Introduction
HIV infection is often associated with a variety of neurological diseases, the most severe being HIV-associated dementia (HAD). The pathogenetic mechanisms of blood-brain barrier disruption and demyelination, which represent the main characteristics of HAD, are yet to be defined. Recent studies have suggested that the development of neurological syndromes in HAD might be a result of alterations in the blood-brain barrier that lead to an increased migration of leukocytes into the central nervous system (CNS) [1]. The entry of leukocytes into the CNS is dependent in several factors including the expression of matrix metalloproteinases (MMP). Likewise, HIV-positive leukocytes transmigrate through the endothelial basement membranes when entering the spleen and/or lymph nodes in a process that also requires MMP.
The MMP form a large family of zinc-containing endopeptidases that degrade extracellular matrix and basement membrane compounds (collagens, gelatine, laminin, fibronectin). They share common structured domains but differ with respect to their cellular sources, inducibility and efficiency of substrate utilization.
The activity of MMP is highly regulated both at the level of gene expression and by activation of latent pro-MMP to active enzymes [2]. In the extracellular milieu, the activity of these enzymes is controlled by four natural tissue inhibitors of matrix metalloproteinases (TIMP). Changes in the fine balance between MMP and their tissue inhibitors drives extracellular matrix turnover and may be critical to inflammation in infection as well as other pathological condition, including neurotoxicity.
Both cells of the myeloid lineage and lymphocytes produce MMP and MMP are involved in many physiological events, including normal tissue remodelling and wound repair, as well as in many pathological processes such as tumour growth and metastasis, rheumatoid arthritis and multiple sclerosis [3]. Recent findings suggest that MMP play an important role in the pathophysiology of tissue damage and dysfunction during HIV infection, leading to dysregulation of T cell and monocyte/macrophage invasiveness. Indeed, in-vitro studies have demonstrated that HIV-1 infection increases T cell and monocyte secretion of gelatinease B (MMP-9) as well as their ability to traverse artificial basement membrane barriers. This suggests a central role for MMP-9 in migration and spreading of HIV-infected mononuclear cells into tissues and consequently in the evolution of HIV infection [4-6]. Targeting MMP enzyme activity, therefore, may constitute a novel therapeutical approach for HAD and other HIV-associated pathologies [7].
The introduction of potent antiretroviral therapy (ART) has dramatically changed the evolution of HIV infection, leading to a reduction in the prevalence of neurological disorders. We have previously demonstrated that two antiretroviral drugs, zidovudine (AZT) and indinavir, inhibit gelatinease A (MMP-2) and MMP-9 expression in glial cells. This finding suggests that the beneficial effect of ART observed in HIV-infected patients may be, at least in part, ascribed to the reduction of MMP secretion and/or expression from glial cells [8].
Given that no in-vivo data are currently available on the putative increased levels of MMP-9 in peripheral blood mononuclear cells (PBMC) of HIV-infected patients, the present study investigated whether or not ART influences the release of MMP-9 in the PBMC of such patients.
Materials and methods
Reagents
RPMI, fetal bovine serum, penicillin and streptomycin were purchased from GIBCO (Paisley, Scotland); gelatine, fluorescein isothiocyanate (FITC), bovine serum albumin and Trypan blue from Sigma (St Louis, Missouri, USA); polystyrene microspheres from Polysciences (Warrington, Pennsylvania, USA); PD-10 columns from Pharmacia (Uppsala, Sweden) and standard proteins and R-250 Coomassie brilliant blue from Bio-Rad (Hercules, California, USA). Primer pairs specific for MMP-2, MMP-9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Sigma Genosys (Cambridge, UK). RNeasy mini kit was from Qiagen (Valencia, California, USA). All the reagents for reverse transcriptase polymerase chain reaction (RT-PCR) were purchased from Invitrogen (San Diego, California, USA). Purified MMP-2 and MMP-9 were purchased from Alexis Biochemicals (San Diego, California, USA). Because the MMP-9 was purified from human neutrophil granulocytes, it also contained covalent MMP-9-NGAL complex.
