INTRODUCTION

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In general, proteinase inhibitors are inhibitory for a single class of proteinases, often resulting in mutual inactivation. On the other hand, proteinases may inactivate many classes of inhibitors without being inactivated themselves and, in the process, may produce bioactive fragments, e.g., complement fragments. The proteinase inhibitor in serum exhibiting the greatest concentration is ₁PI, and the proteinase inhibitor encompassing the broadest spectrum is ₂ macroglobulin (₂M). Multiple molecular conformations of active and inactive ₁PI are known to exist and to be receptor recognized. For example, active ₁PI has been found to react with surface-associated human leukocyte elastase (HLE), and inactive ₁PI has been found to react with the well-characterized scavenger receptor, ₂M receptor-low-density-lipoprotein receptor-related protein (₂MR-LRP) (2, 21). The highly conserved ₂M uniquely inhibits all four classes of proteinases and exhibits a receptor-recognized conformation involved in signaling as well as an ₂MR-LRP-recognized conformation (17). A significant body of evidence suggests that receptor recognition of the multiple forms of circulating proteinase inhibitors results in activation of discrete cellular subsets of the mononuclear phagocyte system (27).

Whereas ₁PI covalently inhibits the catalytic site of HLE, it has been shown that in certain situations the association of HLE with ₂M in plasma is favored (6). When proteinases are complexed with ₂M, proteolysis of low-molecular-weight peptides and cytokines can persist. The balance between available HLE activity, the inhibitor ₁PI, and the substrate-restricting ₂M represents a tightly regulated mechanism for discrete targeting of proteolytic activity in tissues. It has been shown that inter--trypsin inhibitor (II) uniquely acts as a shuttle by transferring HLE to ₁PI or ₂M (25). It is the active site of II which is identical with the principle HIV-neutralizing determinant in the V3 loop, and it is this region of the V3 loop which has been shown to reversibly inhibit proteinases (1). This suggests the possibility that HIV envelope proteins themselves may disrupt the homeostasis between proteinases and proteinase inhibitors.

The importance of maintaining a balance in proteinase inhibitor ratios was dramatically demonstrated more than two decades ago. Ohlsson and Laurell injected two volunteers intravenously with ₁PI or ₂M less than half saturated with radiolabeled HLE or trypsin (20). Rapid clearance of ₂M complexes and ₁PI complexes was shown to be mediated by the mononuclear phagocyte system and to be accompanied by transient circulatory changes in some circumstances. Intravenous injection in dogs resulted in shock and death whenever the concentration of active proteinase exceeded that of ₂M, even though circulating ₁PI was less than half saturated with proteinase (19). The concept that HLE and ₁PI might impact HIV infectivity originated following the observations that gp120 exhibits a proteinase-inhibiting domain (1) and that CD4, the T-lymphocyte antigen receptor, elastase, and ₁PI are functionally associated during antigen-specific lymphocyte activation (8). Evidence that ₁PI and cell surface HLE participate during HIV infectivity (7) (C. L. Bristow, unpublished data) suggested the hypothesis that ₁PI or ₂M concentrations might be altered during HIV disease.

	MATERIALS AND METHODS

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Serum specimens. Sera from healthy volunteers were collected after obtaining informed consent. Sera from HIV-infected patients were excess specimens reserved from previous studies for which informed consent had been obtained. HIV RNA in serum was quantitated by PCR using the Amplicor HIV-1 Monitor Test (Roche Diagnostics, Branchburg, N.J.) by the Retrovirology Core Laboratory, University of North Carolina (UNC)Chapel Hill, using procedures recommended by the manufacturer. Clinical category of disease progression at the time of specimen collection was determined using 1993 Centers for Disease Control and Prevention classification criteria. The clinical parameters examined included HIV RNA, lymphocyte markers, demographic characteristics, known infectious diseases, and antiretroviral therapy. In this study, 44% category A1,A2, 57% category B1,B2, and 58% AIDS patients had or were receiving azidothymidine therapy, and none were receiving HIV-specific protease inhibitors. Characteristics not presented herein were found not to contribute significantly to the analysis. All measurements, chart extraction, and data analysis were performed in a blinded fashion. Data were excluded from analysis if the sera had not been maintained at 80°C prior to analysis, the patient charts could not be located, or the patient charts indicated no evidence of HIV infection.

