Qiagen unless otherwise noted. For ELISA p24 protein was detected in supernatants collected from HIV-infected cells according to the manufacturer's protocol (NEN, Boston, Massachusetts, USA).
Arithmetic means and standard deviations were calculated on a MacIntosh computer using Instat 1.14 (GraphPad, San Diego, California, USA) software. Data were analyzed using Student's t test for unpaired samples. Statistical significance was defined as P < 0.05.
PMP highly express CXCR4 and can transfer it to CXCR4-null cells
After activation, peripheral blood platelets release PMP, which show much smaller side- and forward-scatter as compared to the platelets (Fig. 1a). PMP express several platelet-derived receptors. We previously demonstrated that platelets express CXCR4 [16,20]. In view of this, we next analyzed the expression of surface proteins on PMP. As shown in Fig. 1b, PMP highly expressed CXCR4 and CD41, which is characteristic for the megakaryocytic lineage, but did not express either the erythroid specific marker GPA, CD4, or CCR5 receptor on their surface (data not shown).
We next examined whether PMP can transfer CXCR4 from platelets to the surface of CXCR4-null cells. To address this we used normal human erythroblasts and the UT-7 cell line, which express CD4, but not CXCR4 [21,23], and have been reported to be non-infectible by X4 HIV . Both normal human erythroblasts (Fig. 2a) and UT-7 cells (Fig. 2b) became CXCR4 positive after incubation with PMP.
Based on these observations we asked whether CXCR4 transferred to normal human erythroblasts or UT-7 cells by PMP would render them susceptible to infection by X4 HIV. Both normal human erythroblasts and UT-7 cells were first co-incubated with PMP for 12 h and then exposed to the IIIB strain of X4 HIV. Fig. 3 shows that normal human erythroblasts that expressed CD4, but neither CXCR4 nor CCR5 , contained IIIB (X4 HIV) provirus after incubation with PMP, as assayed by PCR. More importantly, we also found by using a sensitive p24 ELISA that X4 HIV productively infects erythroblasts; however, the low level of detectable p24 in conditioned media harvested from these cells at day 5 after infection (87 ± 19 pg/ml/106 cells) suggests that this infection was relatively low level. As PMP do not express CCR5, and CCR5 is not constitutively expressed by erythroblasts, these cells remained resistant to infection by Ba-L (R5 HIV). Similarly, UT-7 cells which are CD4+CXCR4-null could also be infected by IIIB (X4 HIV) after being co-incubated with PMP as demonstrated by the presence of X4 HIV p24 protein in supernatants harvested from the infected cells and by the presence of gag DNA sequence (data not shown). As UT-7 cells, in contrast to erythroblasts, express CCR5, they were also able to be infected by Ba-L (R5 HIV). The presence of PMP on the surface of these cells did not affect their susceptibility to Ba-L (data not shown).
MegaMP also highly express CXCR4 and transfer it to CXCR4-null cells
We next examined whether MP secreted by in vitro cultured normal human megakaryocytic cells express CXCR4. We found that MegaMP express CXCR4 and CD41 on their surface, but do not express either erythroid (GPA) or monocytic (CD14) markers (data not shown). Moreover, human hematopoietic cells HEL and TF-1, human glioblastoma cells MAGI and U87 were easily attached by MegaMP labeled with PKH26 (data not shown). We also found that MegaMP, like PMP, adhere to lymphoid cells (data not shown) and express megakaryocyte-derived CXCR4 receptors on their surface.
To evaluate whether MegaMP may render CD4/CXCR4 null cells susceptible to infection by X4 HIV, the cell lines U87, MAGI, HEL and TF-1 cells, were incubated with MegaMP in a 12-h pretreatment regimen. Fig. 4a shows PCR data for DNA isolated from cells at 12 h after HIV addition and with or without pre-incubation with MegaMP. None of the cell lines evaluated in this study could be infected by X4 HIV in the absence of MegaMP pretreatment, but were uniformly PCR positive following MegaMP pretreatment. In addition, cells incubated with MegaMP were also able to be infected by DH12, a dual tropic HIV strain (X4R5 HIV) (Fig. 4b).
U87 cells, pre-incubated with MegaMP, began to die shortly after day 14 of X4-HIV exposure (Fig. 5). In contrast, U87 cells that were not pretreated with MP retained normal morphology and viability upon incubation with HIV. We observed similar morphological changes in MegaMP-pretreated, X4 HIV infected MAGI, HEL and TF-1 cells (data not shown). In addition, the supernatants harvested from these cells displayed detectable levels of p24 protein (Fig. 6) that were statistically (P < 0.0001) elevated when compared to non-MegaMP treated cultures.
