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The Journal of Immunology, 2000, 165: 7171-7179.
Copyright © 2000 by The American Association of Immunologists

Tat Protein Is an HIV-1-Encoded ß-Chemokine Homolog That Promotes Migration and Up-Regulates CCR3 Expression on Human Fc{epsilon}RI+ Cells1

Amato de Paulis*, Raffaele De Palma{dagger}, Luisa Di Gioia*, Maria Carfora*, Nella Prevete*, Giovanna Tosi{ddagger}, Roberto S. Accolla{ddagger} and Gianni Marone2,*

* Division of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy; {dagger} Department of Medicine, Second University of Naples, Naples, Italy; {ddagger} Department of Clinical and Biological Sciences, University of Insubria School of Medicine, Varese, Italy; and Advanced Biotechnology Center, Genoa, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. HIV-1 Tat protein is a potent chemoattractant for basophils and lung mast cells obtained from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Tat protein induced a rapid and transient Ca2+ influx in basophils and mast cells, analogous to ß-chemokines. Tat protein neither induced histamine release from human basophils and mast cells nor increased IL-3-stimulated histamine secretion from basophils. The chemotactic activity of Tat protein was blocked by preincubation of Fc{epsilon}RI+ cells with anti-CCR3 Ab. Preincubation of Tat with a mAb anti-Tat (aa 1–86) blocked the migration induced by Tat. In contrast, a mAb specific for the basic region (aa 46–60) did not inhibit the chemotactic effect of Tat protein. Tat protein or eotaxin desensitized basophils to a subsequent challenge with the autologous or the heterologous stimulus. Preincubation of basophils with Tat protein up-regulated the level of CCR3 mRNA and the surface expression of the CCR3 receptor. Tat protein is the first identified HIV-1-encoded ß-chemokine homologue that influences the directional migration of human Fc{epsilon}RI+ cells and the expression of surface receptor CCR3 on these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human immunodeficiency viruses HIV-1 and HIV-2 destroy CD4+ lymphocytes, thus leading to AIDS (1). Entry of HIV-1 into immune cells is mediated by the viral envelope glycoproteins, which are organized into oligomeric, probably trimeric, spikes on the surface of the virion (2). The spike surface consists of gp120 associated by noncovalent interactions with gp41 (2, 3). Entry of HIV-1 viruses into immune cells involves gp120 binding to the CD4 glycoprotein, which serves as the primary receptor (4). CD4 binding induces conformational changes in gp120, involving the exposure and/or formation of a binding site for the specific chemokine receptors CCR5 and CXC chemokine receptor 4 (CXCR4),3 which serve as obligate coreceptors for virus entry (5, 6). Increasing evidence suggests that a subset of primary viruses can use CCR3 as a coreceptor for HIV-1 infection (7, 8, 9, 10). Interaction with coreceptors induces further conformational changes in the envelope protein and exposure of the hydrophobic fusion domain of the transmembrane gp41 subunit, which then mediates fusion of the opposed cell and virus membranes.

Besides the direct cytopathic effect that HIV-1 has on infected cells, several viral products are involved in the profound immunodeficiency in HIV-1-infected patients. HIV-1 Tat, the viral trans-activator protein, has attracted the interest of several research groups. Besides its role in viral gene expression and in replication and control of infection, Tat trans-activates heterologous viral and cellular genes (11), and Tat mutants in the cysteine-rich region down-regulate HLA class II gene expression in macrophages and T cell lines (12). Moreover, Tat protein is secreted by HIV-1-infected cells (13, 14, 15) and can be taken-up by neighboring cells (16) in which it can reactivate latent infection (15). In addition, Tat protein affects the functions of uninfected cells in a paracrine fashion. For example, Tat is chemotactic for human monocytes (17, 18) and monocyte-derived dendritic cells (19). These activities might be mediated by interactions with {alpha}Vß5 integrin (17, 20), Flk-1 tyrosine kinase receptor (21), or binding to chemokine receptors CCR2 and CCR3 (18). There is compelling evidence that Tat protein possesses multiple and distinct binding domains and, consequently, how Tat induces these diverse effects is not clearly understood. However, these findings suggest that extracellular Tat mimics some of the effects of HIV-1 on immune cells and suggest that extracellular Tat plays a role in the pathogenesis of HIV-1 infection.

Basophils and mast cells are the only cells that synthesize histamine and express high affinity receptors for IgE (Fc{epsilon}RI) (22, 23). Immunologic activation of human basophils leads to the synthesis and secretion of a restricted profile of cytokines (IL-4 and IL-13) (24, 25, 26, 27) critical for CD4+ Th2 cell polarization (28, 29, 30). Similarly, immunologic activation of mast cells leads to the synthesis of IL-4 and IL-13 (31, 32, 33, 34). We have recently demonstrated that HIV-1 gp120 from different clades is a potent stimulus for IL-4 and IL-13 release from basophils from healthy individuals seronegative for Abs to HIV-1 and HIV-2 (35). Because HIV-1 enters the body predominantly through mucosal surfaces and because the early phases of infection are associated with high levels of viremia (1, 36), mast cells and basophils can be exposed to shed or virus-bound gp120. This suggests that Fc{epsilon}RI+ cells may be a novel source of Th2 cytokines, thus contributing to the dysregulation of the immune system in HIV-1 infections (37).