Patients
A total of 46 HIV-infected patients (32 males and 14 females (median age, 39 years; range, 26-66) from the Department of Infectious and Tropical Diseases of 'La Sapienza' University of Rome were included in this study. HIV seropositivity was determined by enzyme-linked immunosorbent assay and confirmed by Western blot analysis (Chiron, Emeryville California, USA) according to the manufacturer's instructions. Among the HIV-infected patients, 30 were receiving ART (median CD4 cell count, 268 cells/μl; median CD4/CD8, 0.29; median HIV-RNA, 21 111 copies/ml), while 16 patients with advanced infection were naive for any ART treatment (median CD4 cell count, 126 cells/μl; median CD4/CD8, 0.17; median HIV-RNA, 190 833 copies/ml). All patients were treated with two nucleoside reverse-transcriptase inhibitors (NRTI) and one or two protease inhibitors (PI). Seventeen adult healthy donors (HD) were included as HIV-negative controls. All subjects gave informed consent for the study.
HIV RNA levels and T cell counts
Plasma HIV-1-RNA levels were measured using quantitative RT-PCR (Amplicor HIV Monitor; Roche Diagnostic Systems, Branchburg, New Jersey, USA). The limit of detection was 50 copies/ml. CD4 and CD8 lymphocyte numbers were assessed in blood collected in ethylenediaminetetracetic acid (EDTA)-containing tubes. Two-colour flow cytometric analysis was performed with the Becton Dickinson flow cytometer FACScan using the whole-blood lysis procedure and SimulTEST reagents (Becton Dickinson, San Jose, California, USA).
Specimen collection and cell isolation
Venous blood samples were collected into EDTA-containing tubes and plasma samples and cells were separated immediately. PBMC were isolated from peripheral blood using a Ficoll-Hypaque gradient. After isolation, the cells were washed, counted and suspended in RPMI 1640 containing 10% autologous serum at a concentration of 4 × 106 cells/ml. Samples of PBMC suspension (100 μl, 4 × 105 cells/well) were plated in 24-well microtitre plates in serum-free medium and cultured at 37°C in 5% CO2. After incubation for 24 h, the culture medium was collected, centrifuged at 10 000 g and supernatants were assayed for MMP-2 and MMP-9. Cells were subjected to total RNA extraction for subsequent determination of gene expression by RT-PCR.
Detection of MMP-9 activity by zymography
Gelatinases in cell culture supernatants were detected by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis according to a modification of the method of Heussen and Dowdle [9], as already described by Liuzzi et al. [8]. Briefly, each 50 μl sample of supernatant was supplemented with 30 μl electrophoresis loading buffer containing SDS. The samples were then separated in a 7.5% polyacrylamide gel that had been copolymerized with 0.1% (w/v) gelatine. Stacking gel contained 5.4% polyacrylamide. Electrophoresis was carried out at 4°C for approximately 18 h at 70 V. To remove SDS and reactivate the enzyme after electrophoresis, the gels were washed twice for 20 min in 2.5% (w/v) Triton X-100 in 50 mmol/l Tris-HCl, 10 mmol/l CaCl2, pH 7.4 and then incubated in 1% (w/v) Triton X-100 in 50 mmol/l Tris-HCl, 10 mmol/l CaCl2, pH 7.4.
Gelatinase activity was detected as a white band on a blue background on gels stained with Coomassie brilliant blue R-250. MMP-9 activity was quantified by scanning densitometry and computerized image analysis (Image master ID program, Pharmacia, Biotech, Uppsala, Sweden) and expressed as absorbance × mm2, representing the scanning area under the curves, which takes into account both brightness and width of the substrate lysis zone.
Detection of the net activity of MMP-9 by flow cytometric assay
MMP-9 net activity in cell culture supernatants was determined by a fluorescent activated substrate conversion assay (FASC) based on the digestion of FITC-conjugated gelatine immobilized on microspheres and flow cytometric assay according to the method of St-Pierre et al. [10].
Briefly, 300 μl gelatine, dissolved in carbonate buffer (pH 9.2) to a final concentration of 2 mg/ml was incubated for 24 h at 4°C with 10 μl of FITC dissolved in dimethyl sulphoxide. Free FITC molecules were removed by chromatography on PD-10 columns. Polystyrene microspheres 15.5 μm in diameter were incubated for 2 h at 37°C with FITC-conjugated gelatine (1 mg/ml in phosphate-buffered saline, pH 7,4) to allow non covalent adsorption. After two washes in phosphate buffer containing 0.5% bovine serum albumin and 0.05% sodium azide (PBA), the microspheres were kept at 4°C in PBA (106 beads/ml) in the dark before use.
