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Originally published online as doi:10.1189/jlb.0407251 on August 17, 2007

Published online before print August 17, 2007
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(Journal of Leukocyte Biology. 2007;82:1212-1220.)
© 2007 by Society for Leukocyte Biology

Inhibition of HIV replication by the plasminogen activator is dependent on vitronectin-mediated cell adhesion

Chiara Elia*, Edana Cassol*, Nicolai Sidenius{dagger},{ddagger}, Francesco Blasi{dagger},{ddagger},§, Antonella Castagna||, Guido Poli*,§ and Massimo Alfano*,1

* AIDS Immunopathogenesis and
{dagger} Molecular Genetics Units and
|| Division of Infectious Diseases, San Luigi AIDS Centre, San Raffaele Scientific Institute, Milan, Italy;
{ddagger} Istituto Firc di Oncologia Molecolare (IFOM), Milan, Italy; and
§ Università Vita-Salute San Raffaele University, School of Medicine, Milan, Italy

1 Correspondence: P2/P3 Laboratories, DIBIT, AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Via Olgettina, 58, 20132 Milan, Italy. E-mail: massimo.alfano{at}hsr.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Urokinase-type plasminogen activator (uPA), an inducer of macrophage adhesion, inhibits HIV-1 expression in PMA-stimulated, chronically infected U1 cells. We investigated whether uPA-dependent cell adhesion played a role in uPA-dependent inhibition of HIV-1 replication in these cells. Monocyte-derived macrophages (MDM) were generated from monocytes of HIV-infected individuals or from cells of seronegative donors infected acutely in vitro. U1 cells were stimulated in the presence or absence of uPA in standard tissue culture (TC) plates, allowing firm cell adhesion or ultra-low adhesion (ULA) plates. Moreover, U1 cells were also maintained in the presence or absence of vitronectin (VN)-containing sera or serum from VN–/– mice. Virus production was evaluated by RT activity in culture supernatants, whereas cell adhesion was by crystal violet staining and optical microscopy. uPA inhibited HIV replication in MDM and PMA-stimulated U1 cells in TC plates but not in ULA plates. uPA failed to inhibit HIV expression in U1 cells stimulated with IL-6, which induces virus expression but not cell adhesion in TC plates. VN, known to bind to the uPA/uPA receptor complex, was crucial for these adhesion-dependent, inhibitory effects of uPA on HIV expression, in that they were not observed in TC plates in the presence of VN–/– mouse serum. HIV production in control cell cultures was increased significantly in ULA versus TC plates, indicating that macrophage cell adhesion per se curtails HIV replication. In conclusion, uPA inhibits HIV-1 replication in macrophages via up-regulation of cell adhesion to the substrate mediated by VN.

Key Words: inflammation • AIDS • acute infection • chronic infection • amino-terminal fragment


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Urokinase-type plasminogen activator (uPA), a serine protease, which activates plasminogen to plasmin [1 ], is synthesized as an inactive precursor (pro-uPA), undergoing proteolytic activation. uPA binds to a specific receptor, uPAR, localized at the cell surface [2 ]. uPA and uPAR are expressed by inflammatory cells including neutrophils, monocytes, macrophages, and activated T lymphocytes [2 ], in which they play important roles in cell activation, adhesion, and migration [3 , 4 ].

uPA and uPAR are involved in inflammatory processes [5 ] as well as in the pathogenesis of several diseases, including cancer [6 ], acute myocardial infarction [5 ], and HIV/AIDS. In this regard, high serum levels of soluble uPAR (suPAR) were strongly correlated to HIV disease progression, independently of the numbers of circulating CD4+ T cells or viremia levels in a cohort of HIV-1-infected individuals naïve to antiretroviral therapy [7 , 8 ]. Furthermore, cerebrospinal fluid levels of suPAR were highly correlated to HIV replication in the CNS in individuals with AIDS-dementia complex [9 ]. Therefore, the uPA-uPAR interaction may play an important role in the pathogenesis of HIV-1 infection and its progression toward AIDS. In this regard, we and others [10 , 11 ] have already reported that uPA inhibits HIV replication and expression in acutely infected promonocytic U937 cells and in chronically infected U937-derived U1 cells stimulated by PMA or TNF-{alpha}. These inhibitory effects were mediated by the receptor-binding and signaling-competent amino-terminal fragment (ATF) of uPA but not by its catalytic low molecular weight (LMW) component [11 ]. In addition, we observed inhibitory effects of uPA or ATF in acutely infected promonocytic U937 cell lines and primary lymphoid histocultures but not in mitogen-activated PBMCs [11 ].

