Introduction
The transactivating protein Tat plays a key role in HIV infection. Its ability to induce viral and cellular gene transcription provides a high viral load and promotes a pro-inflammatory status that favours viral spread [1-6]. In addition, Tat released from infected cells may interact with a variety of cells inducing their growth, migration and/or apoptosis [1,7-11]. Tat has been shown to act as a growth factor for endothelial [8,12,13] and Kaposi's sarcoma (KS)-derived cells [7], to induce the migration of KS and endothelial cells [13] and it is angiogenic in vivo [14]. Extracellular HIV Tat also exerts multiple actions on leucocytes, inducing [15] or protecting against [16] apoptosis, stimulating monocyte adhesion to endothelia [17] and inducing monocyte migration [18].
Tat binding to cell surfaces has been shown previously to be mediated by two different domains: a carboxyterminal Arg-Gly-Asp (RGD)-containing domain (amino acids 78-80) which binds to integrins [12,19,20] and a basic, Arginine-lysine-rich domain (amino acids 49-57) [8] which binds and activates the endothelial cell VEGF tyrosine kinase receptor Flk/KDR leading to the angiogenic response [21].
We have analysed the role of the Tat RGD and basic domains in the induction of monocyte migration, and demonstrate that Tat cooperates with the chemokines RANTES and macrophage chemotactic protein (MCP)-1 in inducing monocyte migration. Furthermore, we demonstrate for the first time that Tat is also a chemoattractant for dendritic cells. The dendritic cells responded to both the RGD and the basic peptides. Macrophages were attracted into matrigel sponges by Tat in vivo. Taken together, these data indicate that Tat can influence the directional migration of cells that are critical in the immune response, and that this activity is mediated by at least two distinct functional domains.
Materials and methods
Peptides
Synthetic Tat protein (Tecnogen, Caserta, Italy) was dissolved in phosphate-buffered saline (PBS)-bovine serum albumin (BSA) [PBS containing 0.1% BSA (highly purified fatty acid free; Sigma, Milan, Italy)] and 10 mM dithiothreitol (DTT) in aliquots of 1 µg/10 µl and stored at -80 °C. Synthetic Tat basic peptide (amino acids 46-60: SYGRKKRRQR RRPPQ), RGD peptide (amino acids 65-80: HQVSLSKQPT SQSRGD) and a control Tat peptide (amino acids 56-70: RRPPQ GSQTHQVSLS) (Intracel, London, UK) were dissolved in PBS-BSA (without DTT) in aliquots of 10 µg/10 µl and stored at -80°C. Human recombinant MCP-1 and RANTES (Pepro-Tech, London, UK) were dissolved in PBS-BSA in aliquots of 0.2 µg/10 µl and stored at -80°C. Control N-formyl-Met-Leu-Phe (f-MPL) peptide (Sigma) was dissolved in dimethylsulphoxide to 0.1 M, then diluted in water to 1 µm, divided into 10 µl aliquots and stored at -20 °C.
Isolation of monocytes
Heparinized peripheral blood from normal, HIV-negative healthy donors was carefully layered on to an equal volume of Ficoll (Seromed, Berlin, Germany) and centrifuged at 600 g for 40 min. After centrifugation the peripheral blood mononuclear cells (PBMC) remaining at the sample-medium interface were collected, purified from residual erythrocytes using two rounds of lysis by NH4Cl, resuspended in RPMI containing 0.1% BSA and used immediately for chemotaxis assays. The total PBMC could be used directly as the monocyte source because the contaminating lymphocytes were not able to adhere and migrate through the polycarbonate filter used in the chemotaxis assay. Control experiments were performed throughout using Percoll-purified (Pharmacia-Biotech, Milan, Italy) monocytes (purity 85-95% assessed by flow cytometry of CD14+ cells) for confirmation of the results. Expression of the integrins was determined by flow cytometry using an anti-β1 integrin monoclonal antibody (MAb) or an anti-αvβ3 integrin MAb (Chemicon, Temecula, California, USA).
Generation of dendritic cells from monocytes
Ficoll-isolated PBMC were resuspended in RPMI with 10% fetal calf serum and allowed to adhere to six-well tissue culture plates. After 2 h at 37 °C non-adherent cells were removed and the adherent cells were cultured in serum-supplemented RPMI with 50 ng/ml granulocyte macrophage colony stimulating factor (Pepro-Tech) and 1000 U/ml interleukin (IL)-4 (Pepro-Tech) as described by Sallusto et al. [22]. After 1 week most of the cells displayed a typical dendritic morphology, were positive for CD1a and expressed CD80, CD86, CD54 and high levels of human leukocyte antigen (HLA)-DR as determined by flow cytometry.