The pattern of constitutive expression of chemokine receptors by cells does not always explain their susceptibility to infection by HIV [1,24]. However, the possibility of infection of cells which do not express HIV-entry receptors was observed by several investigators; the molecular mechanisms leading to these infections is not understood [7–9,25–28]. It is now apparent that other mechanisms may play a role in directing the infection of cells that do not constitutively express HIV-1 co-receptors. Evidence is accumulating that HIV entry receptors could be transferred between the cells by small circular membrane fragments (MP) [9,17].
To support this, we have recently demonstrated that the CXCR4 chemokine receptor could be transferred from platelets to the surface of various hematopoietic cells by platelet-derived MP . Similarly, it was also shown that CCR5 chemokine receptors are transferred by monocyte-derived MP to the surface of peripheral blood mononuclear CD4+/CCR5-null cells (homozygous for a 32-base-pair deletion in the CCR5 gene), thus rendering these cells susceptible to infection by R5 HIV . Moreover, it has been postulated that CD4, CCR5 and CXCR4 receptors need not be expressed together on the target cell, but can cooperate in HIV-entry when expressed on neighboring cells in trans . It is very likely that this latter phenomenon could be explained by a MP-mediated receptor transfer between these cells.
In this paper we show that the chemokine coreceptor CXCR4, which is essential for the entry of X4 HIV strains [1,3], may not only be transferred by MP from the surface of platelets and megakaryocytes to the surface of CXCR4 negative cells, but if these recipient cells express CD4, they then become susceptible to infection by X4 HIV.
PMP are a normal component of blood plasma and their concentration in blood increases (from 10 to 500 μg/ml) during various acute and chronic inflammatory processes associated with platelet activation/destruction [14,30–34]. As the plasma level of PMP is elevated in patients suffering from HIV related thrombocytopenia [35,36], transfer of CXCR4 to CXCR4 negative cells may play an important role in advanced stages of HIV, which is characterized by the predominance of X4 HIV. In this way PMP could ‘expand’ the population of CXCR4 cells that could be susceptible to X4 HIV-1 infection. Accordingly, in our study we observed that normal human erythroblasts which express CD4, but neither CXCR4 nor CCR5 , were infected by X4 HIV only after being pre-incubated with PMP. The potential actual contribution of this latter mechanism to the pathogenesis of HIV-related anemia requires further studies.
Another important observation is that MegaPMP may also play a role in increasing the susceptibility of CXCR4 negative marrow cells to HIV infection. Because platelets and megakaryocytes are separated by the blood : bone marrow barrier, MP derived from platelets may play a more important role in the infectability of cells outside the bone marrow (astrocytes and cardiomyocytes), whereas MegaPMP could play an important role in the infectability of cells which reside inside the bone marrow, such as erythroblasts, stromal and endothelial cells. Of note, in HIV related autoimmune thrombocytopenia the number of MP-releasing megakaryocytic cells in the bone marrow cavities increases, as result of a compensatory response to the destruction of platelets [11–13]. Moreover, as MP are derived from various cell types we can not exclude the possibility that CCR5 MP derived from monocytes  or CD4 MP derived from lymphocytes  may also adhere to target cells and render them susceptible to infection by HIV. Thus, cells could be either separately or simultaneously exposed to the platelet-derived MP expressing CXCR4, monocyte-derived MP expressing CCR5  as well as lymphocyte-derived MP expressing CD4 .
Another important mechanism by which PMP could potentially modulate the infectability of cells by HIV is the transfer of CD41 antigen and other platelet-derived adhesion molecules to their surface [17,34]. We demonstrated here that hematopoietic cells exposed to PMP express significant amount of CD41. As the integrin CD41 is a receptor for fibrinogen, cells co-cultured with PMP may become ‘trapped’ in inflammatory areas which are enriched by fibrinogen deposits. This could facilitate the contact of ‘healthy’ hemato/lymphopoietic cells with infected T lymphocytes and macrophages. In addition we recently reported that PMP, which transfer several platelet-derived adhesion molecules to the surface of target cells, increase the homing of early hemato/lymphopoietic cells into the bone marrow . Thus, we hypothesize that infected lymphocytes co-cultured with PMP could home better to the lymph nodes, which might contribute to the spreading of HIV infection to the lymphopoietic organs.
In conclusion we provide evidence that both PMP and MegaMP transfer CXCR4 to the surface of target cells. Such transfer may play an important and under-appreciated role in the infectability of CXCR4-null cells by X4 HIV. The precise mechanism responsible for integration of MP-derived HIV receptors with target cell membrane remain to be elucidated, as does the pleiotropic role of MP derived from different cell types in various aspects of HIV infection.
Sponsorship: Supported by a NIH grant R01 HL61796-01 to MZR and A14083 to GNG.
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Keywords:© 2003 Lippincott Williams & Wilkins, Inc.
platelets; megakaryocytes; microparticles; HIV infection; CXCR4