As ATF is known to mediate uPA-induced cell adhesion, migration, and proliferation in different cell types [12 , 13 ], we here investigated the potential correlation between these biological effects of uPA and its anti-HIV activity. In particular, we have tested the influence of uPAR-mediated cell adhesion on HIV replication in primary monocyte-derived macrophages (MDM) and chronically infected U1 cells stimulated with PMA. We demonstrate that uPA/ATF inhibits HIV replication in acutely infected MDM differentiated in the presence of uPA in association with an increased cell adhesion to the plastic substrate. We further demonstrate that the uPA-inhibitory effect in PMA-stimulated U1 cells is highly correlated to an increased cell adhesion mediated by vitronectin (VN), a serum component known to bind to the uPA/uPAR complex [14 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
HIV+ individuals
PBMC were isolated from five HIV-infected individuals on a Ficoll-Hypaque (Pharmacia, Piscataway, NJ, USA) density gradient and their monocyte cultured in the presence or absence of uPA as specified later. Clinical parameters of HIV-infected individuals at the time of cell isolation are reported in Table 1 . No HIV isolation was obtained concomitantly by cocultivation of the PBMC isolated from these individuals with allogeneic T cell blasts.


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  		Table 1. Clinical Parameters of HIV+ Individuals

 
Reagents
Dr. Jack Henkin (Abbot Laboratories, Abbott Park, IL, USA) provided endotoxin-free (≤2x10–5 EU/IU corresponding to <10–10 EU/mg endotoxin), human pro-uPA (52 kDa), whereas the uPA ATF and the LMW fragment were purchased from American Diagnostica (Stamford, CT, USA). Pro-uPA, ATF, and LMW were used at 10 nM, based on previous studies [11 ]. Pro-uPA is converted immediately in cell culture in its active form, uPA, by cleavage mediated by plasmin [15 ]. PMA, used at 10–8 M, crystal violet, goat anti-mouse FITC, and rabbit anti-goat FITC antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anti-CD11b [(integrin {alpha}M (44)] mAb and its isotype control were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-integrin {alpha}Vβ3 mAb LM609 was obtained from Chemicon International (Temecula, CA, USA). Recombinant (r)IL-6 (used at 10 ng/ml, based on previous observations [16 17 18 ]) was purchased from R&D Systems (Minneapolis, MN, USA). Human-denatured VN was purchased from Promega (Madison, WI, USA) and stored at 2 µg/µl at –80°C. Tissue culture (TC) 96-well microtiter plates were purchased from Falcon (BD Biosciences, Bedford, MA, USA), whereas ultra-low adhesion (ULA) microtiter plates were bought from Corning Inc. (Corning, NY, USA). Dr. Flavio Curnis (San Raffaele Scientific Institute, Milan, Italy) kindly provided the Arg-Gly-Asp (RGD)-containing peptide, highly specific for {alpha}Vβ3 integrin (RGD-4c peptide) after biological testing for interference with cell adhesion of {alpha}Vβ3-positive EA.hy926 human cells [19 ]. Sera from wild-type (WT) C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA), whereas sera from VN–/– C57BL/6J mice were a kind gift of Dr. David Ginsburg (University of Michigan, Ann Arbor, MI, USA) [20 ]. Sera from WT and VN–/– C57BL/6J mice, as well as FCS and normal human AB serum (NHS; B-4800, Cambrex BioScience, Verviers, Belgium) were heat-inactivated before use. All reagents used for culture mediums were endotoxin-free, i.e., below the limit of sensitivity of the Limulus amoebocyte lysate assay (0.25 EU/ml), as certified by the manufacturers. rIL-6 was reported by the manufacturer to contain up to 100 pg LPS per µg protein, resulting in a final endotoxin concentration of 1.0 pg/mL when used in U1 cells. However, as unstimulated or IL-6-stimulated U1 cells are CD14-negative, virus replication was not influenced by LPS, as reported previously [21 , 22 ] and confirmed by us. Finally, LPS contamination in culture supernatants was not detectable in culture supernatants of U1 cells and MDM.

