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The Journal of Immunology, 2001, 166: 1380-1388.
Copyright © 2001 by The American Association of Immunologists

HIV-1 Tat Protein Stimulates In Vivo Vascular Permeability and Lymphomononuclear Cell Recruitment

Marco Arese*, Chiara Ferrandi*, Luca Primo*, Giovanni Camussi{dagger} and Federico Bussolino*

* Institute for Cancer Research and Treatment and Department of Genetics, Biology and Biochemistry, School of Medicine, University of Torino, Candiolo. Italy; and {dagger} Department of Medical and Surgery Sciences, School of Medicine, University of Torino, Torino, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HIV-1 Tat protein released by infected cells is a chemotactic molecule for leukocytes and induces a proinflammatory program in endothelial cells (EC) by activating vascular endothelial growth factor (VEGF) receptors expressed on both cell types. Its potential role in causing vascular permeability and leukocyte recruitment was studied in vivo following its s.c. injection in mice. Tat caused a dose-dependent early (15 min) and late (6 h) wave of permeability that were inhibited by a neutralizing Ab anti-VEGF receptor type 2. Tissue infiltration of lymphomononuclear cells, mainly monocytes (76%), was evident at 6 h and persisted up to 24 h. WEB2170, a platelet activating factor (PAF) receptor antagonist, reduced the early leakage by 70–80%, but only slightly inhibited the late wave and cell recruitment. In vitro, Tat induced a dose-dependent flux of albumin through the EC monolayer that was inhibited by Ab anti-vascular VEGF receptor type 2 and WEB2170, and PAF synthesis in EC that was blocked by the Ab anti-VEGF receptor type 2. Lastly, an anti-monocyte chemotactic peptide-1 (MCP-1) Ab significantly reduced the lymphomononuclear infiltration elicited by Tat. In vitro, Tat induced a dose-dependent production of MCP-1 by EC after a 24-h stimulation. These results highlighted the role of PAF and MCP-1 as secondary mediators in the onset of lymphomononuclear cell recruitment in tissues triggered by Tat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The host inflammatory response is a key event in early and late HIV-1 infection. Inflammatory cytokine secretion by activated immune cells favors virus replication, regulates lymphocyte traffic, and is instrumental to injury of lymphoid tissue (1, 2, 3). Similarly, leukocyte infiltration and abnormal cytokine response are typical features of HIV-1-associated tissue disorders, including tumors, opportunistic infections, and CNS degeneration (4).

In addition to the classic response of the immune system to foreign organisms, the altered host response is directly triggered by HIV-1 proteins, including envelope proteins (5, 6, 7), Nef (8), and the transactivating factor Tat, which up-regulates viral gene expression in infected cells (9) and modulates the expression of cellular genes, including those of IL-6 and TNF-{alpha} and {beta} (10, 11, 12), known to be inductive of HIV replication (13, 14, 15). Furthermore, Tat may alter cellular behavior when released by infected cells in the microenvironment (16, 17). Tat easily enters different cell types and contributes to transactivation of the HIV-1 long-terminal repeat promoter in latently infected cells (18, 19). Alternatively, it acts as a soluble mediator acting on T and B cells (20, 21, 22, 23, 24) and on those of the CNS (25, 26), thus favoring the progression of AIDS and its associated brain damage. Furthermore, Tat profoundly affects functions of vascular endothelial cells (EC)3 and the monocytic/macrophage system. It is an angiogenic inducer through activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2) and integrin system (27, 28) and induces a proinflammatory program characterized by the release of proteolytic enzymes (29), the up-regulation of adhesion molecules (30, 31), and vasopermeability (32). This results in the leukocyte extravasation essential for homing of infected lymphomononuclear cells into lymphoid organs and for the tissue injury typical of some features of the progression of AIDS. In monocytes, subnanomolar concentrations of Tat enhance the expression of CCR5 (33) and the release of inflammatory cytokines, TGF-{beta} and metalloproteinase-9 (34, 35, 36), and increase chemotaxis, chemokinesis (37, 38), and adhesion to EC (36). These activities are mediated by the activation of VEGFR-1 (38, 39) or by CCR2 and CCR3 (40). Tat also recruits monocytes into tissues when injected in the lateral ventricle of the brain (41). Furthermore, macrophage stimulation by Tat results in the immunosuppressive induction of Fas ligand (42) and impairment of the natural host response by inhibition of NO synthesis (43).

EC are strategically located at the interface between the bloodstream and tissues. They are thus both producers and the targets of autacoids and chemokines that regulate leukocyte traffic and vascular permeability during tissue injury (44).

In light of these in vitro effects of Tat on lymphomononuclear cells and EC, we studied its potential role in causing vascular permeability and leukocyte recruitment in a murine model. We demonstrate that s.c. injection of Tat causes accumulation of lymphomononuclear cells preceded by increased vascular permeability. These activities appear to be mediated by the autacoid platelet-activating factor (PAF) (45, 46, 47) and the chemokine monocytic chemotactic peptide-1 (MCP-1) (48).