Human basophils express the chemokine receptor CCR3, which binds eotaxin and RANTES (38). We and others have recently demonstrated that CCR3 receptor is also expressed in situ by human mast cells in gut, skin, and lung tissues (39, 40). Moreover, eotaxin and RANTES act as chemoattractants for human mast cells (40) suggesting that these cells, like human basophils (38) and eosinophils (41), express functional CCR3. The expression of CCR3 on human Fc{epsilon}RI+ cells is important in the induction of migration, and the active presence of these cells in tissues where they exert fundamental roles in the host response to infectious agents (42, 43, 44).

Here, we demonstrate that HIV-1 Tat protein induces basophil and mast cell chemotaxis and calcium fluxes. Fc{epsilon}RI+ cell chemotaxis induced by Tat protein is abolished by preincubation of basophils with a mAb against CCR3 and by an Ab against a conformation-dependent epitope of Tat protein, but not by an Ab against an epitope specific for the basic region of the viral trans-activator. Incubation of basophils with Tat protein up-regulates CCR3 expression on these cells. These data demonstrate that HIV-1 Tat protein influences the directional migration of Fc{epsilon}RI+ cells that are critical in the immune response to HIV-1 and that these activities are mediated by interaction with the chemokine receptor CCR3 through a discrete region of the molecule outside its basic region.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The following were purchased: 60% HClO4 (Baker Chemical, Deventer, The Netherlands); human serum albumin (HSA), {alpha}-chymotrypsin, PIPES, hyaluronidase, chymopapain, collagenase, elastase type I, 1,4-dithio-L-threitol, and PMSF (Sigma, St. Louis, MO); HBSS, FCS, TRIzol, and murine Moloney leukemia virus transcriptase (Life Technologies, Grand Island, NY); DNase I and pronase (Calbiochem, La Jolla, CA); RPMI 1640 with 25 mM HEPES buffer and Eagle’s MEM (Flow Laboratories, Irvine, Scotland); and dextran 70 and Percoll (Pharmacia, Uppsala, Sweden). The mAb 7B11 (IgG2a), which blocks CCR3, was provided by Leukosite (Boston, MA); the mAbs against HIV-1 Tat protein (6.3G12 and 6.15F4) were produced by immunizing BALB/c mice with synthetic 1–86 Tat protein, and characterizing them for their epitope specificity as recently described (45). Ab 6.3G12 recognizes a conformational epitope of 1–86 Tat protein, whereas Ab 6.15F4 recognizes the epitope RRQR (45) of the Tat basic region domain. IL-3 was donated by Novartis (Basel, Switzerland). Rabbit polyclonal IgG from nonimmunized animals was obtained from Sigma. Rabbit anti-human Fc{epsilon} Ab was a gift from Drs. Teruko and Kimishige Ishizaka (La Jolla Institute for Allergy and Immunology, La Jolla, CA). A mouse monoclonal IgG anti-{alpha} chain of the high affinity receptor for IgE (anti-Fc{epsilon}RI) was donated by Dr. John Hakimi (Hoffmann-LaRoche, Nutley, NJ). The HIV-1 Tat protein used in these experiments was obtained by solid phase synthesis using the F-moc/dextran-coated charcoal/HOBL strategy (45). HIV-1 Tat was dissolved at 10 µg/ml in PBS buffer containing 0.1 mM DTT and was frozen in aliquots at -80°C. Tat preparations were screened and were found to be negative for endotoxin contamination.

Buffers

The PIPES buffer used in these experiments was made up of 25 mM PIPES (pH 7.4), 110 mM NaCl, and 5 mM KCl. The mixture is referred to as P. PCG contains, in addition to P, 5 mM CaCl2 and 1 g/L D-glucose (46). PACGM contains, in addition to P, 3% HSA, 1 mM CaCl2, 1 g/L dextrose, and 0.25 g/L MgCl2·6H2O (pH 7.4); PGMD contains 0.25 g/L MgCl2·6H2O, 10 mg/L DNase, and 1 g/L gelatin in addition to P, pH 7.4. PBS contains 8 g/L NaCl, 1.15 g/L Na2HPO4, 200 mg/L KCl, and 200 mg/L KH2PO4, pH 7.4.

Purification of peripheral blood basophils

Basophils were purified from peripheral blood cells of healthy volunteers seronegative for Ab to HIV-1 and HIV-2, aged 20–39 years (mean, 33.6 ± 4.9 years). Buffy coat cell packs from healthy volunteers, provided by the Immunohematology Service at the University of Naples Federico II, were reconstituted in PBS containing 0.5 g/L HSA and 3.42 g/L sodium citrate, and loaded onto a countercurrent elutriator (model J2-21; Beckman, Fullerton, CA). Several fractions were collected, and fractions containing basophils in large numbers (>20 x 106 basophils) and of good purity (>15%) were enriched by discontinuous Percoll gradients (40). Basophils were further purified to near homogeneity (>98%) by depleting B cells, monocytes, NK cells, dendritic cells, erythrocytes, platelets, neutrophils, eosinophils, and T cells using a cocktail of hapten-conjugated CD3, CD7, CD14, CD15, CD16, CD36, CD45RA, and anti-HLA-DR Abs and MACS MicroBeads coupled to an anti-hapten mAb. The magnetically labeled cells are depleted by retaining them on a MACS column in the magnetic field of the MidiMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Yields ranged from 3 to 10 x 106 basophils, with purity usually >98%, as assessed by basophil staining with Alcian Blue and counting in a Spiers-Levy eosinophil counter (46).

