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Changes in Membrane Enzymes and Glycosphingolipids in Lymphocytes from HIV-1-Infected and Noninfected Intravenous Drug Users

Leoni, Lorenzo M.; Losa, Gabriele A.

Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology: February 1st, 1996 - Volume 11 - Issue 2 - p 188-197
Epidemiology
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Summary: The amounts of cell-surface glycosphingolipids and plasma membrane enzymes produced on the peripheral blood mononuclear cells (PBMNCs) isolated from 101 intravenous drug users (IDUs), of whom 91 were HIV-1 seropositive, were examined. Seronegative IDUs and age-matched healthy donors served as controls. The numbers of circulating CD3+, CD4+, and CD8+ T lymphocytes decreased during the advanced stages of the infection. There were also fewer CD4+ T-helper cells in HIV-1-seronegative IDU drug addicts. PBMNCs from HIV-1-seropositive subjects had abnormal surface enzyme kinetics. The phospholipase C had two pH optima, whereas the enzyme on normal cells has only one. The specific activity in cells from AIDS subjects was 4 times lower than that in normal PBMNCs. 5'-Nucleotidase showed a similar trend, whereas neutral endopeptidase activity did not correlate with the amounts of surface common acute lymphoblastic leukemia antigen (CALLA). These enzyme activities were decreased in HIV-seronegative IDUs. The subcellular distribution of enzymes and the profile of surface glycosphingolipids were also markedly changed, indicating the profound alterations in the membranes of PBMNCs from HIV-1-seropositive IDUs. These data suggest that intravenous drug use compromises the biochemical and structural integrity of the membrane surface of PBMNCs even before the onset of HIV.

Laboratory of Cellular Pathology, Institute of Pathology, Locarno, Switzerland

Address correspondence and reprint requests to Dr. G. A. Losa, Laboratory of Cellular Pathology, Institute of Pathology, CH-6604 Locarno, Switzerland.

Manuscript received March 9, 1995; accepted August 15, 1995.

Several studies have revealed that individuals infected by the HIV-1 virus have circulating lymphocytes with phenotypically abnormal plasma membranes (1,2). However, there have been fewer studies on the functional and ultrastructural changes in the membranes of HIV-1-infected lymphocytes (3) than on leukemia cells and related pathologic conditions (4,5). Apart from the ecto-5'-nucleotidase (5'-NT), attention has focused on the enzymes of purine metabolism that lie in the cytoplasm of peripheral blood mononuclear cells (PBMNCs) from asymptomatic and AIDS homosexual patients (6,7). Recent studies have included the role of the cell surface, because they have shown that most membrane-associated enzymes, which are hematopoietic differentiation antigens, are involved in T-cell activation and proliferation, the inflammatory response, hematopoiesis, antigen processing, and virus entry (5,8). Cell-surface heparan sulfate proteoglycans participate in HIV-cell attachment and virus entry into lymphoblastic T-cell lines (9). Other complex molecules, such as glycosphingolipids, which have a hydrophobic ceramide portion and an external glycoside tail, have also been found to modulate structural and functional features of the plasma membrane in human lymphocytes (10,11). There have been reports of increased amounts of acidic GM1 and GM3 gangliosides on the surface of peripheral blood lymphocytes and monocytes from AIDS patients (12,13). The neutral glycosphingolipid galactosylceramide was recently found to act as an alternative receptor for HIV virus in neural and gastrointestinal cells lacking CD4 surface receptors (14,15). To our knowledge, there have been no experimental studies on the effects on and possible interactions of morphine and cocaine opiates with the ultrastructural-functional membrane constituents involved in surface recognition and extracellular signal transmission in circulating immuno-competent lymphocytes. We therefore investigated changes in activity and subcellular distribution of membrane glycosphingolipids and enzymes lying on external and internal membrane bilayers in the PBMNCs of intravenous drug users (IDUs) with or without HIV-1 infection. The ectoenzymes examined included the glycosylphosphatidylinositol membrane-anchored 5'-nucleotidase (5'-NT), which catalyzes the cleavage of phosphate from 5'-nucleotides such as adenosine monophosphate (AMP) and inosine monophosphate (IMP), γ-glutamyltransferase (γ-GT), which catalyzes the first step in the extracellular transpeptidation of the antioxidant glutathione into glutamyl amino-acid intermediates that are later used in the de novo synthesis of intracellular glutathione (16), and neutral endopeptidase (NEP) or enkephalinase. NEP has an amino acid sequence identical to that of common acute lymphoblastic leukemia antigen (CALLA, CD10; 17), cleaves a variety of biologically active peptides (18,19) and is affected by various opiates (20). NEP also is implicated in the growth of cancer cells, but its effect appears to be mediated by the specific environment generated by the tumor (21,22). Last, we measured the activity of phospholipase C (PLC), a key endoenzyme for membrane signal transduction, which catalyzes the formation of the second messengers inositol 1,4,5-triphosphate (IP3) and sn-1,2-diacyglicerol (DAG) after the binding of agonists to the cell surface (23).