Isolation, culture, and HIV infection of primary human MDM
PBMC from healthy, seronegative donors or from HIV-infected individuals were separated on a Ficoll-Hypaque (Pharmacia) density gradient. Suspensions of 8 x 106 cells/ml in complete medium (DMEM, supplemented with 10% FCS, 5% NHS, 1% penicillin, 1% streptomycin, and 1% glutamine) were allowed to adhere to plastic for 2 h at 37°C. Floating cells were removed, and adherent cells were incubated for an additional 24 h. The average of CD14+ monocytes estimated by FACS analysis was always >85%. Then, adherent cells were collected and resuspended in complete medium at 5 x 105 cells/ml, and 200 µl cell suspension was seeded in 96-well plates. MDM differentiation was allowed to occur for 7 days before infection; half of the kinase culture supernatant was replaced with fresh complete medium at the 4th day of culture. HIV seronegative-derived MDM were infected with the CCR5-dependent (R5) HIV-1BaL strain at a multiplicity of infection (MOI) of 0.1, as described previously [23 ]. Fifty percent of the culture supernatants were collected every 3–4 days postinfection and stored at –20°C for determination of RT activity content, and the MDM cultures were fed with an equivalent volume of fresh complete medium.

Vesicular stomatitis virus G protein (VSVg)-Env-pseudotyped HIV-1 was prepared as described previously [24 ] and used at a MOI of 0.1. Briefly, to generate Env-pseudotyped virus, 293T cells were cotransfected by the Fugene 6 transfection protocol (Boehringer Mannheim, Germany) with NL4-3 proviral constructs carrying Env-defective reading frames, followed by an internal ribsosome entry site, the enhanced GFP gene, and a plasmid (pHIT-G) expressing the Env protein of the VSVg [25 ]. Titer of viral stock was determined on MDM (2x105 infectious particles/ml).

Chronically HIV-infected, promonocytic U1 cell line
This cell line was obtained from limiting dilution cloning of U937 cells surviving the cytopathic effect of acute, CXCR4-dependent HIV-1LAI/IIIB infection; each U1 cell contains two copies of integrated proviruses [26 ]. High levels of virus expression are induced rapidly by stimulation of U1 cells with PMA or several cytokines, including IL-6 [27 ]. U1 cells are a faithful indicator of regulatory pathways influencing HIV morphogenesis and production in monocytic cells [28 ]. U1 cells were always seeded at the density of 2 x 105 cells/ml in RPMI-1640 medium containing 10% FCS and incubated with pro-uPA, ATF, or LMW just prior to stimulation.

HIV replication
Virus replication was monitored by determination of Mg++-dependent reverse transcriptase (RT) activity in culture supernatants, as described [24 ].

Western blot analysis of cell-associated HIV-1 proteins
Cell-associated viral proteins and actin were separated as reported [24 ], and chemiluminescence was revealed with a digital high resolution cooled charged coupled-device camera system (UVP BioImaging Systems, Upland, CA, USA) on top of the Epi Chemi II darkroom (UVP BioImaging Systems) in conjunction with LabWorks Image Acquisition (UVP BioImaging Systems) and quantified by Analysis software (UVP BioImaging Systems).

Cell adhesion assay
The cell adhesion assay described here has been used previously and validated for the study of the uPA/uPAR system regulating U937 cell adhesion [29 ]. Briefly, U1 cells or monocytes were resuspended in medium containing 10% FCS or 10% FCS plus 5% NHS, respectively. U1 cells and monocytes were in suspension at the time of seeding but became adherent upon PMA stimulation or during differentiation to MDM. U1 cells were incubated in the presence or absence of pro-uPA and stimuli (PMA and IL-6) and seeded in triplicate cultures per each experimental condition in 96-well microtiter plastic plates. Cells were washed with warm (37°C) medium, fixed for 15 min with 3% paraformaldehyde in PBS, stained for 10 min with 0.5% crystal violet (20/80 methanol/water), washed three times, and lysed with 100 µl SDS (1% in water). The results were quantified by measuring the absorbance at 570 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). In certain experiments, a 96-well microtiter TC plate was coated overnight at 4°C with different concentrations of VN (from 10 up to 1000 ng/well) in PBS. The plates were then left 30 min at 37°C and then incubated with BSA (1% in PBS) for 1 h at 37°C before cell seeding.

