INTRODUCTION

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We previously demonstrated that cell surface human leukocyte elastase (HLE) specifically and reversibly binds the HIV fusion domain (Bristow et al. [3]). We have recently found that human immunodeficiency virus (HIV) preferentially binds to copatches of HLE, CD4, and chemokine receptors (C. L. Bristow et al., unpublished data). HIV receptors were found to copatch in response to ₁proteinase inhibitor (₁PI) potentially explaining the requirement for ₁PI during HIV entry. These studies led to the hypothesis that ₁PI might impact HIV disease progression. Indeed, in a study of HIV-seropositive patients, we recently found that decreased circulating ₁PI is significantly correlated with decreased viral load (3a). The prognostic value of measuring ₁PI was found to be comparable to measuring CD4 and considerably better than measuring HIV RNA. During the asymptomatic category of HIV disease, 100% patients were found to manifest deficient levels of active ₁PI. In contrast, circulating levels of the proteinase inhibiting ₂ macroglobulin (₂M) in this patient population were not significantly different from those of normal controls. Individuals with the inherited form of ₁PI deficiency, especially males, are notably susceptible to respiratory infections, and 80% who survive to adulthood succumb to respiratory failure between the fourth and sixth decades of life (1). This suggests that the ₁PI deficiency acquired during HIV infection could provide a mechanistic explanation for the onset of attendant infections and inflammation which subsequently initiate an increase in ₁PI. Recent evidence has suggested that increased HIV is produced by blood cells from individuals having the inherited form of ₁PI deficiency (6). To further investigate the possibility that variation in circulating ₁PI concentrations might directly impact HIV infectivity outcome of peripheral blood mononuclear cells (PBMC), in vitro infectivity of PBMC from HIV-nonexposed individuals having the inherited form of ₁PI deficiency was compared in the presence and in the absence of exogenous ₁PI in autologous serum.

	MATERIALS AND METHODS

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Subjects. Blood was collected from eight different HIV-seronegative healthy volunteers after we obtained informed consent. Subjects were selected to represent a range of ₁PI concentrations in serum. All subjects were normally healthy; however, the female counterpart from a pair of siblings homozygous for the ₁PI-deficient genotype PI_ZZ (2) was included for comparison despite her history of systemic lupus erythematosis.

Quantitation of ₂M and ₁PI. Sera were measured at the time of collection for ₂M, as well as for active- and inactive-₁PI concentrations. Methods for quantitating active and total ₁PI have been described elsewhere (2). Active ₂M and ₁PI concentrations were determined by elastase inhibitory capacity. Total ₁PI was determined by enzyme-linked immunosorbent assay. Inactive ₁PI was expressed as the difference between total ₁PI and active ₁PI.

Active-site standardization of exogenous ₁PI. Active-site standardization was performed as previously described (2). One mole of active-site titrated porcine pancreatic elastase, type 1 (EC 3.4.21.36; Sigma) was found to saturate 3.06 moles ₁PI (Sigma), suggesting that this preparation was 32.7% active.

In vitro HIV infectivity of mononuclear cells. PBMC were isolated by Ficoll-Hypaque gradient centrifugation from whole blood collected in tubes containing ACD (Becton Dickinson, San Jose, Calif.). Three primary non-syncytium-inducing HIV type 1 (HIV-1) clinical isolates were generously provided by the Retrovirology Core Laboratory, UNC-Chapel Hill. In vitro infectivity outcome was determined by quantitating p24 accumulation or reverse transcriptase (RT) activity as previously described (3, 5). Isolated PBMC were resuspended and maintained in the wells of a 96-well tissue culture plate at 2 × 10⁶ cells/ml in RPMI 1640 containing 20% autologous serum and 10% interleukin-2 (Cellular Products, Buffalo, N.Y.). PBMC in 100 µl in autologous medium were stimulated by the addition of 5 µg of phytohemagglutinin (PHA; Sigma) per ml for 3 days at 37°C in humidified 5% CO₂. Cells were washed and resuspended in fresh autologous medium containing various concentrations of exogenous active-site standardized ₁PI (Sigma).