The enzymatic reaction was carried out in a final volume of 100 μl using serum-free RPMI as reaction buffer; 50 μl cell supernatant was incubated with 10 μl FITC-gelatine-coated microspheres at 37°C for 18 h. The enzymatic reaction was stopped by adding 1 ml PBA, followed by two washes of the microspheres. The sedimented beads were then resuspended in 0.5 ml PBA and analysed by FACScan with standard optics for the detection of FITC fluorescence. Microsphere fluorescence was measured on a single-parameter histogram on a wide range. Between 1000 and 5000 events were analysed for each histogram. The net activity of MMP-9 was calculated as the decrease in fluorescence detected in each sample in comparison with a negative control, represented by FITC-gelatine-coated microspheres incubated in serum-free RPMI. For each assay, the MMP-9 net activity was quantified using a standard curve constructed using known amounts of MMP-9 standard.
RNA analysis
Total cellular RNA was isolated from PBMC using an RNeasy mini kit according to the manufacturer's instructions. RNA was precipitated with ethanol, resuspended in RNase-free water and its quantity and quality was determined spectrophotometrically at 260/280 nm. RNA samples were stored at -70°C. Complementary DNA (cDNA) was prepared by reverse transcription using random primers. The cDNA was used to amplify a 517 bp fragment using specific primers (sense 5′-AGGCAGCTGGCAGAGGAATAC-3′; antisense 5′-TGCCCAGGGACCACAACT-3′) for the human MMP-9 sequence. After preliminary experiments to determine the optimal PCR cycling conditions, 25 cycles of PCR were performed each consisting of denaturation at 94°C for 45 sec, annealing at 59°C for 1 min and extension at 72°C for 1 min in a thermal cycler (PTC-100 Programmable Thermac Controller, MJ Research, Walthan, Massachusetts, USA). PCR products were separated electrophoretically on 1.5% agarose gels and visualized by ethidium bromide staining. Gels were then processed for densitometric analysis as described above for protein gels. Standard molecular size markers, negative control (PCR mix without sample cDNA) and positive controls were run with each PCR assay. Amplification of a 308 bp fragment of human GAPDH (sense 5′-CTTCACCACCATGGAGAAGGC-3′; antisense 5′-GGCATGGGACTGTGGTCATGAG-3′), a relatively invariant internal reference RNA, was performed in parallel, and cDNA amounts were standardized to equivalent GAPDH mRNA levels. The primer sets specifically recognized only the genes of interest as indicated by amplification of a single band of the expected size of the PCR products.
Statistical analysis
Parametric one-way analysis of variance, ANOVA, or the non-parametric Kruskall-Wallis test were used to compare the three groups, as appropriate. In cases of statistical significance, Tukey test or Dunn's multiple comparison post-test were used for the pair-wise comparison of groups. The correlation between MMP-9 activity and HIV viral load was performed using the Spearman rank order correlation test.
Reported P values were considered statistically significant at P < 0.05. Data were analysed by the statistical software SigmaStat for Windows (version 3.0. SPSS, Chicago, Illinois, USA).
Results
The effect of antiretroviral therapy on the elevated MMP-9 levels in HIV-positive patients
In PBMC supernatants analysed by zymography, MMP-9 was observed in all samples but at different levels, whereas only traces of MMP-2 were detected in both HIV-infected subjects and HD without significant variations (Fig. 1a).
Levels of MMP-9 were significantly elevated in 24 h culture supernatants from PBMC of HIV-positive, ART-naive patients (mean, 36.7; SD, 6.0) in comparison with HIV-negative controls (mean, 23.0; SD, 6.3). By contrast, in the ART-treated patients, MMP-9 levels were significantly lower (mean, 23.8; SD, 7.5) than those measured in the untreated HIV-infected individuals (Fig. 1b).
MMP-9 gene expression in HIV-positive patients
To determine whether the decreased MMP-9 production in PBMC of ART-treated patients resulted from inhibition of mRNA transcription, RT-PCR analysis of MMP-9 mRNA expression was performed after normalization with GAPDH mRNA, a housekeeping gene product used as internal control (Fig. 2). The expression of MMP-9 was significantly elevated in ART-naive individuals (mean, 8.7; SD, 1.1; P < 0.05) compared with HD (mean, 5.4; SD, 1.7). By contrast, ART-treated subjects showed a significant inhibition of MMP-9 (mean, 4.1; SD, 1.6; P < 0.05) in comparison with ART-naive subjects. No significant differences were found in the mRNA expression of MMP-9 between HD and ART-treated patients.
