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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2002 Jun 25;99(13):8862–8867. doi: 10.1073/pnas.142078099

Urokinase–urokinase receptor interaction mediates an inhibitory signal for HIV-1 replication

Massimo Alfano *, Nicolai Sidenius †, Barbara Panzeri *, Francesco Blasi †,‡, Guido Poli *,‡,§
PMCID: PMC124389  PMID: 12084931

Abstract

Elevated levels of soluble urokinase-type plasminogen activator (uPA) receptor, CD87/u-PAR, predict survival in individuals infected with HIV-1. Here, we report that pro-uPA (or uPA) inhibits HIV-1 expression in U937-derived chronically infected promonocytic U1 cells stimulated with phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-α (TNF-α). However, pro-uPA did not inhibit PMA or TNF-α-dependent activation of nuclear factor-kB or activation protein-1 in U1 cells. Cell-associated HIV protein synthesis also was not decreased by pro-uPA, although the release of virion-associated reverse transcriptase activity was substantially inhibited, suggesting a functional analogy between pro-uPA and the antiviral effects of IFNs. Indeed, cell disruption reversed the inhibitory effect of pro-uPA on activated U1 cells, and ultrastructural analysis confirmed that virions were preferentially retained within cell vacuoles in pro-uPA treated cells. Neither expression of endogenous IFNs nor activation of the IFN-inducible Janus kinase/signal transducer and activator of transcription pathway were induced by pro-uPA. Pro-uPA also inhibited acute HIV replication in monocyte-derived macrophages and activated peripheral blood mononuclear cells, although with great inter-donor variability. However, pro-uPA inhibited HIV replication in acutely infected promonocytic U937 cells and in ex vivo cultures of lymphoid tissue infected in vitro. Because these effects occurred at concentrations substantially lower than those affecting thrombolysis, pro-uPA may represent a previously uncharacterized class of antiviral agents mimicking IFNs in their inhibitory effects on HIV expression and replication.

Urokinase-type plasminogen activator (uPA), a serine protease that activates plasminogen to plasmin (1), is synthesized as an inactive precursor (pro-uPA) that undergoes proteolytic activation. Both pro-uPA and uPA bind to a specific plasma membrane receptor (uPAR) localized at the cell surface (2). Under physiological conditions, both uPA and uPAR are predominantly expressed by immune cells including neutrophils, monocytes, macrophages, and activated T lymphocytes (3) in which they play important roles in cell activation, adhesion, migration, and extravasation (4, 5). Recently, it has been demonstrated that uPAR becomes a signaling receptor once activated by uPA under conditions in which its catalytic activity is not required. Binding of uPA to uPAR induces cell migration, cell adhesion, and proliferation (6, 7). The migration-promoting activity of uPAR is mediated by integrins such as CD11b/CD18 and requires the activation of both an Src-family tyrosine kinase and of mitogen-activated protein kinases (7, 8). uPAR and integrins seem to act as a single functional unit in several cells, as confirmed in uPAR-deficient mice in which CD11b/CD18-dependent cell adhesion and functions are deficient (9).

uPA and uPAR are involved in the pathogenesis and malignancy of cancer. In this regard, high levels of uPA or uPAR in tissues or serum represent a major negative prognostic marker in human cancer (10). Of interest, the serum levels of soluble uPAR (suPAR) in a cohort of HIV-1-infected individuals studied before initiating anti-retroviral therapy, were highly correlated to the severity of HIV disease, thus representing a negative prognostic indicator independent from and as indicative as low numbers of circulating CD4+ T cells or high viremia levels (11). This observation suggests that the uPA–uPAR interaction may play an important role in the pathogenesis of HIV-1 infection and its progression toward AIDS. This hypothesis is supported by the observation that increased levels of uPAR were found expressed on the surface of CD8+ T cells of AIDS patients (12), whereas HIV-1 has been shown to induce the synthesis of uPAR in CD4+ T cells (13). Recently, a decreased expression of uPAR has been demonstrated in granulocytes of HIV-infected individuals, and its levels were significantly correlated to the number of CD4+ T lymphocytes of those individuals (14). In addition, uPA has been shown to cleave in vitro the HIV-1 gp120 envelope (Env) molecule in its hypervariable V3 loop, a region involved in determining cell tropism and chemokine coreceptor usage and representing a crucial determinant of immune response and virus variability (15).