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

[3H]acetyl-CoA (2.2 mCi/mmol), [3H]acetate (2 Ci/mmol), and [3H]acetyl-CoA (180 mCi/mmol) were obtained from Amersham Pharmacia Biotech (Buchs, U.K.); [125I]albumin (3 µCi/mg) was purchased from NEN (Boston, MA). Human LPS-free serum albumin was obtained from Farma Biagini (Lucca, Italy); TLC plates were purchased from Merck (Darmstadt, Germany); PAF (1-O-octadecyl-2-acetyl-(R)-glycero-3-phosphocholine) and lyso-PAF (1-O-octadecyl-2-lyso-glycero-3-phosphocholine) were obtained from Bachem Feinkemikalien (Bubendorf, Switzerland); WEB2170 was purchased from Boehringer Ingelheim (Ingelheim, Germany). Plastic materials and products for cell culture were obtained from Falcon (Becton Dickinson, Milano, Italy) and Life Technologies (Paisley, U.K.); VEGF-A, Ag affinity-purified goat polyclonal Ab anti-mouse MCP-1, Ag affinity-purified goat polyclonal anti-mouse VEGFR-2, and mouse MCP-1 were obtained from R&D Systems (Wiesbaden-Nordenstadt, Germany); anti-mouse CD8 (53.6.72 hybridoma, Lyt 2), anti-mouse CD4 (GK 1.5 hybridoma, L3T4), and anti-mouse Mac-3 (M37/84, 6, 34 hybridoma) were purchased from PharMingen (San Diego, CA); rabbit anti-rat IgG was purchased from Zymed (San Francisco, CA); and rat peroxidase-antiperoxidase was obtained from Abbot Laboratories (North Chicago, IL). Other reagents were obtained from Sigma (St. Louis, MO). LPS-free Tat86 and Tat101 proteins and Tat peptides covering aa 24–51 corresponding to cysteine and core regions (CysL24–51: NH2-NCY CCK KCC FHC QVC FIT KAL GIS YGR KK-COOH) (9) were synthesized by Tecnogen (Caserta, Italy), purified by HPLC, and quality controlled by mass spectroscopy (38). Tat peptide for aa 46–60 (Basic46–80: NH2-SYG RKK RRQ RRR PPQCOOH) corresponding to the basic region (9) was purchased from Technogenetics (London, U.K.). Two scrambled peptides of CysL24–51 (NH2- NCY KKC ISY CFC VIT KAL GCQ CFG RRKK-COOH) and Basic46–80 (NH2- KKG RRR SQP RYR PQR-COOH) peptides were purchased from Prinn (Milano, Italy). Recombinant wild-type HIV-1 Tat86 amino acid length was expressed in Escherichia coli as maltose-binding fusion protein (MBP), indicated throughout the text as Tat-MBP. MBP alone was the control in all experiments. Tat-MBP was purified to homogeneity from bacterial cell lysates by affinity chromatography on amylose resin and used as fusion protein (49). Synthetic and recombinant Tat molecules were stored at -80°C in aliquots of 5 µg/10 µl of PBS containing 0.1% human serum albumin, 0.1 mM ZnCl2, and 1 mM DTT. Biological activity of Tat-MBP was checked as the ability to induce transcriptional activation of the HIV-1 long-terminal repeat in HL3T1 cells containing the bacterial gene of chloramphenicol acetyltransferase directed by this repeat, as previously described (49). In this assay Tat-MBP inactivated by heating (10 min at 90°C) did not activate chloramphenicol acetyltransferase, and was used as negative control in all experiments.

Cells

EC from human umbilical cord veins and murine microvascular H.end endothelioma cells (H.end) were prepared, characterized, and grown as previously described (50, 51). H.end cells immortalized with the middle T Ag of Polyomavirus maintain the in vitro features of EC, including normal cell growth rate, cobblestone-like morphology, expression of vascular endothelial-cadherin, CD-31, and von Willebrand factor-related Ag, and the ability to respond to inflammatory cytokines IL-1 and TNF-{alpha} and to produce chemokines and PAF (51, 52, 53). Human dermal fibroblasts and human epidermal keratinocytes were purchased from Clonetics-BioWhittaker (Walkersville, MD) and grown according to the manufacture’s instructions. Human PBMC and monocytes were prepared from buffy coats of healthy donors as previously detailed (38). Murine spleen cells (spc) and peritoneal macrophages were prepared from BALB/c mice (Charles River, Calco, Italy) according to Refs. 54, 55 .