Detection of MMP-9 net activity in culture supernatants from peripheral mononuclear cells
The functional activity of MMP-9 in cell supernatants was detected by a FASC assay [9]. As illustrated by the sharp decrease of mean fluorescence intensity (MIF), a high level of MMP-9 functional activity was found in PBMC supernatants of the ART-naive patients. No significant decrease of MIF was observed when FITC-gelatine-coated microspheres were incubated with PBMC supernatants from both HD and ART-treated subjects (Fig. 3a).
As shown in Fig. 3b, the mean percentage of MIF in ART-naive subjects was significantly decreased (65%) compared with that observed in HD (16%) and ART-treated subjects (20%).
The amount of functional activity of MMP-9 was further quantified in all samples and for each assay, using a standard curve constructed with known amounts of MMP-9 standard (Fig. 4). Significant levels of free active MMP-9 were found only in PBMC supernatants from ART-naive patients (mean, 104.0 ng; SD, 2.6; range, 93-105), but not in supernatants from ART-treated individuals (mean, 2.2 ng; SD, 0.5; range, 1.8-2.5) nor in HD (mean, 2.0 ng; SD, 0.5; range, 1.4-2.6) (Fig. 5).
MMP-9 activity is not correlated to viral load
In order to study whether the elevation of MMP-9 and the downregulation by therapy could be correlated with HIV viral load, the relationship between MMP-9 activity and plasma HIV-1 RNA levels was calculated in both ART-naive and ART-treated patients. No significant correlation was found between HIV viral load and MMP-9 activity in either ART-naive (r = 0.262; P = 0.339) and ART-treated individuals (r = -0.336; P = 0.0797).
Discussion
In the era of potent ART, HAD still represents an important cause of morbidity and mortality in patients with HIV infection. Pathological evidence suggests that HAD may occur as a consequence of an alteration of the blood-brain barrier. Another potential link to AIDS pathology is the finding of HIV reservoirs in the lymphoid organs, including spleen, lymph nodes and mucosa-associated lymphoid tissues. Increased blood-brain barrier permeability could contribute to the development of dementia by facilitating the entry of activated and infected mononuclear cells, as well as toxic serum proteins, into the CNS. One mechanism by which blood-brain barrier permeability may be altered is through increased activity of selected MMP.
Viral infection of host cells has been shown to trigger the expression of MMP, in particular MMP-9 [11]. Similarly, in-vitro studies indicate that HIV-1 infection of T cells and monocytes leads to increased secretion of extracellular degrading MMP and enhances the ability of these cells to traverse the basement membrane [5]. It has been shown that the HIV viral proteins Tat and gp120 can upregulate MMP-9 secretion from monocytes and T cells [12,13]. In addition, the intracisternal injection of the HIV Nef protein in an experimental rat model of HIV caused a disruption of the blood-brain barrier that correlated with the changes in cerebrospinal fluid MMP-9 levels [14]. Furthermore, pretreatment with an MMP inhibitor abrogated the increased vascular permeability.
There are no ex-vivo data pertaining to the production of MMP from mononuclear cells of HIV-1-infected individuals or on the effect of ART on MMP and TIMP release. In the present study, we demonstrated that PBMC from HIV-infected patients secrete elevated levels of MMP-9 compared with HD. This finding highlights the contribution of MMP in the pathogenesis of HAD. Preventing inflammatory cells from infiltrating the CNS may, therefore, represent a reasonable therapeutic strategy to control HIV-related neurological diseases.
In this context, it is worth mentioning our recent demonstration that zidovudine and indinavir inhibit the in-vitro expression of MMP-2 and MMP-9 in glial cells [8].
Here we have investigated the effect of ART on MMP-9 production from PBMC isolated from HIV-infected patients and have provided the first experimental demonstration that ART significantly downregulates the expression of MMP-9 in naturally infected PBMC.