In the present study, we have analyzed the effect of pro-uPA on in vitro HIV-1 infection of U937-derived, chronically infected U1 cells, of acutely infected U937 cells, and of different primary cells. Our results indicate that uPA–uPAR can interfere with HIV replication in both acutely and latently infected cells and provide evidence of functional mimicry between uPA–uPAR and the antiviral effects of IFNs.

Materials and Methods

Reagents.

Human grade low molecular weight uPA (LMW-uPA), aminoterminal fragment of uPA (ATF), and active two-chain uPA were provided by John Berryman (American Diagnostica, Greenwich, CT); human grade pro-uPA was a gift of Jack Henkin (Abbot Laboratories, Chicago). Phorbol-12 myristate-13 acetate (PMA; Sigma) was used at 10−8 M; recombinant tumor necrosis factor-α (TNF-α) was used at 1 ng/ml. Polyclonal anti-TNF-α Ab and isotype control Ab (R & D Systems) were used at 1 μg/ml, whereas IFN-α (IFN-α2b) (R & D Systems) was used at 500 units/ml.

Cell Lines.

Promonocytic U937 cell clones were previously defined as “plus” or “minus” in relationship to their efficient or inefficient capacity to sustain CXCR4-dependent (X4) HIV-1LAI/IIIB replication (16–18). The U1 cell line was originally obtained from a population of U937 cells surviving the cytopathic effect of acute HIV-1LAI/IIIB infection and contains two copies of integrated provirus (19). High levels of virus expression are rapidly induced by U1 cell stimulation with PMA or TNF-α (20). U1 and U937 cells were cultivated at 2 × 105 cells per ml in RPMI medium 1640 (BioWhittaker) containing 10% (vol/vol) FCS (GIBCO/BRL) in the presence or absence of various uPAR ligands for 20 min at 37°C before either infection or stimulation.

Cytofluorimetric Analysis.

U937 cells were washed twice with PBS plus 2% (vol/vol) FCS and stained for uPAR and CD11b/CD18 expression by using R4 and anti-MAC-1 (Integrin αM 44; Santa Cruz Biotechnology) monoclonal antibodies (mAbs), respectively, and goat anti mouse-FITC Ab (Sigma). After fixation in 2% (wt/vol) formaldehyde/PBS, 15,000 cells were acquired by using a FACScan apparatus (Becton Dickinson) and then analyzed with CELLQUEST software (Becton Dickinson).

Viruses.

Two laboratory-adapted R5 strains (HIV-1ADA and HIV-1BaL) and the X4 strain HIV-1LAI/IIIB were used. The HIV-1 stocks were obtained by infection of phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) maintained in IL-2 enriched medium (T cell blasts) by pooling the culture supernatants corresponding to the peak levels of Mg2+-dependent reverse transcriptase (RT) activity (16). The infectious titer was determined on T cell blasts by the Reed and Muench formula, and the viral stocks were stored at −80°C, as described (21).

Infection of Primary Cells and of Human Lymphoid Tissue.

T cell blasts and IL-2-stimulated PBMC (22) were infected with R5 and X4 viruses in the presence or absence of different concentrations of pro-uPA; productive infection was monitored by RT activity content of their culture supernatants. Monocyte-derived macrophages (MDM) were obtained from PBMC by plastic adherence, after removal of nonadherent cells. Infection of MDM by R5 HIV-1BaL was performed after 5–7 days of maturation in vitro, as reported (23).

Human tonsils or adenoids were obtained within 3 h from tonsillectomy or surgical removal, and tissue blocks were infected with 300 TCID50 of filtered virus-containing medium to the top of each tissue block (24). Tissue cultures were checked every day for bacteria contamination by direct light microscopy, and aliquots of medium were collected every 2 days and stored at −80°C. Medium was completely removed and exchanged every 2–3 days with pro-uPA-enriched fresh medium. Productive HIV infection was assessed by RT activity accumulated in 24 ml of culture medium bathing 54 tissue blocks.

Electrophoretic Mobility Shift Assay (EMSA) for Nuclear Factor κB (NF-κB), Activation Protein-1 (AP-1), and Signal Transducer and Activator of Transcription (STATs).