In vivo studies

Tat molecules and VEGF-A were s.c. injected into the lower back (right side) of BALB/c mice in PBS for different lengths of time. The negative control was injected into their left side. Thirty minutes before the end of the experiment, [125I]albumin (2 x 106 cpm/mouse) was injected i.v. Mice were then sacrificed, and a skin area of 25 mm2 around the injection point was excised and its radioactivity was counted in a beta counter. Histological skin alterations were studied by omitting the [125I]albumin injection. In selected experiments anti-VEGFR-2 Ab, anti-MCP-1 Ab, goat serum (20 µl of 1:50 dilution), or the PAF receptor antagonist WEB2170 (250 ng) (56) was coinjected with Tat-MBP or VEGF-A. Alternatively, 10 mg/kg WEB2170 in PBS was injected i.p. 30 min before the Tat-MBP treatment.

[125I]Albumin endothelial permeability assay

EC permeability was determined by calculating the ratio of [125I]albumin in the upper and lower chambers of a filter chamber assembly (pore diameter 0.2 µm, 24-mm diameter dish) (50). The upper chamber contained 200 mM BSA and 0.2 µCi [125I]albumin in 2 ml of M199 medium. The baseline permeability was determined after a 1-h incubation at 37°C in 5% CO2 (5.6 ± 0.9% of [125I]albumin added to the upper chamber was recovered in the lower chamber, mean ± SD of three experiments performed in triplicate). The baseline permeability of the membrane without EC was 61.0 1% (mean ± SD of three experiments performed in triplicate). Stimuli were added to the upper chamber, and incubation was continued for 1 h. Radioactivity was counted in the lower chamber, and the results are expressed as follows: Percent change albumin clearance: (cpm [125I]albumin after stimulus - cpm [125I]albumin control/cpm [125I]albumin control) x 100.

In some experiments EC were preincubated for 15 min with WEB2170 (5 µM), Ab anti-VEGFR-2, or goat serum (1:50).

PAF synthesis

EC were used within V passages without coating the plastic dishes with proteins, and growth medium was reefed the day before the experiment (57). EC (6 x 104/cm2), human fibroblasts (1.2 x 105/cm2), human keratinocytes (8 x 104/cm2), murine EC line H.end (7 x 104/cm2), spc (2 x 106/ml), murine macrophages (2 x 105/35-mm diameter well), and human PBMC and monocytes (2 x 106/ml) were washed twice with M199 containing 0.25% BSA and incubated in 5% CO2 for different times with Tat molecules. In some experiments cells were preincubated for 15 min at 37°C with Ab anti-VEGFR-2 or with goat serum (1:50). Cell-associated PAF was extracted, purified by TLC, and measured by washed rabbit platelet aggregation as described (57, 58). PAF was identified from its physicochemical characteristics and sensitivity to lipases as previously described (57, 58). In some experiments PAF synthesis was also quantified by a radiometric assay measuring the uptake of [3H]acetate into lipids comigrating with synthetic PAF (57). In this case, 3 x 105 human EC were stimulated in DMEM supplemented with 0.25% BSA. [3H]Acetate (100 µM) was added 1 h before the reaction was stopped. After extraction, labeled phospholipids comigrating with synthetic PAF on the TLC were counted in a Packard beta counter (Packard, Meriden, CT).

Assay of acetyl-CoA:lyso-PAF acetyltransferase in human EC

Cell extracts from stimulated or unstimulated EC were prepared as previously described (58). The assay was conducted for 10 min at 37°C in 0.2 ml of Tris-HCl 0.1, pH 6.8, containing 50 µg of lysate protein, 40 µM lyso-PAF, 100 µM acetyl CoA, and 1 µCi [3H]acetyl-CoA. Lipid purification and calculation of enzymatic activity was performed exactly as previously reported (58).

Light microscopy and immunohistochemistry

For microscope examination mouse skin was fixed in 10% buffered formalin and embedded in paraffin. Sections were cut at 5 µm and stained with hematoxylin and eosin. For immunohistochemistry, samples were embedded in OCT compound (Miles Laboratories, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -80°C. Five-micrometer cryostat sections were fixed in acetone, preincubated with rabbit serum, and subsequently with optimal dilutions of rat anti-mouse CD8 (1:100), anti-mouse CD4 (1:50), and anti-mouse Mac-3 (1:50). After washes, sections were incubated with rabbit anti-rat IgG and rat peroxidase-antiperoxidase. Each step lasted 30 min and was followed by a 10-min wash in TBS (pH 7.4). Sections were then incubated with 0.03% H2O2 and 0.06% 3,3'-diaminobenzidine for 5 min at room temperature, washed, and counterstained by hematoxylin. The number of positive cells was determined under a light microscope at x400 in five fields on a 1-mm2 grid and is given as cells/mm2.