Isolation and purification of human lung mast cells (HLMC)

Lung tissue was obtained from patients undergoing thoracotomy and lung resection. Macroscopically normal parenchyma was dissected free from pleura, bronchi, and blood vessels and minced into a single-cell suspension as previously described (47). Yields with this technique ranged between 3 x 106 and 18 x 106 mast cells, and purities were between 1 and 8%. Lung mast cells were purified by countercurrent elutriation (J2/21; Beckman) and then by discontinuous Percoll density gradient as previously described (47). Mast cells were further purified to near homogeneity by positive selection by incubation with anti-Fc{epsilon}RI (IgG1) followed by exposure to magnetic beads coated with MACS goat anti-mouse IgG. Labeled cells were enriched by positive selection columns (MACS system; Miltenyi Biotec). The final preparations contained >95% viable cells, assessed by the trypan blue exclusion method, and purity was >90% mast cells.

Flow cytometric analysis of surface molecules

Flow cytometric analysis of cell surface molecules was performed as described previously (40). Briefly, after saturation of nonspecific binding sites with total rabbit IgG, cells were incubated for 20 min on ice with specific or isotype control Abs. For indirect staining this step was followed by a second incubation on ice with an appropriate anti-isotype-conjugated Ab. Finally, cells were washed and analyzed on an FACScan cytofluorometer using either LYSIS II software or Win MDI (Becton Dickinson, San Fernando, CA). A total of 104 events for each sample were acquired in all cytofluorometric analyses.

Histamine release

Basophils (~6 x 104 basophils/tube) or mast cells (~3 x 104 cells/tube) were resuspended in PCG, and 0.1 ml of the cell suspension was placed in 12 x 75-mm polyethylene tubes (Sarstadt, Princeton NJ) and warmed to 37°C; 0.1 ml of each prewarmed releasing stimulus was added, and incubation was continued at 37°C for 45 min (35). At the end of this step, the reaction was stopped by centrifugation (1000 x g, 22°C, 2 min), and the cell-free supernatants were stored at -20°C for subsequent assay of histamine content with an automated fluorometric technique (48). Total histamine content was assessed by lysis induced by incubating the cells with 2% HClO4 before centrifugation. To calculate histamine release as a percentage of total cellular histamine, the spontaneous release of histamine from mast cells (2–14% of the total cellular histamine) was subtracted from both the numerator and the denominator (40). The percentage of histamine release was calculated according to the equation (A - B)/(T - B) x 100, where A is the sample, B is the spontaneous histamine release, and T is the total histamine content. All values are based on means of duplicate or triplicate determinations. Replicates differed in histamine content by <10%.

Chemotaxis assay

Basophil and mast cell chemotaxis was performed using a modified Boyden chamber technique as previously described (40, 47). Briefly, 25 µl of PACGM buffer or various concentrations of the chemoattractants in the same buffer were placed in triplicate in the lower compartment of a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD). The lower compartments were covered with polycarbonate membranes with 5-µm pores (basophils) or with a two-filter sandwich constituted by 5-µm (lower) and 8-µm (upper) pore size polycarbonate membranes (mast cells; Nucleopore, Pleasanton, CA). Fifty microliters of the cell suspensions (5 x 104/well) resuspended in PACGM were pipetted into the upper compartments. The chemotactic chamber was then incubated for 1 h (basophils) or 3 h (mast cells) at 37°C in a humidified incubator with 5% CO2 (automatic CO2 incubator, model 160 IR, ICN/Flow Laboratories). At the end of basophil incubation, the membrane was removed, washed with PBS on the upper side, fixed, and stained with May-Grunwald/Giemsa. When mast cells were used, the upper polycarbonate filter was discarded, while the lower nitrate cellulose filter was fixed in methanol, stained with Alcian Blue, and then mounted on a microscope slide with Cytoseal (Stephen Scientific, Springfield, NJ). Basophil and mast cell chemotaxis was quantitated microscopically by counting the number of cells attached to the surface of the 5-µm cellulose nitrate filter. In each experiment 10 fields/triplicate filter were measured at x40 magnification. The results were compared with buffer controls. Check board analysis was performed to discriminate between chemotaxis and nondirected migration (chemokinesis) of basophils or mast cells. In these experiments basophils or mast cells were placed in the upper chambers, and various concentrations of Tat (6–60 nM), eotaxin (10–100 nM), RANTES (10–100 nM), or PACGM buffer were added into the upper or lower wells or both. Spontaneous migration (chemokinesis) was determined in the absence of chemoattractant or when stimuli were added to either the lower or upper chambers. The basophil or mast cell migratory responses to Tat, eotaxin, and RANTES were largely due to chemotaxis and not to chemokinesis. Indeed, a check board analysis, in which chemoattractants above and below the filters were varied, resulted in significant migration only when there was a gradient of the factor below the filters (data not shown).