Whole-cell extracts (WCE) were prepared from 3 × 106 cells 30 min and 4 h after U1 cell stimulation in the presence or absence of pro-uPA, as described (16). For supershift analysis, parallel aliquots of WCE were incubated with 1 μg of anti-p50 (N-terminal) or anti-p65 (N-terminal) mAbs (Santa Cruz Biotechnology) for 30 min at room temperature. AP-1, STAT-1, and STAT-2 activation were analyzed by EMSA according to published protocols (25–27).

Western Blot Analysis of Cell-Associated HIV-1 Proteins and of Estrogen Receptor Kinase 1/2 (ERK1/2).

Cell-associated viral proteins were analyzed as reported (23). For ERK1/2 phosphorylation, 10 μg of protein from nuclear cell extracts (NCE) were loaded onto an SDS/10% PAGE and detected by the anti-p-ERK D4 mAb (Santa Cruz Biotechnology). The filter then was stripped and reblotted with the anti-ERK2 D2 mAb (Santa Cruz Biotechnology).

Ultrastructural Studies.

U1 cells were washed twice in PBS, fixed in 4% (vol/vol) glutaraldehyde/2% (wt/vol) paraformaldehyde/0.12 M cachodilate, pH 7.4 and postfixed in 1% (vol/vol) OsO4/0.12 M cachodilate buffer. Cells were dehydrated in graded ethanol, washed in propylene oxide and infiltrated for 12 h in a 1:1 mixture of propylene oxyde:epoxydic resin (Epon). Cells then were embedded in Epon and polymerized for 24 h at 60°C. Slides were cut with ultramicrotome (Ultracut Uct, Leica, Deerfield, IL), stained with uranyl acetate and lead citrate, and metallized.

Results

Pro-uPA and ATF Inhibit HIV-1 Expression in Chronically Infected U1 Cells Stimulated with PMA.

The potential antiviral effect of pro-uPA was first investigated in the U937-derived chronically infected U1 cell line characterized by a relatively latent state of viral expression overcome by PMA and various cytokines (20). Pro-uPA did not activate virus expression in unstimulated U1 cells (not shown), but it caused a concentration-dependent decrease of PMA-induced HIV-1 production, as measured by RT activity (Fig. 1A). To dissect out whether pro-uPA anti-HIV effect required the enzymatic activity or its binding to uPAR, we compared the effects of LMW-uPA and ATF in PMA-stimulated U1 cells. LMW-uPA, which does not bind to uPAR, did not affect virus production, whereas ATF, devoid of enzymatic activity, fully reproduced the inhibitory effect of pro-uPA (Fig. 1A). An excess of exogenous suPAR reversed the inhibitory effect of pro-uPA (data not shown). In addition, two anti-uPAR mAbs (R3, R5), preventing the interaction between uPA and uPAR but not the R4 mAb, that does not interfere with pro-uPA/uPA binding to uPAR, reversed the inhibition of HIV-1 expression by uPA (data not shown). These results together indicate that pro-uPA inhibition of HIV-1 expression requires its interaction with uPAR, and that this effect is independent from its catalytic activity.

Figure 1.

Figure 1

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Pro-uPA and ATF, but not LMW-uPA, inhibit HIV-1 expression in PMA-stimulated U1 cells. Additive suppression by pro-uPA and anti-TNF-α Ab. (A) U1 cells were stimulated with PMA in the presence or absence of pro-uPA, LMW-uPA, or ATF; values represent the peak levels of RT activity achieved after 72 h of stimulation. A similar pattern of inhibition was observed after 6 days of stimulation (not shown). (B) Anti-TNF-α Ab (1 μg/ml), isotype Ab, and pro-uPA (10 nM) were added, either alone or in combination, 20 min before PMA stimulation of U1 cells. The results represent the means ± SD of three independent experiments.

Suppression of PMA-Induced HIV Expression in U1 Cells by Pro-uPA and Anti-TNF-α Ab.

The HIV-inductive effect of PMA in U1 cells depends in part on the induction of endogenous TNF-α secretion (28). In contrast to its effect on virus expression, pro-uPA did not inhibit PMA-induced TNF-α secretion (data not shown), thus providing a potential explanation for the incomplete inhibitory effect on HIV expression under this stimulatory condition (Fig. 1A). Furthermore, anti-TNF-α mAb and pro-uPA showed additive inhibitory effects on virus expression induced by PMA (Fig. 1B). In addition, pro-uPA partially inhibited HIV expression in TNF-α-stimulated U1 cells (Fig. 2A). Of interest, only about 10% of U1 cells expressed CD11b/CD18, that was, however, superinduced by both stimuli (data not shown).