MCP-1 production

EC (6 x 104/cm2), fibroblasts (1.2 x 105/cm2), keratinocytes (8 x 104/cm2), and monocytes (2 x 106/ml) were starved overnight in M199 supplemented with 2% FCS and 3% human serum albumin. Cells were stimulated at 37°C with Tat molecules in M199 containing 10% FCS for different periods. In some experiments performed on human EC, WEB2170 was used to check its inhibitory effect. The amount of MCP-1 released by human cells was measured with the Quantikine ELISA Kit (sensitivity 2 pg/ml; R&D Systems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo effects of Tat on vascular permeability and lymphomononuclear infiltration

Increased vascular permeability is an early event in inflammatory injury and often precedes or parallels leukocyte infiltration of tissues. To study the effects of Tat on vascular permeability and leukocyte traffic from bloodstream to tissues, we set up an in vivo model based on Tat molecules injected into mouse skin and on i.v. [125I]albumin injection 30 min before mice were sacrificed. Fig. 1Go shows that Tat-MBP caused a dose-dependent early (15 min) and a late (6 h) peak of albumin leakage. The maximal effect was obtained with 30 ng of Tat-MBP, but a significant leakage was obtained with 10 ng of Tat-MBP (corresponding to 0.7 pmol). MBP alone and heat-inactivated Tat-MBP were devoid of this activity. To further rule out the possibility that the effect observed was due to protein fusion instead of Tat, mice were injected with synthetic Tat86 and Tat101, and the albumin leakage was monitored. These Tat molecules, too, retained their vasopermeability activity (Table IGo).



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  		FIGURE 1. Time- and dose-dependent effects of Tat-MBP on skin permeability. Abdominal BALB/c mouse skin was injected with different concentrations of Tat-MBP, MBP (30 ng/30 µl), or Tat-MBP heat-inactivated (30 ng/30 µl) for 15 min, 90 min, 6 h, and 24 h. Thirty minutes before the end of the incubation time, mice were i.v. injected with [125I]albumin (2 x 106 cpm/mouse). Skin (an area of 25 mm2 around the injection point) was then excised, and the corresponding radioactivity was counted in a beta counter. Results shown are the mean ± SD of six animals. ANOVA gave the following results: mice treated for 15 min, F = 32.06; mice treated for 90 min, F = 6.34; mice treated for 3 h, F = 83.34; mice treated for 6 h, F = 3.31. *, p < 0.05 vs MBP-injected animals by Student-Newman-Keuls test.

 

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  		Table I. In vitro and in vivo effects of Tat86 and Tat101

 
VEGF receptors and CCRs are responsible for several of the extracellular effects of Tat (28, 38, 39, 40, 59, 60). To determine the type of receptor involved in albumin leakage, we used two peptides encompassing the basic (Basic46–80) and cysteine-rich and core (CysL24–51) domains of Tat; the former activates VEGFR-2 (28), and the latter CCR2 and CCR3 (40). As shown in Table IIGo, Basic46–80 strongly induced both leakage peaks, whereas CysL24–51 or a scrambled peptide did not. A further experiment with a neutralizing Ab anti-VEGFR2 indicated the operative role of this receptor in our model. The early effect of Tat-MBP and VEGF-A on [I125]albumin leakage was markedly reduced when this Ab was coinjected with the two molecules, whereas the nonimmune serum had no activity (Fig. 2Go). Inhibition of the late leakage by this Ab efficacy was less effective (32 ± 8% inhibition of [125I]albumin leakage induced by 30 ng Tat-MBP, n = 5), suggesting that other activation pathways played a role or that the Ab was degraded.


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  		Table II. Effect of Basic 46–80 and CysL24–51 on [I125]albumin leakage in mouse skin1

 


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  		FIGURE 2. Effects of the anti-VEGFR-2 Ab on Tat-MBP- and VEGF-A-induced skin permeability. Anti-VEGFR-2 Ab or an irrelevant Ig (20 µl of 1:50 dilution) were coinjected with Tat-MBP (30 ng/30 µl) or VEGF-A (40 ng/10 µl) in mouse skin for 15 min. Tat-MBP or VEGF-A alone were used as controls. [125I]Albumin (2 x 106 cpm/mouse) was injected i.v. 30 min before the end of the incubation. Skin (an area of 25 mm2 around the injection point) was then excised, and the corresponding radioactivity was counted in a beta counter. Results shown are the mean ± SD of six animals. ANOVA gave F = 62.50. *, p < 0.05 vs irrelevant Ig by Student-Newman-Keuls test.