Calcium fluxes

Intracellular Ca2+ changes were measured using an MSIII spectrophotofluorometer (Photon Technology International, South Brunswick, NJ) in human basophils purified (>98%) from peripheral blood. Briefly, 2 x 106 cells were washed twice with 10 mM HEPES buffer, pH 7.4, supplemented with 1x HBSS. Basophils were then loaded with 5 µM fura-2/AM in the above buffer supplemented with 0.8 mM MgCl2, 1.8 mM CaCl2, and 20 mM glucose at 22°C for 30 min; washed twice; and resuspended at 0.5 x 106 cells/ml. Two milliliters of the cell suspension were placed in a stirred, water-jacked quartz cuvette at 37°C. Appropriate loading of the Ca2+ indicator into the cells was determined by means of excitation scans between 300 and 400 nm. Intracellular Ca2+ was determined from fluorescence ratios at 510 nm emission wavelength upon excitation of the sample at 340 and 380 nm using a standard calibration curve for fura-2.

Isolation of cellular mRNA and RT-PCR

Total RNA was extracted from basophils using a single-step method with TRIzol according to the manufacturer’s instructions. One microgram of total RNA was converted into cDNA in a standard reverse transcriptase reaction, using Moloney murine leukemia virus at 200 U/µl and oligo(dT) as primer. cDNA was then titrated for ß-actin message, and equivalent templates of cDNAs, obtained from different cultures, were amplified for CCR3 message using the specific primers described previously (49). Amplification was performed using a 9600 Thermocycler (Perkin-Elmer, Monza, Italy). The amplification protocol consisted in 30 cycles as follows: denaturation, 1 min at 94°C; annealing, 1 min at 56°C; and extension, 72°C for 1 min. A final extension at 72°C for 10 min was performed. The PCR products were loaded on 2% agarose gel and run in a submarine gel apparatus. After the run the gel was stained with ethidium bromide, and bands were visualized on UV sources. Bands were quantified by scanning the gel in a Fluorimager (Molecular Dynamics, Sunnyvale, CA) and measuring relative fluorescence units with ImageQuant software.

Statistical analysis

The results are expressed as the mean ± SEM. Statistical significance was analyzed by one-way ANOVA and, when the F value was significant, by Duncan’s multiple range test (50). Differences were considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of HIV-1 Tat protein on chemotaxis of human basophils

It has been recently demonstrated that the HIV-1 Tat protein is chemotactic for human monocytes through the interaction of a chemokine-like region with the ß-chemokine receptors CCR2 and CCR3 (18). In our experiments we evaluated the effect of increasing concentrations of HIV-1 Tat protein on chemotaxis of basophils purified (> 98%) from peripheral blood from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Fig. 1GoA shows the results of six experiments demonstrating that Tat protein (6–60 nM) caused a concentration-dependent increase in basophil chemotaxis. In a parallel series of experiments we compared the chemotactic activity of Tat with that of eotaxin and RANTES, which are potent chemoattractants of human basophils (38). Fig. 1GoB shows that eotaxin and RANTES induced strong attraction of human basophils. In the same experiments Tat promoted migration of basophils from healthy individuals.



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  		FIGURE 1. A, Effect of increasing concentrations of HIV-1 Tat protein on chemotaxis of human basophils purified from normal donors negative for HIV-1 and HIV-2 Abs. Basophils were allowed to migrate toward the indicated concentrations of Tat protein for 1 h at 37°C in the humidified incubator with 5% CO2. Values are the mean ± SEM obtained from six experiments with different human basophil preparations. *, p < 0.01 compared with control. B, Effect of increasing concentrations of eotaxin ({square}), RANTES ({blacksquare}), and Tat protein () on human basophil chemotaxis. Basophils were allowed to migrate toward the indicated concentrations of eotaxin, RANTES, and Tat protein for 1 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM obtained from four experiments with different human basophil preparations.

 
Induction of Ca2+ mobilization in human basophils by Tat protein

Because changes in free cytoplasmic Ca2+ are a prerequisite for intracellular transduction of chemotactic signals by chemokines (51), we analyzed whether HIV-1 Tat protein induces Ca2+ influx in human basophils. Fig. 2Go shows that the addition of Tat protein (60 nM) to human basophils loaded with the fluorescent Ca2+ indicator fura-2 produced a rapid and transient increase in intracellular Ca2+ concentrations. The pattern of Ca2+ mobilization caused by Tat protein is typical of chemokines (52), as shown in the positive control with eotaxin (100 nM). This activity was specific for Tat, because anti-Tat1–86 mAb blocked the induction of Ca2+ mobilization induced by Tat in basophils (data not shown).



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  		FIGURE 2. Effect of HIV-1 Tat protein on Ca2+ mobilization in human basophils purified from normal donors negative for HIV-1 and HIV-2 Abs. Tat protein (60 nM) induced rapid and transient Ca2+ mobilization in human basophils. The response of these cells in the same experiment to the chemokine eotaxin (100 nM) is shown for comparison.