Chemotaxis assay
Chemotaxis was performed in a 48-microwell chemotaxis chamber (Costar-Nucleopore, Milan, Italy), using a 5 µm pore-size polyvinylpyrrolidone-free polycarbonate filter (Costar-Nucleopore), which divides each well into two compartments. To the lower compartment was added 27 µl of chemoattractant diluted in RPMI 0.1% BSA. Concentrations of Tat were: 1 × 10-8, 2.5 × 10-8, 1 × 10-7, 2.5 × 10-7 and 5 × 10-7 M.
RGD, basic and control Tat peptides were compared with Tat using 1 × 10-8, 1 × 10-7, 1 × 10-6 M concentrations as indicated. MCP-1 and RANTES were used at the concentrations exerting the higher stimulatory activity according to the product supplier specifications (respectively 20 ng/ml for MCP-1 and 50 ng/ml for RANTES). The concentration of f-MLP, 1 × 10-8inducing maximal migration, was used as the positive control; it has previously been shown that 2 log10 higher or lower concentrations give minimal activity [23]. RPMI with 0.1% BSA was the negative control used to evaluate background migration. In some experiments, an anti-β1 integrin MAb or an anti-αvβ3 integrin MAb (Chemicon) was added to the cell suspension 1 h before setting up the test. Each data point was repeated six times. The upper compartment was filled with 50 µl of either monocytes (1 × 107 cells/ml), or dendritic cells (6 × 105cells/ml) suspended in RPMI with 0.1% BSA. The chamber was incubated at 37 °C in a 5% CO2 humidified atmosphere for 120 min. The filter was then removed and the cells fixed with absolute ethanol and stained with toluidine blue. Cells which had not migrated were removed from the upper surface of the filter using filter paper. Migrated cells were counted in five microscopic fields per well.
Polarization assay
The ability of Tat and Tat peptides to induce monocyte polarization was assessed according to Locati et al. [24] with minor modifications. Freshly Percoll-purified (Pharmacia-Biotech) monocytes (5 × 105/500 µl) were pre-incubated at 37 °C in polypropylene tubes for 5 min and then treated with polarizating stimuli for 10-30 min. The assay was stopped by adding an equal volume of cold formaldehyde: PBS (10: 90). The percentage of polarized monocytes was determined by seeding 10 µl of cell suspension in a Bürker haemocytometer and counting the number of polarized cells and the total number of cells by microscopy.
In vivo angiogenesis
The matrigel sponge model of angiogenesis in vivo introduced by Passaniti [25], and modified by Albini [14] was used. Tat (20 ng/ml) and heparin (20 U/ml) were mixed with unpolymerized liquid matrigel at 4 °C to a final volume of 600 µl, then injected subcutaneously into the flank of C57/bl6 mice, where it quickly polymerized. Matrigel with buffer and heparin alone was used as negative control. Four days later, gels were collected and weighed. Samples were embedded in paraffin or snap-frozen and used for histology or electron microscopy respectively.
Electron microscopy
Samples were fixed with 2.5% glutaraldehyde (Polyscience, Warrington, Pennsylvania, USA) in 0.1 M sodium cacodylate buffer, pH 7.3, and post-fixed with 1% OsO4 (Polyscience) in the same buffer. Following staining with 1% uranyl acetate and dehydration with ethanol and propylene oxide, samples were embedded in LX112 (Polyscience). Grey-silver sections were stained with uranyl acetate and lead citrate, and observed with a Zeiss EM 10C electron microscope (Zeiss, Oberkocken, Germany) [26].
Results
Chemotaxis of monocytes to Tat peptides
Tat induced the chemotaxis of primary monocytes in vitro in a dose-dependent manner (Fig. 1a). Monocyte migration induced by 5 × 10-7 nM Tat was approximately 80% of the maximal response induced by the powerful chemoattractant f-MLP peptide. Migration was found to be chemotactic with a chemokinetic component, as previously reported [18,27]. Two peptides encompassing domains found to mediate many extracellular responses to Tat, the RGD domain and the basic domain, were tested for their ability to induce monocyte migration. Peptides encompassing either the RGD domain or the basic domain both had a concentration-dependent chemotactic effect on monocytes (Fig. 1b). A control Tat peptide (amino acids 57-70) had no effect on monocyte migration (Fig. 1c). Basic or RGD Tat peptides (1 × 10-7 M) showed approximately 50% of the activity of the intact Tat protein used at the same concentration (Fig. 1b). Surprisingly, when the basic and RGD peptides were combined together as monocyte chemoattractants, they showed an additive effect only at the lower dose (1 × 10-8 M) and at higher concentrations the use of both peptides together resulted in a decrease in migration to a level below that of the background (approximately 20 cells per field). This decrease was in sharp contrast to the potent activity of the intact Tat molecule at the same dose (Fig. 1b).