 
Two hours after its injection, Tat-MBP induced leukocytes to arrest in capillaries (Fig. 3GoB, inset). Cell migration into tissue was evident after 2 h, peaked after 6 h, and persisted up to 24 h (Figs. 3Go and 4Go). This effect was dose dependent and maximum with 30 ng/30 µl Tat-MBP (Fig. 4Go). MBP alone or Tat-MBP inactivated by heating was ineffective (Figs. 3GoD and 4). Synthetic Tat86 and Tat101 were also active (Table IGo). Light microscopy indicated that the number of infiltrating polymorphonuclear cells was negligible and that the infiltrate was mainly composed of lymphomononuclear cells (Fig. 3Go). Skin sections from animals sacrificed after 3 and 6 h after 30 ng/30 µl Tat-MBP were stained with specific rat mAb anti-mouse CD4, CD8, and Mac-3. Mac-3-positive cells were the most abundant (76 ± 18%), whereas CD8 and CD4 accounted for 8 ± 5 and 16 ± 9% of total infiltrating cells, respectively (Fig. 5Go).



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  		FIGURE 3. Hematoxylin and eosin staining of mice skin injected with Tat-MBP. Skin was injected with Tat-MBP for 2 (B) and 6 (C) h or with MBP (A) or heat-inactivated Tat-MBP (D) for 6 h (30 ng/30 µl) (A). In MBP-injected skin the presence of lymphomononuclear cells was negligible and capillaries contained red cells (A, inset). After 2 h of Tat-MBP treatment, lymphomononuclear cells remained in capillaries (inset) and infiltrated s.c. tissue (B). After 6 h of Tat-MBP treatment, their number dramatically increased (C), whereas few cells were present at the heat-inactivated Tat-MBP injection site (D). A–D, x200; inset, x1000.

 


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  		FIGURE 4. Time- and dose-dependent effects of Tat-MBP on leukocyte infiltration in mouse skin. Mouse skin was injected with different concentrations of Tat-MBP, MBP (30 ng/10 µl), or Tat-MBP heat-inactivated (30 ng/30 µl) for 15 min, 3 h, 6 h, and 24 h. Skin sections were then excised, fixed in 10% buffered formalin, and embedded in paraffin. Then, sections were cut at 5 µm and stained with hematoxylin and eosin. The number of infiltrating cells was determined by light microscopy at x400 in five fields of a 1-mm2 grid and given as cells/mm2. Results shown are the mean ± SD of seven animals. ANOVA gave the following results: mice treated for 15 min, F = 3.39; mice treated for 3 h, F = 58.02; mice treated for 6 h, F = 172.23; mice treated for 24 h, F = 159.24. *, p < 0.05 vs MBP-injected animals by Student-Newman-Keuls test.

 


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  		FIGURE 5. Cell type-specific tissue infiltration elicited by Ta-MBP in mouse skin. Frozen skin sections from animals sacrificed after 3 and 6 h after Tat-MBP injection (30 ng/30 µl) were incubated with optimal dilution of rat anti-mouse CD8 (open columns), anti-mouse CD4 (hatched columns), and anti-mouse Mac-3 (filled columns). After washes, sections were incubated with rabbit anti-rat IgG and rat peroxidase-antiperoxidase, and the reaction was developed. The number of positive cells was determined by light microscopy at x400 in five fields on a 1-mm2 grid. Results shown are the mean ± SD of four animals. *, p < 0.05 by Student’s t test.

 
Role of PAF in Tat-induced skin permeability

VEGFR-2 stimulation by VEGF-A triggers PAF synthesis in human EC (61), and PAF induces vasopermeability (62) and alters in vitro EC barrier function (50). Therefore, we investigated the role of PAF in Tat-induced vascular leakage by i.p. injection of the specific PAF-receptor antagonist WEB2170 (56) or its coinjection with Tat-MBP in the skin. Table IIIGo shows that WEB2170 reduced the early phase of albumin leakage by 70–80%, but only slightly (0–2%) inhibited the late wave, indicating that other indicators were involved.


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  		Table III. Effect of WEB2170 on Tat-MBP-induced [I125]albumin leakage in mouse skin1

 
A direct effect of Tat on EC toward vascular permeability was demonstrated with an in vitro system in which confluent EC monolayers were cultured on transwell cell culture inserts. [125I]Albumin (2 x 106 cpm) was loaded in the upper chamber of the well along with Tat-MBP at different concentrations. After 1 h at 37°C the radioactivity of the lower well was counted. As shown in Fig. 6Go, Tat-MBP induced a dose-dependent trans-endothelial flux of albumin that was reduced by 90% by an anti-VEGFR-2 Ab. Irrelevant Ig did not change the response to Tat-MBP, whereas boiled Tat-MBP or MBP alone failed to reduce the flux. Basic46–80 peptide, but not CysL24–51 (Fig. 6Go) or the scrambled peptides (data not shown), induced an increase of albumin clearance through the EC monolayer. WEB2170 (5 µM) also inhibited albumin flux induced by Tat-MBP and by Tat86, as well as that induced by PAF (Fig. 6Go).