 
CCR3 on human basophils is involved in their chemotaxis to Tat protein

CCR3 expression occurs in the majority of basophils enriched from peripheral blood of normal donors (38, 40). To establish whether CCR3 expression on basophils is responsible for the chemoattractant effect of Tat protein, purified basophils were preincubated with anti-CCR3 Ab and then assessed for their ability to migrate in the presence of Tat protein. Fig. 3GoA shows the results of three experiments demonstrating that preincubation of human basophils with anti-CCR3 Ab completely blocked the chemoattractant effect of Tat. In contrast, the chemotactic effect of the formylated tripeptide FMLP, which activates a specific seven-transmembrane receptor independent of the CCR3 receptor (53), was not affected by anti-CCR3 Ab. In a series of three parallel experiments, preincubation of basophils with anti-CCR3 Ab completely suppressed the chemotactic activity of eotaxin and RANTES on these cells (Fig. 3GoB).



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  		FIGURE 3. A, Chemotactic responses of human basophils to HIV-1 Tat protein (60 nM) or FMLP (100 nM) incubated with ({blacksquare} or ) or without ({square}) 5 µg/ml anti-CCR3 added to the cells 10 min before loading into the chemotaxis chamber. Values are the mean ± SEM of three distinct experiments with basophils obtained from different donors negative for HIV-1 and HIV-2 Abs. *, p < 0.01 compared with basophils preincubated with anti-CCR3 Ab. B, Chemotactic responses of human basophils to Tat protein, eotaxin, and RANTES incubated with ({blacksquare}) or without ({square}, , or ) 5 µg/ml anti-CCR3 added to the cells 10 min before loading into the chemotaxis chamber. Basophils were allowed to migrate toward the indicated concentrations of eotaxin, RANTES, and Tat protein for 1 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM obtained from three experiments with different human basophil preparations. *, p < 0.01 compared with basophils preincubated with anti-CCR3 Ab.

 
Effects of mAb specific for distinct epitopes of Tat protein on human basophil chemotaxis

HIV-1 Tat protein contains several distinct domains (17, 18, 19, 20, 21). Peptides corresponding to the basic domain (aa 46–60) and the Arg-Gly-Asp (RGD)-containing domain (aa 65–80) activate tyrosine kinase receptor (54) and integrin receptor (55), respectively. The cysteine-rich domain (aa 24–51) presumably interacts with the ß-chemokine receptors CCR2 and CCR3 and is responsible for Ca2+ mobilization and monocyte chemotaxis (18).

The availability of mAbs specific for distinct epitopes of the Tat molecules (12, 45) prompted us to investigate the effects of these Abs on basophil chemotaxis induced by Tat protein. Fig. 4Go shows that the mAb 6.3 G12 against a conformation-dependent epitope of Tat (Tat1–86) strongly inhibited the chemotactic activity of Tat on human basophils. In contrast, mAb 6.15 F4 recognizing the epitope RRQR of the basic region of Tat was only marginally effective, if at all, in inhibiting the chemoattractant capacity of Tat protein. In the same experiment anti-CCR3 Ab completely blocked the chemoattractant effect of the Tat protein (data not shown).



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  		FIGURE 4. Effects of mAbs specific for distinct epitopes of HIV-1 Tat protein on basophil chemotaxis. Basophils were incubated with ({blacksquare} or ) or without ({square}) 20 µg/ml of mAb 6.3G12 against a conformation-dependent epitope of Tat protein (Tat1–86 or 20 µg/ml of mAb 6.15F4 against the epitope RRQR of the Tat basic region (Tat46–60) added to the cells 10 min before loading into the chemotaxis chamber. Values are the mean ± SEM of three distinct experiments with basophils obtained from different donors negative for HIV-1 and HIV-2 Abs. *, p < 0.01 compared with basophils preincubated with anti-Tat1–86.

 
Cross-desensitization between HIV-1 Tat protein and eotaxin

The relationship between CCR3 receptors and Tat protein was examined using eotaxin to induce desensitization of chemotaxis specific for CCR3-mediated stimuli. Purified basophils (>98%) were incubated with PIPES buffer containing EDTA (4 mM), Tat protein (60 nM), or eotaxin (100 nM) in PIPES buffer containing EDTA (4 mM) for 30 min at 37°C. At the end of incubation, basophils were washed twice, resuspended in PACGM, and rechallenged with the chemotactic stimuli (60 nM Tat protein, 100 nM eotaxin, or 10-8 M FMLP). Fig. 5Go shows the results of three experiments in which the response to Tat protein was significantly desensitized by preincubation with either Tat or eotaxin. Similarly, preincubation with either Tat or eotaxin significantly reduced the chemotactic activity of eotaxin. In contrast, the chemotactic response to FMLP, which activates a specific receptor independent of the CCR3 receptor (53), was unaffected by desensitization with either Tat protein or eotaxin.



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  		FIGURE 5. Effects of cross-desensitization between eotaxin and Tat protein. Human basophils obtained from normal donors negative for HIV-1 and HIV-2 Abs were incubated with PIPES buffer containing EDTA (4 mM; {square}), Tat protein (60 nM; {blacksquare}), or eotaxin (100 nM; ) in PIPES buffer containing EDTA (4 mM) for 30 min at 37°C. At the end of incubation, cells were washed twice, resuspended in PACGM, and challenged with the chemotactic stimuli (60 nM Tat protein, 100 nM eotaxin, or 10-8 M FMLP). Values are the mean ± SEM of three distinct experiments obtained from different donors. *, p < 0.01 compared with basophils preincubated in the absence of chemotactic stimuli.