Optical microscopy
Pictures of cells were acquired with an optical microscope Leica DHIL at magnification of 40x and analyzed with Leica FireCam software.

Statistical analysis
All data are expressed as mean ± SD. Paired t-test with two tails was applied where appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
uPA inhibits HIV-1 replication in primary MDM infected in vivo or in vitro
We and others [10 , 11 ] have shown previously that uPA and ATF inhibit HIV replication in promonocytic U937 cell lines acutely infected with an X4 viral strain and virus expression in U937-derived, chronically infected U1 cells stimulated with PMA. Thus, we here evaluated the potential anti-HIV effect of these molecules in primary MDM, derived from monocytes of HIV-infected individuals or infected acutely in vitro with an R5 HIV-1 strain. Primary monocytes were isolated from PBMC of five infected individuals and induced to differentiate into MDM in the presence or absence of uPA before infection. Productive HIV replication was detected in two out of five MDM cultures established from HIV-infected individuals. Of interest, cultivation of these monocytes in the presence of uPA abolished virus replication in the two HIV+ MDM cultures (Fig. 1A ), and no virus replication was observed with MDM established from three additional HIV+ individuals in the presence or absence of uPA.


Figure 1
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  		Figure 1. uPA inhibits HIV replication in MDM established from monocytes of HIV+ individuals or infected in vitro. (A) Monocytes were purified from PBMC of two HIV-infected individuals (Pts. 1 and 2) and seeded in TC microtiter plates. Cells were cultured in the absence or presence of uPA, which was added back every 3–4 days up to the end of the cell culture period. uPA suppressed HIV replication completely in these two independent MDM cultures, and it had no effect on four other MDM cultures established from monocytes of HIV-infected individuals listed in Table 1 . (B) Monocytes from healthy, HIV-seronegative individuals were seeded in 96-well TC microtiter plates and cultured for 7 days in the presence or absence of uPA. MDM were then washed and infected in the absence of uPA ({blacksquare}) or were incubated with fresh uPA added every 3 days throughout the cell culture period ({square}amp;). The percentage of viral replication in uPA-stimulated versus unstimulated MDM (defined as 100%) per time-point is reported, indicating the means ± SD of all tested cultures (n=11, assayed in triplicate). (C) MDM differentiated in the presence or absence of uPA were infected and then cultured again in the presence or absence of uPA, as described in B ({square}amp;). Seven days after infection, levels of cell-associated HIV proteins (left, arrows) were revealed by Western blot. (Left) Results from an individual experiment, representative of four experiments, conducted with cells of four independent donors; (right) the ratio between the expression of HIV proteins and β-actin. Columns and vertical bars represent the average ± SD of samples from four independent experiments. (D) MDM were cultured in 96-well TC or ULA microtiter plates in the presence or absence of uPA before and after infection. uPA inhibited HIV-1 replication (peak virus production observed 21 days after infection) in TC but not in ULA plates (left panel). Higher levels of virus production were observed when MDM were cultured in ULA rather than TC plates (right), where each symbol represents an independent infection of MDM established from different seronegative donors.

 
When monocytes from HIV-seronegative individuals were maintained in the presence of uPA during their differentiation to MDM a 7- to 10-day delay in the kinetics of HIV replication was observed, although virus production then bounced to control levels (Fig. 1B) . However, a persistent inhibition of HIV replication was observed when uPA-differentiated MDM were maintained in culture in the presence of uPA-enriched medium throughout the cell culture for several weeks (Fig. 1B) . We did not detect any modulation of CD4 and CCR5 expression at the time of MDM infection, after the 7 days of differentiation in the presence of uPA (data not shown). Furthermore, uPA inhibited virus expression in MDM infected with VSVg-Env-pseudotyped HIV-1 (52–57% inhibition up to 13 days postinfection), bypassing the requirement for receptor/coreceptor-dependent viral entry [30 ]. Consistent with our previous findings in an acutely infected promonocytic U937 cell line and chronically infected U1 cells [11 ], uPA did not inibit the levels of viral proteins in infected MDM (Fig. 1C) , therefore indicating that its primary mechanism of antiviral action influences a late event in the viral life cycle also in primary macrophages.