Figure 2.

Figure 2

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Pro-uPA inhibits HIV-1 expression but not NF-kB activation induced by TNF-α or PMA stimulation of U1 cells. (A) U1 cells were preincubated for 20 min with different concentrations of pro-uPA and then stimulated with TNF-α (1 ng/ml). The results represent peak RT activity observed after 72 h of stimulation. (B) WCE prepared 30 min and 4 h after PMA or TNF-α stimulation of U1 cells were analyzed for NF-kB activation. U1 cells express a banding pattern typical of minus U937 cell clones, in that truncated p65 heterodimerizes with p50, as reported (16). The last two lanes on the right indicate supershifting of the bound complexes by anti-p50 and anti-p65 Abs.

Pro-uPA Inhibits HIV Expression in U1 Cells by Acting at a Posttranslational Level.

Stimulation of U1 cells with either PMA or TNF-α induces HIV-1 expression by activating both the NF-kB and AP-1 families of transcription factors (28–30). The two stimuli induced NF-kB-binding activity in U1 cells with different kinetics (30 min and 4 h, respectively; Fig. 2B); the induced complexes were mostly composed of p50-p65 heterodimers and p50-p50 homodimers, as determined by supershift analysis (16). Pro-uPA neither inhibited NF-kB activation nor modified the complex composition as determined by supershift analysis (Fig. 2B). Likewise, pro-uPA did not interfere with AP-1 activation induced by TNF-α or PMA stimulation of U1 cells and did not alter the activation pattern of ERK1/2 (data not shown). In addition, pro-uPA did not inhibit the synthesis of viral protein in U1 cells stimulated by either TNF-α or PMA (Fig. 3A). These results together suggest that pro-uPA inhibits a posttranslational step in the virus life cycle, thus resembling the antiviral effect of IFNs on chronically infected cell lines, including U1 cells (31, 32).

Figure 3.

Figure 3

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Posttranslational inhibition of HIV-1 expression from U1 cells by pro-uPA. (A) HIV-1 protein synthesis was analyzed by Western blotting 20 h after stimulation in the presence or absence of pro-uPA. Molecular weight markers and the main viral proteins are indicated on the left and on the right, respectively. (B) Reversion of pro-uPA (10 nM) mediated inhibition of HIV expression in stimulated U1 cells by cell disruption. F/T indicates that the cells were disrupted by five cycles of freezing and thawing.

To investigate this hypothesis, U1 cells were then stimulated by PMA or TNF-α in the presence or absence of pro-uPA or IFN-α, and the supernatant-associated RT activity was tested on intact cells or after cell disruption by freezing and thawing, as described (31). Indeed, cell disruption completely reversed the inhibitory effect of both pro-uPA and IFN-α on stimulated U1 cells (Fig. 3B), suggesting that HIV virions were assembled but not efficiently released.

Ultrastructural analysis then was performed on U1 cells after 48 h of PMA stimulation in the presence or absence of pro-uPA. Most virions were detected associated to the plasma membrane in PMA-stimulated cells, with the remainders budding from and accumulating in intracellular Golgi-derived vacuoles (Fig. 4 A and B), as reported in IFN-treated cells (31, 32). In the presence of pro-uPA, the number of cells producing virus was not decreased in comparison to control cells, but a substantial increase in the fraction of intracellular (from 27 to 79% in the absence and presence of pro-uPA, respectively) vs. plasma membrane-associated virions was observed (Fig. 4 C and D). This observation provides an explanation of the apparently discrepant findings of the reduced levels of RT activity observed after pro-uPA treatment, because the enzyme is associated mostly with the viral particles (33) and the lack of inhibitory effects on transcription factors and translation of HIV genes. In addition, a substantial increase in the vacuolization of U1 cells was noted when U1 cells were stimulated with PMA in the presence of pro-uPA (Fig. 4C), likely reflecting an increased state of macrophage differentiation (34). In support of this interpretation, the binding but not the catalytic component of uPA has already been shown to induce differentiation and vacuolization of the promyelocytic cell line HL60 after PMA stimulation (35).

Figure 4.