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Immunofluorescent staining and flow cytometric analysis. Three-parameter flow cytometric analysis using direct immunofluorescent staining of whole blood was performed on a FACScan flow cytometer (Becton Dickinson) using fluorescein isothiocyanate (FITC), phycoerythrin (PE), and peridinin chlorophyl protein (PerCP) by using methods recommended by the manufacturer. To detect chemokine receptors, blood was stained simultaneously in a single step with three monoclonal antibodies which included (i) anti-CD14-FITC, anti-CCR5-PE, and anti-CD4-PerCP (PharMingen, San Diego, Calif.); (ii) anti-CD14-FITC, anti-CXCR4-PE, and anti-CD4-PerCP (PharMingen); or (iii) anti-CD14-FITC, immunoglobulin G2a (IgG2a)-PE, and anti-CD4-PerCP (PharMingen). To detect cell surface HLE, blood was stained stepwise with three antibodies, including polyclonal sheep anti-HLE-FITC (Biodesign, Inc., Kennebunkport, Maine), monoclonal anti-CXCR4-PE (PharMingen), and monoclonal anti-CD4-PerCP (Becton Dickinson). The isotype-matched controls were IgG2a-PE, IgG1-PerCP, or nonspecific IgG-FITC. Blood was washed in 2 ml of phosphate-buffered saline between each staining step.

	RESULTS AND DISCUSSION

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Influence of ₁PI on HIV-1 produced by PBMC infected in vitro. To determine whether ₁PI might influence HIV infectivity, healthy volunteers were selected to represent a range of circulating ₁PI levels (Table 1). PBMC from these individuals were infected in vitro using three concentrations of a non-syncytium-inducing clinical isolate of HIV-1. PBMC were maintained at all times in autologous serum containing various concentrations of exogenous ₁PI. We have observed that circulating ₁PI significantly impacts the adherence of PBMC to tissue culture tubes (r² = 0.81, P < 0.0001; data not shown). To avoid exclusion of adherent cell populations, isolated PBMC were stimulated, infected, and cultured without removal from their original tissue culture wells. Cultures were performed in duplicate, and supernatants were collected and replaced with fresh medium every other day for the determination of RT activity.

View this table: [in this window] [in a new window]   TABLE 1.   Coreceptor levels and circulating 1PI and 2M in HIV-seronegative volunteers

View larger version (24K): [in this window] [in a new window]   FIG. 1.   In vitro HIV infectivity of PBMC in the presence of exogenous 1PI. (a) Infection outcome using a non-syncytium-inducing clinical isolate of HIV-1 was determined in duplicate by measuring the RT activity produced by PBMC from subjects 1 (), 2 (), 3 (), 4 (), 5 (), and 6 (), represented in Table 1 in the absence or presence of 3 or 30 µM exogenous 1PI in autologous serum. Mean values are depicted. (b) On day 8, the RT activity in the culture supernatants increased as the exogenous 1PI concentration increased.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Correlation between the rate of HIV p24 production and the circulating 1PI concentration. (a) PBMC were infected with 10 or 40 TCID50, and the infectivity outcome was determined by measuring p24 accumulation. PBMC from subjects 1 (), 3 (), 4 (), 7 (), and 8 () represented in Table 1 were infected with a non-syncytium-inducing clinical isolate of HIV-1 and cultured in autologous serum. (b) The rate of p24 accumulation was calculated as the difference in p24 between consecutive measurements. The computer-fit linear regression curve for 10 TCID50 is log [y] = 1.29  0.03 log [x] (r2 = 0.93, P < 0.008) and for is 40 TCID50 is log [y] = 1.81  0.04 log [x] (r2 = 0.95, P < 0.005), where y represents p24 and x represents active serum 1PI.