Of interest, MDM differentiated in the presence of serum and uPA showed a significant increase in their adhesion to the plastic substrate versus control MDM (140±10%, P=0.01; n=5), as described previously [31 32 33 ]. Based on the fact that increased cell adhesion was associated with decreased viral replication, we investigated the possible contribution of uPA-mediated cell adhesion to HIV replication. uPA-inhibitory effects on HIV replication in primary cells were observed when MDM were cultured in adherent conditions, i.e., in TC plates, but were abolished completely in ULA microtiter plates characterized by low binding of serum proteins [34 ] (Fig. 1D , left). Of note is the fact that viral replication in control cells was increased in MDM cultured in ULA versus TC plates (Fig. 1D , right), as reported independently [35 ].

Cell adhesion inhibits HIV-1 expression in PMA-stimulated U1 cells
Thus, to better characterize the anti-HIV mechanism of uPA, we next investigated whether the uPA, anti-HIV effect exerted on U1 cells, stimulated with the well-known, differentiating agent PMA (inducing a macrophage-like phenotype in U1 cells) [36 ], was also correlated to an up-regulation of cell adhesion. A later time-point (i.e., 24 h stimulation) was explored previously to characterize the mechanism of uPA/uPAR in mediating adhesion of U937 cells [29 ], the parental (uninfected) clone of U1 cells. In our experimental conditions, cell adhesion was typically measured after 48 h of stimulation, a time-point in which the number of adherent cells reached a plateau. Moreover, 48 h of U1 stimulation was also the first time-point in which HIV expression and anti-HIV activity of uPA and ATF are deleted firmly and consistently. Therefore, we cultured U1 cells in medium containing 10% FCS in TC or ULA plastic plates. Indeed, PMA stimulation induced U1 cell adhesion in standard TC plastic plates, and this effect was increased in the presence of uPA; in contrast, PMA failed to induce U1 cell adhesion in ULA plates (Fig. 2A ). In parallel, morphological changes upon uPA treatment of U1 cells were also evaluated. uPA enhanced U1 cell adhesion (Fig. 2A) and prevented PMA-induced cell clustering (Fig. 2B) . In contrast, when U1 cells were cultured in ULA plates, uPA did not prevent PMA-induced cell clustering (Fig. 2B) . Of note is the fact that uPA failed to inhibit PMA-induced viral expression in ULA plates (Fig. 2C) . Results similar to those here described with ULA plates were observed when cells were cultured in Teflon-coated wells, also preventing monocytic cell adhesion (data not shown). As already observed with HIV-infected MDM, PMA-stimulated U1 cells also expressed higher levels of virus when cultured in ULA versus TC plates (Fig. 2C and 2D) , supporting the concept that cell adhesion per se mediates an inhibitory signal for HIV expression in macrophages [35 ]. Common to PMA-stimulated U1 cells and MDM cultured in TC or ULA plates is the fact that both cell types express similar levels of uPAR (CD87), as evaluated by means of cytofluorimetric analysis and confocal microscopy, respectively (data not shown). Therefore, we excluded the possibility that the lack of the anti-HIV effect of uPA in nonadherent cells was a result of a lower expression of CD87.


Figure 2
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  		Figure 2. Cell adhesion inhibits HIV expression in PMA-stimulated U1 cells, which were stimulated with PMA in the presence or absence of uPA and then seeded in TC or ULA microtiter plastic plates. Cell adhesion (A) as well as cell clustering (B) were measured 48 h after stimulation, whereas virus expression was followed up for 5 days (C). (D) Statistical analysis of RT activity levels in PMA-stimulated U1 cells (n=5; paired t-test with two tails).

 
IL-6 is a well-known up-regulator of HIV expression in U1 cells [37 ], although unlike PMA, it does not induce cell adhesion to the plastic substrate in the presence or absence of uPA, ATF, or LMW (Fig. 3A ). Consistently with a tight interdependence between cell adhesion and inhibition of HIV production in monocytic cells, neither uPA nor ATF inhibited IL-6-dependent up-regulation of virus production in U1 cells (Fig. 3B) .