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MATERIAL AND METHODS

Materials

L-Phosphatidylinositol 4,5-bisphosphate and D-myoinositol trisphosphate from bovine brain, bovine intestinal mucosa alkaline phosphatase (type VII-T), cetyltrimethylammonium bromide (CTAB), γ-glutamyl-p-nitroanilide, glycylglycine, propidium iodide, ribonuclease, dansyl-D-Ala-Gly-Phe-(pNO2)-Gly (DAGNPG), Triton X-100, L-phosphatidylinositol-4,5-biphosphate from bovine brain (PIP2), alkaline phosphatase (type VII-T) from bovine intestinal mucosa, and adenosine 5'-monophosphate (5'-AMP) from equine muscle A were purchased from SIGMA Chemical (Sigma Chemie, Buchs, Switzerland). The anti-CD10 monoclonal antibody (MoAb) Lau A12 was provided by Prof. S. Carrel (Ludwig Institute for Cancer Research, Lausanne, Switzerland). Other MoAbs were purchased from Becton-Dickinson (Becton-Dickinson Immunocytometry Systems, San Jose, CA, U.S.A.). All other chemicals used at finest grade of purity were purchased from Merck, Darmstadt, Germany (Bender & Hobein AG, Zürich, Switzerland).

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Probands

The study included 91 intravenous drug users (IDUs), from 18 to 48 years (mean age of 30) examined during the period from 1988 to 1993. All had circulating HIV antibodies confirmed twice by reference laboratories. The examined cases included 61 men and 30 women. Some patients were examined several times and received chemotherapy and immunotherapy after the first blood examination. Blood samples or buffy coats from normal healthy male and female donors (n = 30; mean age, 30) and from HIV-1 seronegative IDUs (n = 10; mean age, 28) served as controls. According to the stage of infection (25) diagnosed by physicians, the distribution of IDU HIV+ patients was as follows: 23% constituted the group of asymptomatic CDC II (ASY); 6% CDC III; 23% CDC IVA; and 48% CDC IVC (AIDS). CDC III was not investigated because of the scarcity of blood samples. CDC IVA was analyzed only for the immunophenotype.

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Isolation of PBMNCs

Human PBMNCs were isolated, as previously described (26), from heparinized blood samples of HIV-1+, seronegative IDUs and from buffy coats of blood units of nontransfused healthy donors aged between 19 and 58. The cell viability, determined by trypan blue exclusion, always exceeded 95%.

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Flow Cytometry, Immunophenotype

The immune phenotype of PBMN cells was assessed by flow cytometry (FACScan, Becton-Dickinson Immunocytometry Systems, San Jose, CA, U.S.A.) after staining with fluorescent conjugated anti-CD3, CD4, CD8, CD10 (W8E7, Becton-Dickinson and/or Lau A12, from Prof. Carrel, Ludwig Institute for Cancer Research, Lausanne), CD19 and HLA-DR monoclonal antibodies, as previously described (27).