Influence of circulating ₁PI on HIV coreceptor density. The relationship between infectivity and coreceptor densities was compared using blood collected from the same six volunteers as those for which RT activity was measured. We have observed that cell surface HLE on promonocytic cells appears to be increased when the cells are interacted with antibodies specific for CD4 first and specific for HLE secondarily and then decreased when the order of antibody addition is reversed (C. L. Bristow, unpublished data). When blood was first reacted with anti-HLE and secondarily with anti-CD4 or antibodies specific for CXCR4 or CCR5, HLE density was negligible on CD4⁺ lymphocytes or CD4⁺ CD14⁺ mononuclear phagocytes (Fig. 3). When blood was reacted with antibodies in the reverse order, considerable levels of HLE were detected on both CD4⁺ lymphocytes and CD4⁺CD14⁺ mononuclear phagocytes. In contrast, the order of coreceptor ligation had no influence on the fluorescence intensities of CD4, CD14, CCR5, or CXCR4 (data not shown). These results suggest a dynamic functional association between membrane-associated HLE and the HIV coreceptors CD4, CXCR4, and CCR5. As would be expected, receptor densities were found to vary in the subjects examined here without any obvious pattern (Table 1). For example, a low receptor density for one receptor was not related to a concordantly low or high receptor density for any other receptor. Nor was RT activity found to be related to the receptor densities of any HIV coreceptors examined (Fig. 3). Since the fractions of CD4⁺ lymphocytes and CD4⁺ mononuclear phagocytes varied considerably between individuals, the fluorescence intensity of coreceptors was normalized to CD4⁺ cells and was expressed as RFI. Increased HLE RFI, but neither the CXCR4 nor the CCR5 RFI value on CD4⁺ CD14⁺ mononuclear phagocytes was found to be directly correlated with increased RT activity (r² = 0.81, P = 0.01; Fig. 4). These results suggest increased cell surface HLE molecules associated with each CD4 molecule resulted in increased RT activity. In contrast, RT activity was not related to the RFI for any coreceptors on CD4⁺ lymphocytes. Nor was RT activity related to RFI when coreceptors were expressed relative to CXCR4 or CCR5 (data not shown). Consistent with these results, when coreceptors were expressed as the fluorescence intensity relative to HLE, increased RT activity was found to be related to CD4 on CD4⁺ CD14⁺ mononuclear phagocytes but not to either CXCR4 or CCR5 (data not shown). In this case, increased numbers of CD4 molecules associated with each HLE molecule resulted in decreased RT activity. These results suggest that, although all coreceptors may participate during HIV entry, the ratio of cell surface of HLE and CD4, but neither CXCR4 nor CCR5, is determinant during HIV infectivity outcome.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Relationship between RT activity and HIV coreceptor expression. PBMC from subjects 1 to 6 represented in Table 1 were analyzed for coreceptor levels using three-color flow cytometry. (a) Whole blood first interacted with anti-CD4 and second with anti-HLE (tightly dotted line) exhibited considerable HLE on the cell surface of CD4+ CD14+ mononuclear phagocytes (left panel) and CD4+ lymphocytes (right panel) compared with isotype controls (loosely dotted line). In contrast, blood first interacted with anti-HLE and secondly with anti-CD4 (solid line) exhibited diminished HLE. Two representative subjects are depicted. (b) The RT activity produced by subjects 1 (), 2 (), 3 (), 4 (), 5 (), and 6 () showed no relationship to the cell surface densities of any coreceptors examined on CD4+ lymphocytes or CD4+ CD14+ mononuclear phagocytes.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Relationship between RT activity and HIV coreceptor expression relative to CD4 levels. (a) The RT activity produced by PBMC from subjects 1 (), 2 (), 3 (), 4 (), 5 (), and 6 () represented in Table 1 was correlated with the HLE relative to CD4 (RFI) on CD4+ CD14+ mononuclear phagocytes but not with other coreceptors. The computer-fit linear regression curve predicts y = 770 + 80x (r2 = 0.81, P = 0.01), where y represents the RT activity and x represents the HLE RFI. (b) Circulating 1PI, but not 2M, was correlated with HLE RFI on CD4+ CD14+ mononuclear phagocytes but not with other coreceptors on these cells. The computer-fit linear regression curve predicts y = 137  9x, where y represents the 1PI and x represents the HLE RFI relative to CD4 (r2 = 0.88, P = 0.006). (c) The RT activity decreased as the circulating 1PI concentration increased on day 8 as represented in Fig. 1. Computer-fit linear regression of these data predict that log [y] = 3.0  0.69 log [x], where x represents the 1PI concentration in serum and y represents the in vitro HIV RT activity (r2 = 0.95, P < 0.001).