Figure 3
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  		Figure 3. Serum dependency of uPA/ATF up-regulation of cell adhesion and inhibition of HIV expression in U1 cells stimulated with PMA but not IL-6. U1 cells were incubated with uPA, ATF, or LMW and left unstimulated or stimulated with PMA or IL-6. Neither uPA nor its derivatives affected adhesion of otherwise unstimulated U1 cells. uPA and ATF but not LMW enhanced cell adhesion, promoted by PMA-induced differentiation of U1 cells (A). This effect was associated with a similar pattern of inhibition of HIV expression (B). IL-6 induced HIV expression but not cell adhesion and was insensitive to the inhibitory effects of uPA. Cell adhesion (C) and HIV expression (D) were evaluated in U1 cells resuspended in medium with different concentrations of FCS and then incubated or not with uPA and PMA. These results were maximal after 48 h, as shown here, and were confirmed up to 5 days after stimulation. Columns and symbols represent the means ± SD of samples run in triplicate. One representative experiment out of seven (A and B) or out of four (C and D) performed independently is shown. **, P = 0.05; *, P < 0.001.

 
Serum VN mediates the HIV-inhibitory effects of uPA in PMA-stimulated U1 cells
As serum proteins are known to mediate cell adhesion [38 ], we have investigated the potential role of serum in mediating the modulatory effects of uPA in PMA-stimulated U1 cells. A concentration-dependent decrease of uPA-induced cell adhesion was observed at lower serum concentrations (Fig. 3C) . In parallel, uPA-suppressive effects on HIV expression were reduced substantially in the presence of 2% serum (Fig. 3D) .

Among other major serum proteins, VN binds to the uPA/uPAR complex, inducing increased cell adhesion [14 ]. Therefore, we next investigated whether VN was involved in the modulatory effects of uPA in PMA-stimulated U1 cells. TC wells were then coated with different amounts of human rVN, and U1 cells were seeded in medium with 2% FCS, a condition in which uPA failed to inhibit HIV expression (Fig. 3D) . However, addition of increasing concentrations of VN restored uPA anti-HIV activity in PMA-stimulated U1 cells (Fig. 4A ). The VN dependence of the anti-HIV effects of uPA and ATF was demonstrated further by cultivating U1 cells in medium supplemented with serum from WT or VN–/– mice. As observed with FCS, the anti-HIV activity of uPA and ATF in the presence of 20% of WT mouse serum was higher than those exerted at 10% serum (Fig. 4B) . It is striking enough that uPA and ATF lost their suppressive effects when PMA-stimulated U1 cells were cultured in medium containing serum from VN–/– mice (Fig. 4B) , indicating the central role of VN in mediating uPA/ATF effects on HIV expression.


Figure 4
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  		Figure 4. VN mediates uPA/ATF anti-HIV effects in PMA-stimulated U1 cells (A), which were washed and resuspended in medium with 2% FCS and seeded in plastic wells coated with increasing amounts of human VN and were left unstimulated or stimulated with PMA in the presence or absence of uPA. Control U1 cells were cultured in standard conditions (medium with 10% FCS) in the absence of coated VN. No significant effects of VN coating were observed when U1 cells were stimulated in medium with 10% FCS. (B) U1 cells were washed and resuspended in medium containing 10% or 20% sera from WT or VN–/– mice. Cells were then stimulated with PMA in the presence or absence of uPA or ATF. Neither uPA nor ATF exhibited inhibitory effects on HIV expression in medium enriched with serum from VN–/– mice. One representative experiment out of three independently performed is shown. Columns and bars represent means ± SD of samples tested in duplicate.

 
It should be underscored that in addition to the uPA/uPAR complex, VN can bind to the {alpha}Vβ3 and {alpha}Vβ5 integrins [39 ], which however, were not expressed in unstimulated or PMA-stimulated U1 cells (data not shown). Furthermore, neither an RGD peptide highly specific for {alpha}Vβ3 (i.e., RGD-4c, 5–150 µg/ml) nor the inhibitory {alpha}Vβ3 (LM609) mAb interfered with uPA-induced events in U1 cells (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we report that uPA is a strong inhibitor of late events in HIV-1 replication in primary MDM, derived from HIV-infected individuals or infected in vitro. This inhibitory effect of uPA, as well as that exerted on PMA-stimulated U1 cells, was linked strongly to the enhancement of cell adhesion to the substrate and was mediated by the serum protein VN. UPA-inhibitory effects on MDM or U1 cells were lost completely when cells were not allowed to adhere firmly to the plastic substrate, as in ULA plates, or when U1 cells were stimulated to produce new progeny virions with IL-6, a cytokine, which unlike PMA, does not induce U1 cell differentiation and adhesion.