Figure 4

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Preferential accumulation of HIV-1 virions in intracytoplasmatic vacuoles in PMA-stimulated U1 cells in the presence of pro-uPA. U1 cells were stimulated for 48 h with (A) PMA (magnification ×11,000) or (B) PMA plus pro-uPA (10 nM) (magnification ×15,000). (B and D) Enlargement of a detail of A and C, respectively. Two independent experiments were performed with similar results. Both unstimulated and pro-uPA-treated U1 cells did not show evidence of virion expression, whereas approximately 50% of PMA-stimulated cells were positive for virion expression, as reported (31, 32).

No evidence of expression of endogenous IFNs was obtained in PMA-stimulated U1 cells in the presence or absence of pro-uPA (data not shown). Furthermore, we did not observe activation of the IFN-related Janus kinase/STAT (JAK/STAT) pathway of signal transduction (36), as reported for smooth muscle cells treated with pro-uPA (37), that we have correlated to the anti-HIV effect of IFNs in U937 cells (ref. 38, and data not shown). Altogether, these findings indicate that pro-uPA activates an HIV inhibitory pathway in PMA or TNF-α stimulated U1 cells that mimics but is clearly distinct from the antiviral effects of IFNs.

Inhibitory Effects of Pro-uPA on Acute HIV-1 Replication in U937 Cells, Primary Cells, and Lymphoid Tissue.

To investigate whether uPA–uPAR interaction could interfere with acutely infected cells, we first determined their levels of expression in promonocytic cell clones of the U937 cell line (parental to U1 cells), previously characterized in terms of their efficient (plus clones) or poor (minus clones) capacity to sustain X4 HIV-1 replication (16, 18). Plus cells (U937-SB and clone 10) expressed lower levels of both uPAR and CD11b/CD18 than minus cells (clone 12 and 34) at the cell surface (Fig. 5A), as confirmed by Western blot analysis (data not shown). Pro-uPA (1 nM) caused a substantial inhibition of virus replication in both U937 cell types in the absence of cellular toxicity (Fig. 5B).

Figure 5.

Figure 5

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Pro-uPA inhibits HIV-1 replication in U937 plus and minus cells. (A) Constitutive expression of uPAR and CD11b/CD18 in U937 cells. Cells were identified based on their light scatter and analyzed for markers expression. Isotype analysis is shown as black histograms. Minus U937 cells constitutively express higher levels of uPAR (bold line) and CD11b/CD18 (dotted line) than plus cells. (B) U937 cell clones were pretreated with pro-uPA (1 nM) and infected with the X4 HIV-1LAI/IIIB virus. Fresh medium supplemented with pro-uPA (1 nM) was added to the cell cultures every 72 h.

To investigate whether pro-uPA exerted inhibitory effects also in primary cells, we tested different model systems, including T cell blasts or IL-2-stimulated PBMCs (22) and MDMs from several seronegative healthy donors. Pro-uPA inhibited HIV-1 production in two of four IL-2-stimulated PBMC but not in T cell blasts, and only in two of six MDM obtained from different donors with different efficacy, ranging from no effect to 90% suppression (data not shown). This high interdonor variability was not reflected by a different pattern of uPAR or uPA expression, although IL-2 and mitogen activation of primary PBMC led to an increase of uPA expression (0.56 ± 0.09 and 0.26 ± 0.02, respectively, vs. 0.08 ± 0.05 ng/mg of protein of untreated PBMCs) and to a partial down-regulation of uPAR (1.10 ± 0.20 and 2.47 ± 0.27, respectively, vs. 3.41 ± 0.14 ng/mg of protein in untreated PMBCs). Also, MDM constitutively expressed uPA (0.17 ± 0.01 ng/mg of protein) and uPAR (3.23 ± 0.45 ng/mg of protein; data not shown).

The potential antiviral effect of pro-uPA also was tested in a model of HIV-1 infection of in vitro unstimulated cultures of lymphoid tissues, likely better reflecting the in vivo intercellular interactions determining cell migration, activation, and viral expression than the above-mentioned models of in vitro infection of isolated T cells and macrophages (39–41). Pro-uPA inhibited HIV-1 replication in lymphoid organ tissue cultures established from adenoids and tonsils in three of three independent donors (Fig. 6). Although the reason for the observed variable effects of pro-uPA on primary-cell infection are unclear at present, these observations in lymphoid tissue cultures and in acutely or chronically infected U937 cells support the hypothesis that uPA–uPAR interaction mediates an inhibitory signal for HIV replication.