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  		FIGURE 6. Effects of Tat molecules on EC monolayer permeability. EC were cultured to confluence on a filter chamber transwell apparatus (pore diameter 0.2 µm, 24-mm diameter dish) and stimuli (Tat-MBP at the indicated concentrations, heat-inactivated Tat-MBP at 30 ng/ml, Tat86 at 10 ng/ml, Basic46–80 and CysL24–51 at 1 µg/ml, and PAF at 50 nM) were added to the upper chamber and incubated for 1 h. The upper chamber also contained 200 mM BSA and 0.2 µCi [125I]albumin in 2 ml of M199 medium. EC permeability was determined by calculating the ratio of [125I]albumin in the upper and lower chamber of the filter assembly. When indicated, EC were preincubated for 15 min with WEB2170 (5 µM), or with Ab anti-VEGFR-2 or goat serum (1:50) and then stimulated with the optimal concentration of Tat-MBP (30 ng/ml), PAF (50 nM), or Tat86 (10 ng/ml). Results shown are the mean ± SD of three experiments performed in triplicate. ANOVA gave F = 66.37. *, §, **, #, and °, respectively, indicate p < 0.05 vs MBP, Tat-MBP (30 ng), none, Tat86, and PAF by the Student-Newman-Keuls test.

 
These data suggest that PAF produced in mouse skin after Tat injection participates in the vasopermeability alteration. To detect the origin of PAF, several human and murine cells were stimulated with Tat86 at 10 ng/ml (an optimal concentration to alter albumin clearance), and their associated PAF was measured by a biological assay on washed rabbit platelets. As shown in Table IVGo only human and murine EC and monocytes/macrophages produced PAF, whereas human dermal fibroblasts and keratinocytes did not synthesize PAF after Tat86 challenge. The small amount of PAF produced by PBMC and spc may be due to the presence of monocytes. Because in our in vivo model lymphomononuclear cells began to be recruited after 3 h (Fig. 4Go) and the infiltrating population was almost entirely composed of monocytes (Fig. 5Go), it is reasonable to speculate that EC are the first to produce PAF. Detailed investigation of PAF production in human EC showed that after 30 min of stimulation 30 ng/ml Tat-MBP induced a peak of PAF synthesis that declined to basal levels within 45 min (Fig. 7Go). This effect was dose dependent up to 50 ng/ml (Fig. 7Go). Heat-inactivated Tat-MBP (30 ng/ml) (data not shown) and MBP (Fig. 7Go) were ineffective. PAF synthesis was also observed when EC were challenged with Basic46–80, whereas CysL24–51 and scrambled basic peptide were inactive (pmol PAF after a 30-min stimulation: None, 0.54 ± 0.21; Basic46–80 (1 µg/ml), 3.65 + 0.92; scrambled basic peptide (1 µg/ml), 0.39 ± 0.12; CysL24–51 (1 µg/ml), 0.41 ± 0.23; n = 3). Similar results were provided by a radiometric assay of the uptake of labeled acetate into PAF molecule (data not shown). PAF synthesis is preceded by activation of the specific acetyltransferase that is the key enzyme of the remodeling pathway (Fig. 7Go) (45, 46, 47). Preincubation of EC with the anti-VEGFR-2 abolished PAF synthesis triggered by 15-min cell stimulation with 30 ng/ml Tat-MBP (Tat-MBP + goat serum, 5.8 ± 0.5 pmol; Tat-MBP + anti-VEGFR-2, 1.2 ± 0.5 pmol; goat serum, 0.6 ± 0.2 pmol; anti-VEGFR-2, 0.8 ± 0.3 pmol; n = 3).


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  		Table IV. PAF production by human and murine cells stimulated by Tat molecules1

 


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  		FIGURE 7. Tat induces PAF synthesis in EC. PAF synthesis, evaluated by a washed rabbit platelet aggregation assay (A and B), and activity of acetyl-CoA:lyso-PAF acetyltransferase (C) were performed on confluent EC (6 x 104/cm2) stimulated with Tat-MBP ({circ}) or MBP (•) at 37°C. A, EC were stimulated with different concentrations of polypeptides for 20 min; B and C, EC were stimulated with 30 ng/ml of polypeptides for the indicated times. Results shown are the mean ± SD of four experiments performed in duplicate.

 
Role of chemokines in Tat-mediated lymphomononuclear infiltration

The PAF receptor antagonist WEB2170 (administered i.p. or coinjected with Tat-MBP) had a negligible effect on lymphomono-nuclear infiltration induced by Tat-MBP. It delayed the cell recruitment at 3 h, but did not reduce the number of infiltrating cells after 6 h (data not shown).