Among the multiple biological effects induced by uPA, some are not accounted for by its catalytic activity displayed by its C-terminal LMW fragment but by its receptor-binding and signaling component ATF [6 ]. Relevant to the present study, it has been demonstrated previously that only ATF, but not LMW, was able to promote VN binding to uPAR [40 ]. The binding site of uPAR in VN has been located in the N-terminal somatomedin B domain of VN [41 ], in close proximity to the RGD sequence required for integrin binding. Several uPAR-mediated effects have been ascribed to direct binding of uPAR to integrins or to the interference of integrin-binding to the extracellular matrix (ECM) substrate [42 43 44 45 46 47 48 ]. In particular, several studies have identified amino acid sequences in the {alpha} subunit of integrins, which bind uPAR [42 , 44 , 49 ], whereas Domain 2 of uPAR plays a structural and functional role in this interaction [47 ]. However, alanine mutations in Domain 2 of uPAR have no effect on uPAR functions, as mutations at these sites have demonstrated that uPAR binding to VN (but not to integrins) is at the basis of the cell migration effects mediated by uPAR [50 ]. Indeed, cell surface uPAR binding to VN increases RGD-independent cell adhesion [50 ]. Thus, the demonstration of the crucial role of VN in uPA/uPAR-dependent inhibition of HIV replication sheds new light on our original observation [11 ]. According to these findings, the uPA-mediated effects demonstrated in chronically infected U1 cells stimulated with the differentiating agent PMA; i.e., increased cell adhesion and inhibition of HIV replication are likely independent of an RGD/integrin interaction. Moreover, preliminary observations suggest to exclude the contribution of integrin {alpha} chains, in agreement with a recent study [50 ]. Our current observations demonstrate that the anti-HIV effect, dependent on uPA/uPAR interaction, requires VN, a ligand of uPAR, whereas VN binding to uPAR induces a series of effects in cell shape, cytoskeleton remodeling, cell adhesion, and migration [42 , 46 , 50 , 51 ]. Our findings also indicate that macrophage adhesion delivers an inhibitory signal for HIV replication, as reported previously in U937 cells and lymphoid tissue [11 ], and that this pathway is dependent by the formation of a VN/uPA/uPAR complex. Therefore, the VN/uPA/uPAR complex likely drives macrophages toward an activated phenotype, poorly supporting HIV replication [52 ].

Monocyte adherence has been reported to enhance the expression of proinflammatory cytokines such as IL-1β, TNF-{alpha}, IL-6, and IL-8 [53 54 55 ], known to stimulate HIV expression [56 ]. Conversely, other authors have reported that cellular adhesion inhibits expression of G-CSF, GM-CSF, and IL-6 from monocytes [57 ]; TNF-{alpha} expression and activation and nuclear translocation of NF-{kappa}B from PBMC [58 ] upon LPS stimulation; as well as NF-{kappa}B translocation in IL-4-stimulated, adherent macrophages [59 ]. Thus, the above reports indicate that cellular adhesion finely tunes cellular responses of leukocytes, in particular, by decreasing classical (M1 type) activation on monocytes [60 ]. Moreover, M2 macrophages participate in a diversity of events designed to suppress inflammation, increase phagocytic activity, and promote tissue repair and parasite killing [61 ], and few of these characteristics (i.e., tissue repair [62 ] and antimicrobial activity [63 ]) were induced by the uPA/uPAR system. Furthermore, IL-4 has been shown to inhibit NF-{kappa}B translocation and HIV replication in adherent macrophages [59 ]. Thus, a potential link among M2 activation, cellular adhesion, and the anti-HIV activity of the uPA/uPAR system may exist.