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Neutral Endopeptidase

NEP (EC 3.4.24.11) was assayed as described by Florentin et al. (28) by using the synthetic substrate dansyl-D-Ala-Gly-Phe-(pNO2)-Gly at a final concentration of 100 μM. Cells (1 × 106/0.1 ml) or cell-membrane aliquots (100 μg proteins) were incubated for 30 min at 37°C in 0.05 M Tris, pH 7.4, 0.05 M NaCl, and 0.5 μM captopril for a final volume of 0.3 ml. The reaction was stopped by addition of 2 ml ice-cold buffer with 10 mM EDTA, centrifuged at 10,000 r/min for 5 min. The fluorescence of the supernatant was measured using a Perkin-Elmer LS-3B Fluorescence Spectrophotometer with an excitation wavelength of 333 nm and a emission wavelength of 541 nm. A calibration curve was assessed with the product of the reaction, dansyl-D-Ala-Gly, at a concentration range of 0 to 15 μM, to convert the units of fluorescence to pmol/h. The assay was optimized with NALM-6 cells, which are known to express high levels of NEP and high CALLA positivity (95%). NALM-6 cell homogenates were included as internal standard. The product dansyl-D-Ala-Gly was a gift generously provided by Prof. B. P. Roques, Laboratoire de Chimie Organique, Faculté de Pharmacie, Paris.

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PIP2-Phospholipase C

The assay for PIP2-PLC (EC 3.1.4.3) was performed according to the method of Palmer (29) with minor modifications (30). The assay mixture contained 40 mM Tris-HCl (pH 6.5), 0.78 mM PIP2, 2 g/L Triton X-100, 100 mM KCl, 0.5 mM CaCl2, 3.5 mM CTAB, and 150-200 μg protein for a final volume of 200 μl. After incubation at 37°C for 30 min, the reaction was stopped by adding 80 μl of 10 mM cold EDTA (pH 7.0); 20 μl of an alkaline phosphatase solution (IU/μl) were added to the assay system and further incubated for 30 min at 37°C. All reactions were terminated by adding 300 μl of ice-cold 100 g/L trichloroacetic acid (TCA). After centrifugation (900 g, 20 min at 4°C), the inorganic phosphorus (Pi) content was assayed in analysis and blank supernatants by the colorimetric method of Chen et al. (31).

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5'-Nucleotidase (5'-NT)

5'-NT (EC 3.1.3.5) was assayed on homogenates by incubating 50 μg of proteins in a total reaction volume of 4 ml for 20 min at 37°C. The assay mixture contained 50 mM Tris-HCl buffer (pH 7.5), 8 mM MgCl2, and 2 mM 5'-AMP (32). The reaction was stopped with 30% ice-cold TCA, and after centrifugation, the Pi content of the supernatant was evaluated colorimetrically at 820 nm by the method of Chen et al. (31).

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γ-Glutamyltransferase (γ-GT)

γ-GT (EC 2.3.2.2) was measured on intact cells (1-5 × 105 cells) or in homogenates of 50 μg of proteins in 1 ml reaction mixture containing 20 mM Tris-HCl buffer (pH 8.0), 60 mM glycylglycine, 300 mM NaCl, and 2.5 mM γ-glutamyl-p-nitroanilide (27,32). The L-γ-glutamyl-p-nitroanilide released the glutamyl group to form by conversion the products p-nitroaniline and γ-glutamyl-glycylglycine. The reaction was terminated by adding 2 ml 1.5 N acetic acid. The mixture was then cleared by centrifugation (900 g, 20 min at 4°C), and the absorbance of the free p-nitroaniline in the supernatant measured spectrophotometrically at 410 nm.

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Enzymatic Activities

Specific activity was defined as pmol/min/mg protein for NEP, nmol/min/mg protein for 5'-NT and PLC, and nmol/h/106 cells or nmol/min/mg for γ-GT. The relative specific activity of each enzyme (RSA) was defined as the ratio of the absolute specific activity in each fraction to the absolute specific activity of the homogenate. The relative enzyme amount (or enzyme portion) in each fraction is the area of the corresponding column as obtained by multiplying RSA (ordinate) ×% proteins (abscissa). The enzyme recovery was defined as the ratio of the sum of activity in each fraction and the activity of the total homogenate and varied between 84 and 100%.