 
The relationship among CCR3 receptors, Tat protein, and eotaxin was further examined by looking at desensitization of Ca2+ influx in human basophils. Fig. 6Go shows that cells incubated with Tat (60 nM) induced rapid and transient Ca2+ mobilization in basophils. This treatment caused desensitization of cells to a subsequent challenge with eotaxin (100 nM), whereas the Ca2+ ionophore ionomycin (20 µM) caused a remarkable Ca2+ influx. Similar results were obtained in two additional experiments.



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  		FIGURE 6. Tat and eotaxin cross-desensitization of Ca2+ transients. Basophils were loaded with fura-2 as described in Materials and Methods and subsequently stimulated with Tat (60 nM), eotaxin (100 nM), and ionomycin (20 µM). A single trace representative of three experiments is shown.

 
Effect of Tat protein on surface expression of CCR3 on human basophils

To examine whether Tat protein regulated ß-chemokine receptor expression as well as basophil chemotaxis, we analyzed the surface expression of CCR3 in highly purified (>98%) preparations of basophils by flow cytometry. Fig. 7GoA shows that CCR3 expression occurred in the majority of Fc{epsilon}RI+ cells in human basophils. After a 12-h incubation with Tat protein, CCR3 expression was up-regulated in a significant proportion (~20%) of cells with respect to the untreated control (Fig. 7GoB). The Tat-mediated up-regulation of CCR3 was inhibited in presence of the Tat (aa 1–86)-specific mAb (Fig. 7GoC). Therefore, HIV-1 Tat protein up-regulated the surface expression of CCR3, which can be used by a subset of primary HIV-1 strains as a coreceptor (7, 8, 9, 10).



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  		FIGURE 7. Effects of HIV-1 Tat protein on the surface expression of CCR3 in human basophils obtained from normal donors negative for HIV-1 and HIV-2 Abs. Purified basophils (>98%) were incubated with anti-CCR3 mAb and anti-Fc{epsilon}RI mAb or isotype-matched control mAbs, followed by PE- or FITC-conjugated goat anti-mouse isotype. Electronic gates were set on Fc{epsilon}RI+ cells, and CCR3 expression was analyzed by flow cytometry. Expression of CCR3 on freshly isolated (empty area) and 12-h cultured basophils (shaded area) is shown. The isotype control is indicated by the dotted lines. Human basophils were incubated with buffer (A), Tat protein (60 nM; B), and anti-Tat1–86 (20 µg/ml) and Tat (60 nM; C). Results from a single donor representative of three separate experiments are shown.

 
Effect of Tat protein on CCR3 mRNA in human basophils

To examine further whether Tat protein regulates CCR3 expression in human basophils, we analyzed the expression of CCR3 transcript by RT-PCR. Fig. 8Go shows the results of a representative experiment from among three different donors. We found that CCR3 mRNA was expressed in basophils, and the expression was increased after 6 and 12 h of incubation of basophils with Tat protein (60 nM).



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  		FIGURE 8. Kinetics of CCR3 mRNA expression induced by Tat protein in human basophils. Basophils were incubated for 6 and 12 h with Tat protein (60 nM). mRNA transcript for CCR3 was analyzed by RT-PCR (see Materials and Methods).

 
Effect of Tat protein on histamine release from human basophils

Eotaxin, which selectively binds to CCR3, is a weak inducer of histamine release from human basophils (38). Therefore, we compared the effects of Tat and eotaxin alone or in combination with IL-3 on histamine release from human basophils. Fig. 9GoA shows that Tat alone did not elicit mediator release from basophils. Preincubation with IL-3 did not enhance the effect of Tat protein on histamine release. Fig. 9GoB shows that although eotaxin alone elicited a low or no response, preincubation with IL-3 significantly enhanced the releasing activity of eotaxin on human basophils. These results show that there are some differences in the activation of CCR3 receptor by eotaxin and Tat protein with respect to the release of mediators from basophils primed by IL-3.



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  		FIGURE 9. A, Effects of IL-3 (10 ng/ml) and increasing concentrations of HIV-1 Tat protein (6–60 nM) alone and in combination on histamine release from human basophils obtained from normal donors negative for HIV-1 and HIV-2 Abs. Cells were preincubated for 30 min at 37°C with IL-3 and then challenged (45 min at 37°C) with Tat protein. Values are the mean ± SEM of four experiments with different human basophil preparations. B, Effects of IL-3 (10 ng/ml) and increasing concentrations of eotaxin (1–100 nM) alone and in combination on histamine release from human basophils obtained from normal donors negative for HIV-1 and HIV-2 Abs. Cells were preincubated for 30 min at 37°C with IL-3 and challenged (45 min at 37°C) with the indicated concentrations of eotaxin. Values are the mean ± SEM of four experiments with different human basophil preparations.