Under physiological conditions, MMP activity is the result of a careful balance between two processes: activation of proenzymes and inhibition by specific tissue inhibitors of MMP, the TIMPs, which form stable, non-covalent enzyme-inhibitor complexes with active MMP. Therefore, matrix degradation ultimately depends on the ratio of free active gelatinase to TIMP-complexed forms. Since zymography reveals latent and active forms of gelatinases and dissociates the MMP-TIMP complexes, it cannot be used to measure the net activity displayed in cell supernatants in presence of TIMP. In order to complement our zymography data, we measured the functional activity of MMP-9 in PBMC supernatants using the FASC assay [10]. This method allows measurement of the net activity of MMP-9 resulting from the balance between MMP-9 and its inhibitor TIMP-1 by monitoring the decrease of the fluorescence emitted by a FITC-conjugated substrate immobilized on polystyrene microspheres in the presence of the enzyme. Using this procedure, we clearly demonstrated that only samples from ART-naive individuals displayed MMP-9 functional activity; samples from ART-treated subjects had little or no free active MMP-9, which was comparable to the situation observed for HD samples. This indicates that in HIV-infected individuals subjected to ART, MMP-9 levels are lower than in untreated patients and additionally counterbalanced by absence of activation or inhibition. Whether this balance is only through MMP-9 regulation or also reflects an effect of ART on TIMP-1 remains to be established.
There is little information on the regulation of the matrix-degrading enzymes in the brain tissue microenvironment by their TIMP inhibitors. Activated brain mononuclear phagocytes may contribute to the excess MMP activity within the brain, either through directly secreting MMP or reducing secretion of the TIMP that counteracts the MMP [15]. It is noteworthy that elevated MMP-9 and reduced TIMP-1 have been observed in the cerebrospinal fluid of patients with HAD [16,17], implying that the balance between proteases and their inhibitors has important biological effects.
Using RT-PCR, we demonstrated that TIMP-1 expression is unlikely to be regulated in PBMC from HIV-infected patients whether or not treated by ART (data not shown). Indeed, MMP-9 expression was not only found to be increased in ART-naive subjects but was also inhibited by ART.
The mechanisms involved in MMP-9 modulation during the course of HIV infection are not well established. It has been recently demonstrated that the in-vitro treatment of primary T cells with the viral protein gp120 activates MMP-9 transcription via the p38 and SAPK/JNK MAPK pathways, and that inhibition of p38 MAPK activation abolished MMP-9 expression [13], indicating a direct effect of HIV on MMP-9 expression. In our experiments, we did not find any correlation between viral load and MMP-9 activity in either ART-naive or ART-treated patients. Several studies indicate that antiretroviral drugs, particularly HIV protease inhibitors, exert specific effects on cellular turnover, apoptosis and proteolytic systems that are independent from their ability to block HIV replication [18,19]. In a recent study, Lichtner and coworkers showed that the ability of HIV protease inhibitors to reverse cell apoptosis in both infected and uninfected cells is a result of their direct inhibition of cysteine proteases, as calpains [20]. On the basis of these observations, antiretroviral drugs could exert a direct effect on MMP release, regardless of their activity on viral replication. Nevertheless, in a previous study using an in-vitro assay, we demonstrated that the antiretroviral drugs zidovudine and indinavir did not directly inhibit the activity of MMP-2 and MMP-9 [8].
Therefore, we cannot exclude the possibility that ART may modulate MMP release by indirect mechanisms. Indeed, the increased production of MMP in patients with AIDS could be ascribed to the state of exaggerated immune activation seen during the course of HIV infection [21]. This activation could, in turn, lead to upregulation of proinflammatory cytokines responsible of MMP-9 overexpression. In this respect, it has been recently demonstrated that soluble HIV Tat upregulates MMP-9 expression in monocytes by inducing the production of tumour necrosis factor α and interleukin 1β [22]. One of the main immunologic effects of ART is the reduction of this state of immune activation, which is closely related to the degree of viral suppression and immune reconstitution. In this respect, ART could inhibit the MMP release by reducing the HIV-associated immune activation.
In conclusion, the present findings show for the first time that ART can reduce the capacity of PBMC from HIV-infected patients to secrete increased amounts of MMP-9. This provides a novel insight into the modulatory effects of antiretroviral drugs on MMP that could be independent from their ability to block HIV replication. The understanding of the molecular mechanisms of MMP-9 activation in HIV-infected patients and its downregulation by ART may provide new clinical perspectives useful to mitigate the MMP-related damage during HIV infection.
Acknowledgements
The authors extend a special thanks to Dr Guglielmina Chimienti for the statistical analysis of data.
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