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Preparation of Subcellular Fractions

Cells at various conditions were recovered by centrifugation (800 g, for 10 min at 4°C). The pellet was resuspended in 1.5 ml of 0.25 M sucrose containing TKM buffer solution, and the cell suspension homogenized in a glass homogenizer with a Teflon pestle for 5 min (4,000 r/min, 10 strokes). The total homogenate (H) was centrifuged (800 g, for 10 min at 4°C) to obtain a nuclear pellet. The nuclear fraction (N) was carefully separated, resuspended in 0.5 ml TKM-sucrose 0.25 M, and centrifuged as previously. The postnuclear supernatants were pooled and centrifuged at 100,000 g for 30 min at 4°C in a L50 Beckman ultracentrifuge. The 100,000 g microsomal pellet, resuspended with 0.5 ml TKM-sucrose, constituted the particulate fraction (P), whereas the supernatant represented the soluble fraction (S). All the fractions were assayed for protein, with bovine serum albumin as a reference protein, and for specific marker enzymes.

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Total Lipid Extraction (TLE)

TLE was prepared from homogenates of 107 normal or HIV+ PBMNCs by using the chloroform-methanol method (33) as follows. Membrane pellets (100,000 g) were suspended in 20 volumes of chloroform/methanol (2:1, vol/vol), mixed to obtain a single phase. After standing for 15 min, preparations were centrifuged (750 g, 10 min, 4°C) to separate protein and particulate matter. Supernatants were reextracted with chloroform/methanol (1:1) and chloroform/methanol (1:2) solutions. The combined extracts were evaporated, the residues were dissolved in chloroform/methanol (1:1), and insoluble materials were removed by centrifugation. The final clear, total lipid extracts used for the partition step were evaporated to dryness.

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Gangliosides and Glycosphingolipids Isolation

Gangliosides and glycosphingolipids were isolated by the method described by Ladisch and Gillard (34) with few modifications. The TLEs were dispersed in a mixture of two organic solvents, DIPE (diisopropyl ether) and 1-butanol at the ratio 6:4 (vol/vol), vortexed and sonicated for 30 s. To the TLEs opalescent fine suspension, 0.25 M NaCl (half the volume of organic solvent) was added. Samples were vortexed and sonicated for several minutes, until TLEs were completely dissolved. After centrifugation (750 g, 10 min), the upper organic phase that contains neutral lipids and phospholipids was carefully removed. The lower aqueous phase containing the gangliosides, together with the small amount of emulsion present at the interface, was reextracted with an original volume of organic solvent mixture and centrifuged again. A total of two extractions was adequate to obtain clear thin layer chromatography (TLC) patterns of the ganglioside contents. The clear to slightly opalescent aqueous phase was lyophilized to concentrate the sample and to remove the residual.

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Thin-Layer Chromatography

TLC of total lipids extracts or isolated gangliosides was performed using 10 × 10 cm or 20 × 20 cm HPTLC plates (Merck), which were preactivated by heating to 90°C for 45 min. The plates were prerun with methanol for cleaning impurities and developed in chloroform/methanol/aqueous 0.25% CaCl2 (60:40:9) or in n-propanol/aqueous CaCl2 0.25% (8:2) (33).

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Staining

After drying, spots of lipids were revealed by spraying plates with a glacial acetic acid/sulfuric acid/anisaldehyde (50:1:0.3, vol/vol) solution, heated at 120°C for 15 min in a drying cabinet. Gangliosides appeared as green-brown spots. Contrary to oligosaccharides (green), cholesterol (blue), or phospholipids (pale violet), gangliosides changed color to brown after several hours in air.

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Densitometry

GSLs were analyzed quantitatively by densitometric scanning, using Apple OneScanner and Image 1.5.3 software. Known amounts of individual gangliosides and neutral glycolipids were used as standards to define intervals of linearity (calibration curves) of spot intensity. This technique allowed detection of 0.1 optical density (OD) differences over a range of 0-1.8 OD units, and a minimum amount of 0.02 μg gangliosides with a coefficient of variation <5%. For quantitative evaluation of glycosphingolipids from lymphocytes, the intensity of sample spots was referred to the calibration curve established for each glycosphingolipid.