 
Effect of Tat protein on human lung mast cell chemotaxis

We and others have recently shown that a remarkable proportion of human mast cells (~25%) express CCR3 receptor (39, 40). We have also demonstrated that CCR3 receptor is expressed in situ by human mast cells in gut mucosa, skin, and lung tissue (40). More importantly, CCR3 receptor on human mast cells is functionally active and is involved in the chemotactic response to eotaxin (40). Fig. 10Go shows the results of four experiments in which we evaluated the effect of Tat protein on chemotaxis of HLMC. These experiments demonstrated that Tat protein induced a concentration-dependent increase in lung mast cell chemotaxis. Preincubation of mast cells with a mAb against CCR3 completely blocked the chemoattractant effect of Tat protein (data not shown).



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  		FIGURE 10. Effects of increasing concentrations of HIV-1 Tat protein on HLMC chemotaxis. Mast cell-enriched suspensions obtained from human lung parenchyma were allowed to migrate toward the indicated concentrations of Tat protein for 3 h at 37°C in a humidified incubator with 5% CO2. Values are the mean ± SEM of four distinct experiments with different lung mast cell-enriched preparations obtained from donors negative for HIV-1 and HIV-2 Abs. *, p < 0.01 compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that HIV-1 Tat protein is a chemoattractant for human Fc{epsilon}RI+ cells. The chemotactic activity of Tat protein is mediated by interaction with the CCR3 receptor present on human basophils and on a remarkable proportion of mast cells. In addition, Tat protein up-regulates CCR3 mRNA and the level of surface CCR3 receptor in human basophils. This is the first demonstration that Tat protein is an HIV-1-encoded chemokine homologue functionally active on human Fc{epsilon}RI+ cells through interaction with the CCR3 receptor.

The relevance of these findings is 4-fold. First, they suggest that during HIV-1 infection, which is associated with high levels of Tat protein in biologic fluids (14), Tat protein influences the directional migration of human basophils and mast cells exposed to soluble Tat, thus contributing to the recruitment of these cells at sites of HIV-1 infection. Second, the chemotactic activity of Tat on human Fc{epsilon}RI+ cells might contribute to increase the local density of mast cells and basophils available for HIV-1 interaction through the virus bound or shed gp120. In fact, we have recently demonstrated that gp120 from different clades interacts with the VH3 region of IgE present on human Fc{epsilon}RI+ (35). Third, the superantigenic interaction between gp120 and IgE leads to the rapid synthesis and release of IL-4 and IL-13 from human Fc{epsilon}RI+ cells (35). This interaction might represent an initial source of cytokines, thereby favoring a shift from a Th0 toward a Th2 phenotype. The latter observation is relevant because HIV-1 replicates preferentially in Th2 cells (56). Finally, our findings might help to understand the spread of HIV-1 infection. Indeed, there is increasing evidence that CCR3 is a coreceptor for several strains of HIV-1 (7, 8, 9, 10). Therefore, the Tat-mediated up-regulation of CCR3 in human Fc{epsilon}RI+ cells described herein might facilitate the interaction between HIV-1 and Fc{epsilon}RI+ cells through the IgE VH3+-gp120 binding site, thus providing an additional and/or alternative mechanism by which HIV-1 can bind to and possibly infect Fc{epsilon}RI+ cells.

Our findings are compatible with the idea that Tat protein interacts with the CCR3 receptor present on human basophils and mast cells (39, 40). This hypothesis is supported by our finding that a mAb anti-CCR3 completely blocked the chemoattractant effect of Tat protein on human basophils and mast cells. In addition, Tat protein induced a rapid and transient Ca2+ mobilization in human basophils analogous to ß-chemokines (52). Finally, the cross-desensitization of basophil chemotaxis and Ca2+ influx between eotaxin and Tat protein is also compatible with the hypothesis that Tat protein interacts with the CCR3 receptor on human Fc{epsilon}RI+ cells.

It has been demonstrated that HIV-1 Tat protein induces CXCR4 on both lymphocytes and monocytes, whereas CCR3 and CCR5 are induced only on monocytes (57, 58, 59). We now provide the first evidence that Tat protein can both up-regulate CCR3 mRNA and induce overexpression of surface CCR3 receptor in human basophils.

Our results provide some clues as to the site of interaction between Tat protein and CCR3 receptor on human Fc{epsilon}RI+ cells. Tat protein contains at least three characterized binding regions: a basic domain (aa 46–60), an RGD domain (aa 65–80), and a cysteine-rich region. The interaction of the basic domain and the RGD domain with several cell types does not induce calcium flux (18). The RGD peptide interacts with the {alpha}Vß5 integrin receptor (55), whereas the basic domain activates tyrosine kinase receptors (54). Albini et al. have demonstrated that HIV-1 Tat protein acts as a chemoattractant for human monocytes through the interaction between the peptide (aa 24–51) encompassing the chemokine-like region of Tat and CCR2 and CCR3 (60). Our finding that a mAb specific for the basic domain epitope RRQR (52, 53, 54, 55) (45) does not modify the chemotactic activity of Tat indicates that this region of the protein is not essential for its effect on Fc{epsilon}RI+ cells.