We have observed that cells producing Tat under the control of the HIV-long terminal repeat promoter [28] recruit significantly more monocytes (112 ± 6) than equivalent cells which do not secrete Tat (82 ± 6; P < 0.001, Student's t test). These data suggest that Tat released from Tat-expressing cells could contribute to increased monocyte attraction.
Cell migration is often mediated by adhesion molecule receptors, mainly integrins. In particular, α5β1 [29] and αvβ3 [30] dimers mediate migration through recognition of an RGD sequence in their ligands. Both β1 and αvβ3 integrins were expressed on monocyte cell surfaces (Table 1). Function-blocking anti-β1 and anti-αvβ3 antibodies were used to assess the involvement of these molecules in Tat-mediated monocyte chemotaxis (Fig. 1d). Both antibodies reduced the increase in migration induced by Tat relative to the unstimulated control by 40-50%. The migration induced by the RGD peptide was almost completely inhibited by anti-β1 or anti-αvβ3 antibodies, resulting in monocyte migration comparable to the unstimulated control. In contrast, control f-MLP-induced chemotaxis was only slightly affected by the anti-β1 antibody. These data suggest that integrins mediate Tat RGD peptide-induced migration.
Polarization of monocytes to Tat peptides
Cytoskeletal reorganization, involving cell polarization, is a prerequisite for cell migration. Tat was able to induce a rapid polarization of monocytes in a concentration-dependent manner (Fig. 2a). At 1 × 10-7 M the activity of Tat was comparable to the f-MLP-positive control (Fig. 2a) with 68% polarized cells. The basic and RGD peptides in equimolar concentrations (1 × 10-7 M) were able to induce about 50% of the polarizing activity of the intact Tat protein (Fig. 2b). Again, the addition of both peptides did not show any additive effect.
Chemotaxis of dendritic cells to Tat peptides
Dendritic cells migrate into peripheral tissues from a circulating monocyte-like precursor pool and differentiate locally to a stage in which they are specialized for antigen processing and presentation [31]. Both macrophage and T-cell tropic HIV-1 strains efficiently enter dendritic cells [32]. Monocyte-derived dendritic cells also showed a chemotactic response to Tat (Fig. 3a). Migration of dendritic cells in response to Tat varied between 20 and 40 cells per field depending on the donor, but was always twice that of the unstimulated control (10-20 cells per field). The basic or RGD peptides used at 2 × 10-8 M were able to induce approximately 50% of the dendritic cell migration when compared to the intact Tat at the same concentration; no additive effects were observed (Fig 3a). A fivefold higher dose (1 × 10-7 M) gave a similar result (Fig. 3a). However, as already observed for monocytes, the combination of both the RGD and basic peptides at high concentration led to a decrease of migration with respect to either peptide alone (Fig. 3a). The involvement of β1 and αvβ3 integrins in dendritic cell migration was also examined (Fig. 3b). The anti-β1 antibody slightly inhibited f-MLP and Tat-induced migration, but effectively blocked RGD-induced migration. The anti-αvβ3 antibody showed a marked inhibition of f-MLP-, Tat- and RGD-induced migration, reducing migration to background levels. Taken together, these data suggest that the αvβ3 integrin is a major mediator of dendritic cell migration.
Cooperation of Tat with β-chemokines
Chemokines are small chemotactic molecules, many of which regulate cell migration in inflammation [33]. KS spindle cells produce the β-chemokine MCP-1 in AIDS-associated KS lesions [34]. We therefore tested potential interactions between Tat and β-chemokines. Either MCP-1 or RANTES, together with Tat, gave an additive increase in monocyte chemotaxis as compared to the single attractants alone (Fig. 4). These chemokines could potentiate Tat recruitment of new monocytes towards an HIV infected cell.