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Sialic Acid Determination

TLEs of lymphocyte homogenates were assayed for the level of lipid-bound sialic acids (LBSA), by evaporating to dryness with a steam of nitrogen, mixing with 1 ml 0.5 M H2SO4, and heating for 45 min at 80°C to remove the glycosidically bound sialic acids. Samples were kept at room temperature for 1 h, centrifuged at 800 g, and the amount of sialic acids in the supernatant assayed by the fluorimetric method, according to the method of Hammond and Papermaster (35), with slight modifications. Sialic acid-containing supernatants (0.2 ml) were incubated with 0.1 ml of a solution containing 0.2 M sodium-meta-periodate (NalO4) in 9 M phosphoric acid at room temperature for 20 min. The reaction was stopped by adding 0.1 ml 10% sodium-meta-arsenite NaAsO2 in 0.5 M Na2SO4 and 0.5 M H2SO4 solution, shaken until the color turned to brown, and 0.9 ml of the same solution was added until the color disappeared. After addition of 3 ml 0.6% thiobarbituric acid in 0.5 M Na2SO4, the mixture was heated in a boiling water bath for 15 min.

After cooling, the dye was mixed with 1 ml N-butanol/HCl 12 N (95:5, vol/vol) and centrifuged. The fluorescence of the clear solvent upper phase was read with excitation at 532 nm and emission at 570 nm, using a Perkin-Elmer LS-3B Fluorescence Spectrophotometer. Standards with increasing amounts of pure sialic acids were run simultaneously. The sialic acid content in the assay was calculated using a calibration curve. To define the assay specificity, LBSA level was also determined on HSB2, REH, NALM-1, and Raji lymphoblastoid cell lines, with a distinct degree of morphologic differentiation (36).

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Statistics

Cell counts, data about monoclonal antibody (MoAb)-positive cells, absolute and relative specific activities, relative enzyme amounts, and sialic acid and glycosphingolipid values were analyzed by the nonparametric Wilcoxon ranked-sign test for matched pairs.

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RESULTS

Cell Population Immunophenotyping

The immunophenotypes of circulating PBMN cells isolated from patients and controls are summarized in Table 1. Several samples were obtained from some patients, but the data showed no significant changes during the period of the study. There was a gradual reduction in the absolute numbers of CD3, CD4, and CD8 cell subpopulations, and the cell ratio (H/S) in the advanced stages of infection confirming findings generally reported. There was a significant reduction in the absolute number of CD4 T-helper cells in the HIV-1-seronegative subjects compared with those of the control (Table 1).

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Enzymatic Characterization

The second messenger-generating enzyme phospholipase C in mononuclear cells isolated from HIV-1-infected patients had kinetic properties that were different from those of the enzyme in normal cells. The specific activity in HIV+ cells was four times lower than that in normal cells, and PLC had two pH optima (pH0), one at pH0 5.7 and the other pH0 6.2. The optimum in normal cells was pH0 6.0 (Fig. 1). Plasma membrane enzyme activities indicated that the ectoenzyme 5'-NT and the endoenzyme PLC gradually decreased from the control group to the CDC IVC group from 5 to 2.8 nmol/min/mg for 5'-NT and from 11.2 to 3.8 nmol/min/mg for PLC. The activities of both enzymes are significantly reduced in cells from HIV-1-seronegative IDU subjects (Table 2), in line with immunophenotyping findings. Normal lymphocytes had a discontinuous surface CALLA (median value of zero) content by staining with an anti-CD10 MoAb that was significantly lower than CALLA values for cells from HIV-1-seropositive or seronegative IDU (Table 1). In contrast, the enzymatic activity of the NEP was never detected biochemically in normal lymphocytes, although its amino acid sequence is almost identical to that of the CALLA antigen (17; Table 2). The substrate concentration and pH optimum for enzyme activity were established on NALM-6 cells that have a high basal enzyme level (26), because of the absence of NEP from normal lymphocytes and its sporadic occurrence in HIV+ lymphocytes. The NEP activity in HIV+ cells varied from 0 to 162 pmol/min/mg and was maximal in the group of HIV+ with AIDS (CDC IV). These findings indicate an intriguing lack of correlation between CALLA expression and NEP activity at the cell surface of circulating PBMNCs from infected IDU patients.