Figure 6.

Figure 6

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Pro-uPA inhibits HIV-1 replication in lymphoid tissues. Pro-uPA (10 nM) was added either 20 min before or after infection of lymphoid tissue by the R5 HIV-1BaL. Data are shown as a mean ± SD of six independent cultures from one representative of three independent experiments performed.

Discussion

In the present study, we have observed that exogenous pro-uPA and ATF, but not LMW-uPA, inhibited HIV expression in chronically infected U1 cells stimulated with PMA or TNF-α. In these cells, the antiviral effect of pro-uPA was related to the coexpression of both uPAR and the integrin CD11b/CD18 and did not affect HIV gene expression or translation of viral proteins, but induced an IFN-like inhibitory effect on virion release. The signaling pathway triggered by uPA–uPAR interaction leading to this antiviral effect remains elusive, and does not involve either inhibition of NF-κB or AP-1 or activation of JAK/STAT proteins. In addition, pro-uPA potently and consistently inhibited acute HIV-1 replication in U937 cells and in cultures of lymphoid tissue infected ex vivo.

Near-complete suppression of HIV expression induced by PMA stimulation of U1 cells was achieved when pro-uPA was combined with anti-TNF-α mAb. Although pro-uPA inhibited, in part, TNF-α-induced HIV expression, no effects on the activation of NF-κB (the main transcription factor involved in the up-regulation of HIV expression triggered by this cytokine; refs. 29 and 42) or AP-1 were observed. Consistently, the total amount of viral proteins was unchanged in the presence or absence of pro-uPA. Because U1 cells, unlike acutely infected cells, do not undergo cytopathicity as a consequence of virus expression (17), it is possible to conclude that the antiviral effect of pro-uPA is inhibiting a posttranslational step in the viral life cycle in chronically infected cells. In this regard, a strong analogy exists between the antiviral effects of uPA–uPAR and those of IFNs, which are known to mediate similar posttranslational effects in cells chronically infected by different retroviruses, including HIV (31, 33, 43).

Both IFN-α and IFN-γ (a cytokine usually not expressed by monocytic cells) suppress HIV replication in minus U937 cell clones via activation of the JAK/STAT pathway (38). Furthermore, the suppressive effect of IFN-γ in PMA-stimulated U1 cells has been linked to the redirection of virion assembly and budding from the plasma membrane to intracellular Golgi-derived vacuoles (33). Thus, the suppressive effect described here for pro-uPA more closely resembles IFN-γ- rather then IFN-α-mediated inhibition (31), at least in the U1 cell model. The hypothesis that pro-uPA mediates antiviral effects via an IFN-related pathway was further supported by the observation that pro-uPA induced STAT1/STAT2 activation, along with other transcriptional regulators, in smooth muscle cells (37). However, no evidence of either JAK/STAT activation or induction of endogenous IFNs was obtained in U1 cells stimulated with PMA in the presence or absence of pro-uPA. Similar results (i.e., suppression of HIV production without interference at the transcriptional level) were shown in a recent study where ATF has been characterized as a major HIV-suppressive factor released from activated CD8+ T cells (44).

UPAR/CD87 is a glycosylphosphatidylinositol (GPI)-anchored protein predominantly localized to glycolipid-enriched microdomains also known as lipid rafts (45). Rafts are involved in different steps of the HIV life cycle including infection (46), multimerization of HIV-1 Gag proteins (47), and budding of new progeny virions (48). Thus, a potential mechanism of the HIV suppressive effects induced by uPA–uPAR interaction is a modification in the composition of the lipid rafts environment resulting in a diminished efficiency of virion budding from the plasma membrane. In this regard, uPAR interaction with the integrin CD11b/CD18 mediates some intracellular activities of uPA (7, 9, 49, 50). Because uPAR is a GPI-linked protein that requires an accessory adapter protein to signal (45), we cannot exclude that other proteins coregulated by PMA and TNF-α but distinct from CD11b/CD18 are involved in the antiviral effect described here. The integrin–uPAR interaction may modify the membrane solubility of uPAR and, hence, lipid composition of rafts.

Whether the variable effects observed with pro-uPA on acute HIV infection of activated PBMC and MDM are similarly explained by the triggering of an IFN-like pathway, as observed in U1 (and, presumably, U937) cells, is currently unclear. However, pro-uPA consistently inhibited virus production in a lymphoid organ-tissue culture system, in which T cells are mostly in a resting state (39) but are, nevertheless, permissive for both R5 and X4 HIV infection (40) without requiring in vitro activation (41, 51).