Because Tat-MBP up-regulates cytokine production in several cell types, we investigated the role of MCP-1 produced by EC. Coinjection of Tat-MBP with a specific blocking Ab anti-MCP-1 reduced by 65% lymphomononuclear infiltration induced by Tat-MBP (Fig. 8Go), and the anti-MCP-1 Ab resulted in a 90% reduction of the recruitment elicited by MCP-1. MCP-1 seems to act as a downstream mediator of the Tat inflammatory activity. Lastly, the role of EC as producers of MCP-1 upon Tat stimulation was studied by stimulating EC with Tat-MBP and evaluating the amount of MCP-1 released in the medium. As shown in Fig. 9Go, Tat-MBP, at concentrations ranging from 0.1 to 50 ng/ml, induced a dose-dependent production of MCP-1 that peaked after 24 h but was detectable after 6 h. At the optimal Tat-MBP concentration of 50 ng/ml, production of MCP-1 by EC was about one-third of that elicited by TNF-{alpha}, used as positive control (44). Synthetic Tat86 or Tat 101 were also active in terms of MCP-1 production (Table IGo), as well as Basic46–80, whereas CysL24–51 and scrambled basic peptide were ineffective (ng MCP-1/ml after a 24-h stimulation: None, 5.54 ± 2.02; Basic46–80 (1 µg/ml), 18.12 ± 4.03; scrambled basic peptide (1 µg/ml), 4.78 ± 1.15; CysL24–51 (1 µg/ml), 6.32 ± 2.10; n = 3). The presence of 20 µM WEB2170 did not change the MCP-1 production triggered by Tat-MBP (30 ng/ml) after a 12-h stimulation (Control, 3.6 ± 0.4 ng/ml; WEB2170, 4.1 ± 0.9 ng/ml; Tat-MBP, 15.4 ± 0.8 ng/ml; WEB2170 + Tat-MBP, 14.8 ± 1.4 ng/ml; n = 3).



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  		FIGURE 8. Role of MCP-1 in lymphomononuclear infiltration induced by Tat. Mouse skin was injected with Tat-MBP (30 ng/30 µl) or MCP-1 (20 ng/20 µl) for 6 h alone or Tat-MBP plus neutralizing Ab anti-MCP-1. Skin sections were then excised, fixed, and embedded in paraffin, then sections were cut and stained with hematoxylin and eosin. The number of infiltrating cells was determined by light microscopy at x400 in five fields of a 1-mm2 grid and is given as cells/mm2. Results shown are the mean ± SD of four animals.

 


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  		FIGURE 9. Tat induces MCP-1 production in EC. A, Confluent EC (6 x 104/cm2) were stimulated with Tat-MBP ({square}) or TNF-{alpha} ({blacksquare}) at different concentrations for 24 h. B, Confluent EC (6 x 104/cm2) were stimulated with Tat-MBP ({square}) (30 ng/ml) or vehicle ({circ}) for different times. The amount of MCP-1 released was measured with a specific ELISA kit. Results shown are the mean ± SD of three experiments performed in triplicate. *, p < 0.05 by the t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This paper describes the effects of s.c. injection of Tat in mouse skin. An acute inflammatory response marked by protein leakage and lymphomononuclear cell infiltration is induced.

Subnanomolar Tat concentrations induced an early (15-min) and a delayed (6-h) vascular permeability response. The extent of this effect in the early phase was comparable to that elicited by VEGF-A. This finding is quite remarkable because VEGF-A was originally identified on account of its enhancement of vascular permeability and called "vascular permeability factor". We then showed that Tat provokes lymphomononuclear recruitment into tissues. This is evident after 3 h and maximum after 6 h. It was mostly evident on monocytes, whereas the number of CD4- and CD8-positive cells was only slightly increased.

Synthesis of acetylated alkyl ethers of phosphorylcholine, namely PAF, is a response of cells stimulated by VEGF-A or Tat. VEGF-A induces rapid PAF synthesis in bovine EC through activation of VEGFR-2 (61, 63). In human monocytes, VEGF-A and Tat trigger the same biological response, but by activating VEGFR-1 (39). Furthermore, VEGF-A-induced protein extravasation in vivo is abolished by a selective PAF receptor antagonist (63). PAF is a mediator of cell-to-cell communication with a broad range of biological activities on inflammatory and noninflammatory cells (45). Vascular endothelium is a main target for PAF that promotes angiogenesis by inducing cell migration (64) and vascular leakage through modification of cytoskeleton (50, 62, 65, 66). Our results demonstrate that PAF plays a substantial role in the early protein leakage induced by Tat in murine skin and is produced by EC through activation of VEGFR-2. This conclusion is based on the following findings: 1) WEB2170, a PAF receptor antagonist, abrogated Tat-induced in vivo [125I] albumin leakage and 2) Tat-induced increase of [125I]albumin clearance in vitro; 3) by activation of the remodeling pathway, Tat stimulated in vitro PAF synthesis by EC from large vessels or microvasculature; and 4) both [125I]albumin clearance and PAF synthesis were inhibited by EC pretreatment with an Ab anti-VEGFR-2. The second wave of vascular permeability observed in mice injected with Tat was not reduced by WEB2170, suggesting that several mediators participate in the late phase. This is not surprising, because PAF triggers and participates in several inflammatory circuits (47).