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Subcellular Distribution of γ-GT, 5'-NT and PLC in Normal and HIV+ PBMNCs

The subcellular distribution of marker enzymes in the PBMNCs from HIV-1+ subjects was markedly different from that of the control group. HIV-1-infected PBMNCs contained less 5'-NT in the nuclear fraction (N, reduced from 7.2% in normal cells to 4.1%), whereas the membrane (P) and soluble (S) fractions changed little. The fraction of PLC in the N was also reduced from 13.4% (normal) to 6.4%, but it was unchanged in the P fraction and increased in the S fraction (from 63.0 to 70.3%). There was more γ-GT in the N fraction (up to 15.2%) but less in the S fraction (down to 9.1%). The relative specific activity (RSA), defined as the ratio of the absolute specific activity in each fraction to the absolute specific activity of the homogenate, was also changed in lymphocytes from AIDS patients. The RSA of the soluble form of 5'-NT increased from 0.44 to 0.94, whereas the RSA of the membranebound form in the P fraction decreased from 2.84 to 1.86. The RSA of the membrane-bound form of PLC in the microsomal fraction increased from 0.94 to 1.26, whereas that of the nuclear fraction decreased from 0.51 to 0.35; the RSA of the soluble form remained unchanged. The RSA of the ectoenzyme γ-GT increased in both the nuclear (from 0.71 to 1.15) and microsomal fractions (from 3.61 to 4.79). The RSA of the soluble form was reduced from 0.22 to 0.13 (Table 3 and Fig. 2).

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Distribution of Gangliosides, Glycosphingolipids, and Lipid-Bound Sialic Acids

The most frequent ganglioside in membranes of normal PBMNCs was 11-N-acetylneuraminosyllactosylceramide (GM3). Its concentration of 2.1 μg/108 cells accounted for 82% of the total gangliosides (Fig. 3); it was followed by 11-N-acetylneuraminosylparaglobosylceramide (SPG) (10%) and trace amounts of other gangliosides (results not shown). Dihexosylceramide (GL2) was the most frequent glycolipid (50%) of the neutral glycosphingolipids (GSLs) in these cells, whereas the GL1 concentration was lowest (8%) and trihexosylceramide (GL3) (22%) and tetrahexosylceramide (GL4) (18%) were intermediate. PBMNCs from CDC IVC patients had less GM3 (1.2 μg/108 cells), although it still accounted for the majority of the ganglioside pool (75%). The amounts of GL2 and GL4 neutral GSLs also dropped, whereas monohexosylceramide (GL1) totally disappeared and GL3 remained unchanged (Figs. 3 and 4). As expected, the content of total lipid-bound sialic acid (LBSA), measured by fluorimetric assay, was reduced ≈50% in PBMN cells from AIDS subjects (2.6 μg/108 cells or 20 nmol/mg protein in controls and 1.6 μg/108 cells or 13 nmol/mg protein in HIV+ subjects).