The uPA–uPAR system has multiple and bidirectional interactions with chemokines and their receptors. On the one hand, several chemokines, including CCL2, CCL3, CCL4, and CCL5, have been shown to decrease the secretion of uPA from microglial cells (52), whereas CCL2 restored the inhibition of extracellular matrix invasion by dendritic cells determined by anti-uPAR Ab (51). More recently, CCL5 stimulation of monocytes has been shown to up-regulate uPAR expression in the context of a transcriptome analysis (53). On the other hand, uPA–uPAR has been shown to process CCL14 into an active form (54) and to modulate the chemotactic effect of CCL11 (55). In addition, uPA has been shown to increase the number of CD4+ and CD8+ T cells from healthy children in vitro (56), whereas uPA-deficient mice showed a reduced infiltration of CD4+/CD11b+/CD18+ cells in their lung and had an increased mortality by infection of Criptococcus neoformans (57). In vitro, HIV infection of cell lines, PBMC, and/or MDM has resulted in increased expression of uPAR (13) as well as of certain CC chemokines such as CCL2 (58), CCL3, and CCL4 (59, 60). Finally, it has recently been shown that a cleaved, chemotactic form of uPAR can directly activate the chemotactic receptor FPRL1/LXA4R (61) that has been shown to be desensitized by gp120 Env-derived peptides (62). It is possible, therefore, that uPA, in addition to the IFN-like mechanisms described here, may influence the efficiency of viral infection by either directly affecting the expression of CD4 and/or of CCR5 and CXCR4 or, indirectly, by interfering with the biology of related chemokines endogenously released by activated T lymphocytes and macrophages.

Although the functional role of uPA–uPAR in HIV-disease progression needs to be fully elucidated, strong indirect evidence exists for such a connection. In this regard, we have recently shown that high serum levels of suPAR are a major negative prognostic factor in HIV infection independently of the clinical stage, viremia levels, and CD4+ T cell counts (11). In addition, dysregulation of the uPA–uPAR system has been observed in children with AIDS in terms of a decreased number of uPA+ circulating monocytes (56) and, more recently, in granulocytes of HIV-infected individuals, where the levels of uPAR expression reflected the number of peripheral blood CD4+ T lymphocytes (14).

Pro-uPA has been used already as a pharmacological agent (63, 64); the use of enzymatically inactive molecules, i.e., mutant pro-uPA, ATF and growth factor domain-like agents, is feasible in that they are devoid of side-effects such as hemorrhage (65). In this regard, the concentration required for inhibition of virus replication in lymphoid tissues is at least 10 times lower than that affecting thrombolysis, further decreasing the risk of potential side-effects. Thus, pro-uPA is a nontoxic molecule that could be tested for its potential efficacy as an anti-HIV-1 compound in infected individuals.

Acknowledgments

We thank Dr. Chiara Bovolenta (MolMed SpA, Milan) for help and guidance with STATs analysis; Dr. Leonid Margolis and Dr. Jean Charles Grivel (National Institute of Child Health and Human Development, National Institutes of Health, Washington, DC) for teaching us how to study HIV replication in lymphoid hystocultures; Dr. Andrea Ciolli (ASL Melzo, Milan) for providing us with lymphoid specimens; Dr. Gunilla Høyer-Hansen from the Finsen Laboratory for providing anti-uPAR Abs; Dr. Carla Panzeri (Alembic, San Raffaele Scientific Institute, Milan) for electron microscopy; and Dr. Chiara Rizzi (San Raffaele Scientific Institute, Milan) for help with infections. This study was supported in part by Grant n.40.C.73 (to G.P.) of the III° National Program of Research on AIDS of the Istituto Superiore di Sanità, Rome.

Abbreviations

uPA

urokinase-type plasminogen activator

uPAR

uPA receptor

PBMC

peripheral blood mononuclear cells

PMA

phorbol-12 myristate-13 acetate

TNF-α

tumor necrosis factor-α

MDM

monocyte-derived macrophages

ATF

amino-terminal fragment

RT

reverse transcriptase

AP-1

activation protein-1

STAT

signal transducer and activator of transcription

JAK/STAT

Janus kinase/STAT

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

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