HIV infection of monocytes and macrophages is established soon after primary infection, mostly as tissue macrophages, such as brain macrophages and microglia, and is a dominant feature in the pathogenesis of HIV disease and its progression to AIDS [28 ]. The phenomenon of cell adhesion to the extracellular substrate is mediated through the interaction of a variety of cellular and ECM proteins [64 ], such as the interaction among uPA/uPAR and VN [65 ], and it is crucial for monocyte migration [66 ], cytokine production [66 ], and phagocytosis [64 ]. The uPA/uPAR/VN complex has been localized in lipid rafts [67 ], which are also privileged sites for entry and exit of HIV particles [68 ]. VN binding to the uPA/uPAR complex induces cell shaping, cytoskeleton remodeling, adhesion, and migration [42 , 46 , 51 ], possibly by extending the cell-ECM surface area [50 ]. The formation of clusters, highly enriched in signal transduction molecules, associated to lipid rafts, may be crucial to the triggering of the anti-HIV effects of uPA/uPAR/VN [69 ]. In support of this possibility, uPAR clustering has been shown to be essential for uPA to induce calcium fluxes [70 ], and GPI-anchored proteins, other than uPAR, such as CD59, lead to Ca2+ signaling, only after antibody-induced clustering [71 , 72 ]. It is noteworthy that it has been published previously that virion release by HIV-1-infected primary MDM and monocytic THP-1 cells was reduced upon adherence without suppression of HIV-long-terminal repeat-driven transcription or p24-Gag antigen release [35 ], thus interfering with late events in the HIV life cycle, namely, assembly and release of new progeny virions [35 ], as we and others [10 , 11 ] have reported for uPA/ATF, with an IFN-like mechanism [36 ]. Of particular relevance is that fibronectin-mediated cell adhesion also resulted in HIV retention inside infected T cells, as a consequence of adhesion-induced cytoskeleton reorganization [73 ]. In conclusion, the VN/uPA/uPAR system regulates macrophage activation toward a phenotype characterized by enhanced cellular adhesion and poor ability to support HIV replication. This phenotype is consequent to inhibitory event(s) occurring at post-translational level(s), similar to the effect of IFN, which may involve cytoskeleton rearrangement.

In vivo, the uPA/uPAR system is profoundly dysregulated in HIV-infected individuals. In particular, increased expression of uPA has been observed in brain sections of HIV+ individuals, staining negative for HIV-1 p24-Gag and uPAR antigens [74 ]. Conversely, intact and cleaved suPAR were found increased in plasma and cerebrospinal fluid of HIV+ individuals (correlated with refs. [7 8 9 , 74 75 76 ]). These observations suggest that suPAR may favor HIV replication indirectly by complexing uPA, as demonstrated in plasma and cerebrospinal fluid [7 , 9 , 76 ], and in vitro, where suPAR reversed the anti-HIV activity of uPA in PMA-stimulated U1 cells [11 ].

uPA is being used currently as a pharmacological agent for thrombolysis [77 ], although undesired effects on blood-clotting and coagulation have been reported [78 ]. Conversely, enzymatically inactive forms of uPA, such as ATF, are equally effective in their anti-HIV effects in the absence of undesired effects on blood clotting and coagulation [78 ] and usually display an increased plasma half-life, as they are not inactivated by inhibitors of fibrinolysis, such as the serine protease plasminogen activator inhibitor-1 [79 ]. Therefore, our current study reinforces the hypothesis that uPA and/or ATF should be considered lead compounds endowed with relevant pharmacological activities for containment of macrophage-dependent HIV-1 infection and spreading.


   ACKNOWLEDGEMENTS
 
This study was supported partially from la Fundaciò La Maratò de TV3 (Barcelona, Spain) and by the National Institutes of Health grant 1R21MH075670-01A1. The authors declare no competing financial interest. We thank Massimo Resnati and Flavio Curnis (San Raffaele Scientific Institute, Milan, Italy) for helpful discussions and for provision of the RGD peptide, Silvia Ghezzi (San Raffaele Scientific Institute) for collecting clinical data and blood samples from HIV+-infected individuals, Cesare Covino (Alembic, San Raffaele Scientific Institute) for help with optical microscopy, David Ginsburg and Sara Manning (University of Michigan, Ann Arbor, MI, USA) for providing serum from VN–/– mice, and Frank Kirchhoff (University of Ulm, Germany) for providing NL4-3 proviral constructs carrying Env-defective reading frames and a plasmid (pHIT-G) expressing the Env protein of the VSV.

Received April 26, 2007; revised June 20, 2007; accepted July 18, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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