Tat mediated recruitment of monocyte/ macrophages in vivo
The induction of monocyte and dendritic cell migration by Tat in vitro suggested that Tat could have a similar role in vivo. This was confirmed by examination of Tat-loaded matrigel sponges injected subcutaneously in vivo. Electron microscopy of Tat-containing matrigel sponges in vivo showed an infiltration of endothelial, spindle cells [14] and monocytes/macrophages (Fig. 5). Macrophages within the Tat-containing matrigel were mostly in areas adjacent to collagen bundles. Some of the macrophages had engulfed red blood cells (Fig. 5), this has been previously observed in KS lesions [35]. The migration of monocytes/macrophages within the matrigel gel in vivo parallels the Tat-induced invasion observed in vitro [18].
Discussion
Tat has been shown to interact with cellular integrins in vitro [12], including α5β1, αvβ3 and αvβ5 [16,17,20], mediating adhesion and migration of different cell types. Tat binding to integrins is mediated by the RGD-containing domain and by the basic domain for αvβ5 [20]. More recently, Tat has been found to bind and activate the Flk-1/KDR VEGF tyrosine kinase receptor on endothelial cells [21]. This interaction is mediated by the basic domain of Tat, which contains a highly charged basic sequence similar to many angiogenic growth factors interacting with tyrosine kinase receptors [8]. Unlike VEGF, Tat showed specificity for Flk-1/KDR, and did not interact with another VEGF receptor on endothelial cells, Flt-1 [21].
Recently Tat has been shown to be chemotactic for monocytes [18]. A recent report suggests that this activity in monocytes appears to be partially mediated by the VEGF receptor Flt-1 [27], unlike that which occurs in endothelial cells. However the Tat domains involved in monocyte activation and the role of integrins had not yet been investigated. Our studies have demonstrated that RGD and the basic domain, but not a control Tat peptide, show similar chemotactic potential for monocytes. These domains were not able to restore the full potential of the intact Tat molecule, and surprisingly both peptides together not only failed to have an additive effect but appear to inhibit the activity of each peptide alone. These observations suggest that a third Tat domain could be involved in monocyte interactions.
Monocytes from various donors expressed high levels of integrins of the β1 family, as well as αvβ3. Function blocking antibody experiments indicate that these integrins are partially involved in monocyte migration to Tat. Polarization of monocytes, a prerequisite for their chemotaxis, was also induced by both the RGD and basic peptides. Again, the full Tat activity was not achieved by either peptide alone or their combination.
Dendritic cells are highly motile in vivo and accumulate at sites of infection and inflammation [31], and also infiltrate certain tumours [36]. Here we demonstrate for the first time that Tat stimulates the migration of monocyte-derived dendritic cells. The basic and RGD peptides were both active on dendritic cells. However, as observed for monocytes, the basic peptide, along with the RGD peptide did not achieve the activity exerted by Tat on dendritic cells, and the combination of the two peptides was not additive. The anti-αvβ3 antibody effectively reduced the chemotactic response of these cells to Tat down to the basal levels of unstimulated migration. The anti-β1 antibody was as effective as the anti-αvβ3 antibody in blocking RGD-induced migration, whereas it had a limited effect on migration induced by intact Tat. It is interesting to note that anti-αvβ3 antibodies also inhibited f-MLP-induced dendritic cell migration, indicating that blocking of this integrin abolishes stimulus-induced migration. These results are in agreement with the recent observations by Rubartelli et al. [37] that demonstrate the involvement of the αvβ3 integrin in dendritic cell engulfment of apoptotic bodies and the ability of the Tat-RGD domain to inhibit this process [38]. Both chemotaxis and phagocytosis require the polarized interaction of integrins linked to intracellular microfilaments with the substratum. These observations suggest that αvβ3 could be the major mediator of dendritic cell polarized cytoskeletal interaction with the extracellular environment.
Macrophages and dendritic cells can harbour and release HIV for several months after their infection; these cells probably release Tat as well. Initial stages of HIV infection seem to target macrophages or dendritic cells by HIV strains tropic for these cell types, while progression to AIDS appears to involve a switch to T-cell tropic strains within the host [39-42]. It is possible that the dendritic and monocyte cell chemotactic activity of Tat could contribute to recruitment of other dendritic cells and monocytes/macrophages to the area of an infected dendritic cell or monocyte. The observations that HIV-LTR-Tat-producing cells show an increased ability to attract monocytes, and that Tat is able to recruit phagocytic monocytes in vivo, support this hypothesis. This function appears to be partially mediated by receptors recognizing the RGD domain (integrins), in particular αvβ3. Anti-αvβ3 antibodies can inhibit metastasis and angiogenesis [43], it is possible that these antibodies could also act as inhibitors of Tat-induced monocyte migration.
Acknowledgements
We thank C.E. Grossi (University of Genoa) for revision of the manuscript and M. Arvigo for electronmicroscopy.
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