Our in vitro results indicate that monocytes/macrophages stimulated by Tat also produce PAF, whereas skin fibroblasts and keratinocytes do not. However, the role of monocytes/macrophages as a PAF source seems to be negligible in our model. Monocytes appeared later than the early peak of permeability inhibited by the PAF receptor antagonist, and this antagonist did not interfere with the late wave. Furthermore, WEB2170 did not inhibit lymphomononuclear infiltration in vivo even though PAF receptor antagonists abrogate Tat-induced in vitro monocyte migration (39).

Tat is a powerful in vitro chemoattractant of monocytes (35, 38) and induces them to transmigrate across an EC monolayer (33, 35). Recruitment of lymphomononuclear cells by Tat was specifically inhibited by a mAb anti-MCP-1, a chemokine that potently activates monocyte migration at the site of inflammatory injury (48). EC are a substantial source of MCP-1 (44). Tat increased its baseline production, as already reported for a macrophage-derived cell line and for glial cells (33, 67, 68). Furthermore, Tat up-regulates the cognate receptor of MCP-1 on the surface of human monocytes (33, 69), indicating that it may facilitate the infiltration of monocytes into tissues via endothelium-derived MCP-1 production and by rendering them more susceptible to MCP-1.

Our data do not clarify the relationship between PAF and MCP-1 production. PAF up-regulates the MCP-1 transcript in human EC (70). However, the PAF receptor antagonist WEB2170 did not reduce Tat-induced skin lymphomononuclear cell recruitment and did not block the MCP-1 synthesis in EC stimulated in vitro by Tat, which means that PAF is not a secondary mediator of Tat-induced MCP-1 production in human EC. Furthermore, blocking of its receptor does not inhibit MCP-1 release into plasma during endotoxemia in chimpanzees (71).

Tat exerts its extracellular biological activity by activating two receptor families: the tyrosine kinase VEGF receptors and the G protein-coupled CCRs. Structure-activity relationship studies have established that the Tat basic domain binds to and activates VEGFR-2, and the Cys rich and the core domains are the molecular determinants responsible for CCR2 and CCR5 engagement (28, 38, 40). Our in vitro and in vivo results show that stimulation by Tat of VEGFR-2, which is primarily expressed on EC (reviewed in Refs. (72, 73), is crucial for establishment of increased vascular permeability. The dramatic reduction in [I125]albumin leakage elicited by an anti-VEGFR-2 Ab in both Tat-MBP- and VEGF-A-treated skin indicates that both molecules activated in vivo VEGFR-2, as also reported in vitro on EC and Kaposi’s sarcoma cells (60, 74). Similarly, this Ab blocked, in vitro, the augmented albumin clearance and the PAF synthesis triggered by Tat. Mirrored experiments with Basic46–80 and CysL24–51 peptides corroborated the importance of VEGFR-2. Basic46–80 peptide caused in vivo vasopermeability and increased the albumin clearance through the EC monolayer as well as PAF and MCP-1 production by EC. In contrast CysL24–51 did not share this activity, which may exclude the involvement of chemokines receptors.

These data shed new light on the molecular mechanism through which extracellular Tat may be essential to alter the vascular permeability and recruit monocytes into tissues, including lymphoid organs, where they cooperate in inducing injury and represent a cellular reservoir for HIV replication.


   Footnotes
 
1 This study was supported by Istituto Superiore di Sanità, Programma Nazionale di Ricerca sull’AIDS: Patogenesi, immunità e vaccino per l’ AIDS (40B.19), Patologia, clinica e terapia dell’AIDS (30B.9 and 30B.10), and Italian Association for Cancer Research (Associazione Italiana per la Ricerca sul Cancro), Ministero dell’ Università e della Ricerca Scientifica e Tecnologica (60%) and Programmi di Ricerca di Rilevante Interesse Nazionale, 1998, 1999, and 2000 Regione Piemonte and Consiglio Nazionale delle Ricerche (P.F. Biotecnologie). Back

2 Address correspondence and reprint requests to Dr. Federico Bussolino, Institute for Cancer Research and Treatment, strada provinciale 142, kilometer 3,95, 10060 Candiolo (Torino), Italy. Back

3 Abbreviations used in this paper: EC, endothelial cells(s); Basic46–80, Tat peptide for aa 46–60; CysL24–51, Tat peptide covering aa 24–51 corresponding to the cysteine and core region; MBP, maltose-binding fusion protein; MCP-1, monocytic chemotactic peptide-1; PAF, platelet-activating factor; spc, murine spleen cells; Tat-MBP, Tat-MBP fusion protein; VEGF, vascular endothelial growth factor; H.end cells, EC from human umbilical cord veins and murine microvascular H.end endothelioma cells. Back

Received for publication May 12, 2000. Accepted for publication October 24, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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