Intriguingly, many viruses exploit the strategy of using homologues of cellular cytokines and chemokines to shield virus-infected cells from immune defenses and enhance virus survival in the host (61, 62). The existence of these virus-encoded homologues of cellular proteins is indirect evidence of their relevant role in orchestrating the host immune response to invading pathogens (62). Many large DNA viruses, e.g., human herpes viruses, including CMV and HHV-8, as well as the poxvirus molluscum contagiosum, encode several ß-chemokine homologues (virokines) acting on CCR3 or CCR8 receptors (63, 64, 65, 66, 67, 68). HIV-1 Tat protein is the first identified virokine encoded by retrovirus that is functionally active on human Fc{epsilon}RI+ cells through the interaction with CCR3 receptor. This novel observation may have several implications for a better understanding of the pathogenesis of HIV-1 infection.

Tat has been found in the serum of HIV-1-infected patients in concentrations as high as 10 ng/ml; comparable levels of Tat have been found in the media of HIV-1-infected H9 cells (14), and it has been shown that monocyte migration can be induced by supernatants of Tat-producing cells. In tissues where viral replication occurs (the lymph nodes), local levels of Tat could exceed those found in serum. Because the early phases of infection are associated with high levels of viremia (1), and this, in turn, may be associated with high levels of Tat, chemokine-like activity of Tat on Fc{epsilon}RI+ cells might be of clinical relevance in patients with HIV-1 infection.

CXCR4 and CCR5 are the predominant chemokine coreceptors in HIV-1 entry to the cell (5, 6). However, there is compelling evidence that several HIV-1 strains can use CCR3 as coreceptors (7, 8, 9, 10). Because CCR3 is already expressed by resting basophils and mast cells (38, 39, 40), a crucial issue will be to evaluate whether the Tat-mediated increase of surface CCR3 on human Fc{epsilon}RI+ cells could impact HIV-1 infectivity. In this respect we have recently observed that a percentage of human mast cells express CD4 molecules (A. de Paulis, unpublished observations). Thus, it is not inconceivable that extracellular Tat may contribute to render a larger population of Fc{epsilon}RI+ cells more susceptible to HIV-1 in the course of infection.

We have recently demonstrated that the interaction between a gp120 binding site and the VH3 domain of IgE on human basophils and mast cells induces the release of Th2 cytokines (IL-4 and IL-13) from human Fc{epsilon}RI+ cells without synthesizing Th1-type cytokines (e.g., IFN-{gamma}) (35). The presence of CCR3 receptors on the majority of basophils (39, 40) and on a significant percentage of human mast cells (38, 40) and their role as coreceptor for HIV-1 infection (7, 8, 9, 10) suggest that the interplay between Tat protein, which up-regulates CCR3 receptors, and Fc{epsilon}RI+ cells could facilitate HIV infection of these cells. Therefore, it is not unlikely that these cells might be a hitherto unrecognized reservoir of HIV infection.

Due to the pleiotropic effects of Tat protein, this molecule has been considered an immunogen for a HIV-1 vaccine for prevention of HIV-1 infection (69, 70). The effects of Tat protein on human basophils and mast cells and their complex interaction with other immunologic stimuli or cytokines (IL-4 and IL-13) relevant for Th2 polarization (37, 56) raise important issues that will need to be considered when designing clinical trials to evaluate the protective effects of Tat-containing vaccines.

In conclusion, we provide the first evidence that Tat protein is an HIV-1-encoded chemokine homologue for human Fc{epsilon}RI+ cells. Tat protein interacts with the CCR3 receptor on these cells and up-regulates its expression. Because HIV-1 enters the body predominantly through mucosal surfaces and because early phases of infection are associated with high levels of viremia, mast cells and basophils can be exposed to high local levels of Tat protein. This suggests that Fc{epsilon}RI+ cells can contribute, also through this novel mechanism, to the dysregulation of the immune system in HIV-1 infection.


   Acknowledgments
 
We thank Dr. Giacinto Forte for providing lung specimens and Dr. Giorgio Fratellanza for providing buffy coat cell packs. We are grateful to Jean Gilder for editing the text.


   Footnotes
 
1 This work was supported by grants from the Ministero della Sanità-Istituto Superiore Sanità (AIDS Projects 40B.64 and 40.B1), the Consiglio Nationale delle Ricerche (Target Projects Biotechnology 99.00216.PF31, 99.00401.PF49, and 99.03569.PF49), and MURST National Projects "Mechanism of Tumor Escape from Immune Surveillance and Specific Strategies of Intervention" and "Structural Basis and Functional Consequences of Cell Surface Recognition Processes" (Rome, Italy). Back

2 Address correspondence and reprint requests to Dr. Gianni Marone, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy. Back

3 Abbrevations used in this paper: CXCR4, CXC chemokine receptor 4; HSA, human serum albumin; anti-Fc{epsilon}RI, mouse monoclonal IgG anti-{alpha} chain of high affinity receptor for IgE; anti-IgE, rabbit IgG anti-Fc fragment of human IgE; Fc{epsilon}RI, high affinity receptor for IgE; HLMC, human lung mast cells; P, 25 mM PIPES (pH 7.4), 110 mM NaCl, and 5 mM KCl; PACGM, P plus 3% HSA, 2 mM CaCl2, 1 g/L D-glucose, and 0.25 g/L MgCl2·6H2O; PCG, P plus 5.0 mM CaCl2 and 1 g/L D-glucose; PGMD, P plus 0.25 g/L MgCl2·6H2O, 10 mg/L DNase, and 1 g/L gelatin. Back

Received for publication May 22, 2000. Accepted for publication September 5, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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