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DISCUSSION

The surface immunophenotype of circulating PBMNCs from HIV-1+ IDUs was abnormal. The PBMN cells of HIV-1-seronegative patients also appeared to be phenotypically different from PBMNCs from healthy donors. These results confirmed the results of other studies, indicating that IDU HIV-1-seronegative patients have fewer total lymphocytes (37,38), CD4+ and CD8+ T cells (39) and a lower CD4/CD8 (H/S) ratio (40,41). These findings may be considered in light of the “opiate cofactor hypothesis” (42), which postulates that the immunosuppressive properties of opiates can produce changes in cell-mediated immunity and foster the development of opportunistic infections in HIV-1-infected drug users. The influence of opiates on the immune system is controversial; there are reports that opiates can activate the immune status (39,43,44) and others that they stimulate HIV-1 replication in PBMNCs (45) or in nervous system cells (46). This study provides evidence that HIV-1 infection perturbs the subcellular distribution or the kinetic parameters of the plasma membrane enzymes examined. The relative specific activity of PIP2-PLC in HIV-1-infected cells was increased in the microsomal fraction but decreased in nuclear fractions. The microsomal increase yielded an enzyme subcellular localization similar to that found in Con A-stimulated lymphocytes, whereas the enzyme decrease was comparable to that found in PHA-stimulated lymphocytes (26). Confirmation of an abnormal plasma membrane in the PBMNCs from HIV-1+ patients was provided by the changes in the activity of the signal-transmission enzyme. The PLC had two pH optima (pH0) at pH 5.7 and pH 6.2 in HIV+ patients, but only a single optimum at pH 6.0 in normal cells, suggesting activation of isoenzymes in infected cells. The activity at pH 6.0 was 3 to 4 times lower in infected cells than in control cells or in HIV-negative cells. The lowest 5'-NT activity was in the HIV-1+ cells from IDUs, in line with results reported for HIV-1+ homosexuals (6). The 5'-NT activity was also reduced in HIV-1-seronegative IDU patients. This functionally impaired behavior is similar to that observed in lymphocytes from patients with sex-linked agammaglobulinemia and common variable immunodeficiency (47). In contrast, the γ-GT activity in lymphocytes from HIV-1+ patients was normal, a finding similar to that for cells from congenital immunodeficiency and agammaglobulinemia (32). Last, the neutral endopeptidase activity in PBMN cells of HIV-1+ IDUs was sporadic and low, but was not correlated with the increased phenotypic expression of CALLA detected by anti-CD10 MoAbs. The results were not totally unexpected, in light of experiments carried out with cultures of PBMNCs. In these, the NEP activity was high only in presence of concanavalin A and cocaine, whereas the CALLA positivity remained very low (26). The amounts of lipid-bound sialic acids and glycosphingolipids in the membranes of PBMNCs from IDU AIDS patients were subnormal. These data on glycosphingolipids do not agree with those in a recent report (13) but are consistent with data from another study indicating a reduction of neutral GL1 and GL2 glycolipids and GM3 ganglioside in human cells infected with human retroviruses (48). The changes in PBMNC glycosphingolipids reported here could be related to general changes in the synthesis of membrane lipids and phospholipids in HIV-1-infected cells, as documented by others (49). An increase in unsaturated fatty acids, followed by a decrease in the content of saturated fatty acids, has also been observed in lymphocytes cultured with HIV (50). Others have shown that the HIV-1 envelope glycoprotein gp120, which binds to the CD4 receptor, has no effect on the metabolism of inositol phosphate and arachidonic acid, on protein kinase C translocation, or on tyrosine phosphorylation in human T-and tumor-cell lines (51). A marked alteration in the membrane-dependent steps of phospholipid synthesis and a decrease in the intensity of fatty acid staining has been observed in several strains of lymphoblastoid cells soon after HIV-1 infection (52). Changes in inositol phospholipid metabolism due to impaired phospholipase C have been reported after adding HIV-1 to human lymphocytes (53). Taken together, the data indicate that there are profound changes in the structural and functional features of the plasma membrane of circulating lymphocytes from both HIV-1+ and HIV-1-seronegative IDUs. The decrease of plasma membrane enzymes may reduce the availability and the entry in lymphocytes of essential molecules such as nucleosides and impair the reception of regulatory molecules and their transmission. This would affect the de novo synthesis of nucleotides and nucleic acids (32,47), the purine pathway salvage (7,54), as well as other immune-specific pathways (42). Drug-mediated changes of chemicophysical properties (26) might alter the molecular integrity and the functionality of the plasma membrane, maintain part of lymphoid cells in an immature and labile state (3,55), and, finally, diminish the capacity to respond to antigenic stimuli.

Acknowledgment: We thank Dr. Damiano Castelli of the Centro Trasfusioni Croce Rossa Lugano, Switzerland, for providing blood samples of HIV-1-seronegative intravenous drug users and normal healthy donors. We thank also Dr. Owen Parkes (Paris) for editorial help.

This work was supported by grant no. 32-26600.89 of the Swiss National Science Foundation.

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    Keywords:

    Human immunodeficiency virus type 1; Peripheral blood mononuclear cells; Intravenous drug users; Immunophenotype; Plasma membrane enzymes; 5'-nucleotidase; Phospholipase C; Neutral endopeptidase; CALLA; γ-Glutamyltransferase; Gangliosides; Glycosphingolipids

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