Quantitation of ₂M and ₁PI and T-cell markers. Methods for quantitating active ₂M and ₁PI by determination of elastase inhibitory capacity have been described elsewhere (6). Total ₁PI was determined by enzyme-linked immunosorbent assay (ELISA). Inactive ₁PI was expressed as the difference between total ₁PI and active ₁PI. Measurements of CD4⁺, CD8⁺, and CD3⁺ levels were performed by the Flow Cytometry Laboratory, UNC Hospitals. Normal values were determined from 100 healthy donors (Janet Mantell and Cindy Evoy, unpublished results). Data were not normally distributed, and all comparisons were made using nonparametric methods. Medians among clinical categories were compared by using Kruskal-Wallis one-way analysis of variance on ranks. Comparison of medians between pairs of clinical categories was done by using the Mann-Whitney rank sum test. Correlation was measured using the Pearson product moment correlation.

Detection of anti-₁PI immune complexes. Detection of anti-₁PI immune complexes was performed by modification of the ₁PI-specific ELISA (6). Sera were heat inactivated for 30 min at 56°C. Anti-₁PI immune complexes were captured by diluting an aliquot in 0.05 M Tris(hydroxymethyl)aminomethane-0.15 M NaCl (pH 7.8) in wells of a microtiter plate precoated with the immunoglobulin fraction of chicken anti-human ₁PI (lot 18823680; O.E.M. Concepts, Toms River, N.J.), followed by incubation for 120 min at 37°C. In parallel, to detect anti-₁PI in serum by first dissociating the anti-₁PI immune complexes, the sera were acidified by diluting an equivalent aliquot in 0.05 M glycine-HCl (pH 3.0). After incubation for 60 min at 37°C, acidified sera were adjusted to pH 7.8, and complexes were allowed to reassociate for an additional 60 min at 37°C. Binding was detected by using horseradish peroxidase (HRP)-complexed rabbit anti-human immunoglobulin G (IgG; lot 091H4808; Sigma).

Anti-gp120 ELISA. Immunorecognition of ₁PI by anti-gp120 was determined by ELISA (6). Wells of a microtiter plate (Nunc) were precoated with various concentrations of ₁PI (A6150; Sigma) or soluble CD4 (Biogen, Cambridge, Mass.). Microtiter plates were incubated for 60 min at 20°C with monoclonal anti-gp120 (1C1, lot 704-111; Repligen, Cambridge, Mass.) in the presence or absence of 1 µg of competing recombinant gp120 (lot 1008-25; Repligen) per ml. Binding was detected with HRP-conjugated protein A (Zymed, South San Francisco, Calif.). Immunorecognition of ₁PI by HRP-conjugated monoclonal anti-gp120 (approximate epitope V3 loop, 10 µg/ml; Research Diagnostics, Flanders, N.J.) was performed for comparison.

Statistical analysis. Normality was determined using the Kolmogorov-Smirnov test. Values for CD4, CD8, ₁PI, and ₂M were not normally distributed, and all comparisons were made using nonparametric methods. Medians among clinical categories were compared by using Kruskal-Wallis one-way analysis of variance on ranks. Comparison of medians between pairs of clinical categories was done by using the Mann-Whitney rank sum test. Values for anti-₁PI and HIV-1 RNA were normally distributed, and all comparisons were made by using one-way analysis of variance. Comparison of means between pairs of clinical categories was done by t test. Correlation was measured using Pearson product moment correlation, and linear regression was measured by computer-fit least-squares analysis.

	RESULTS

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₁PI levels in clinical category of HIV disease. The ₁PI and ₂M levels were quantitated in the sera from 68 HIV-seropositive patients and 30 seronegative volunteers (Table 1). None were being treated with HIV-specific proteinase inhibitors at the time of blood collection. In the normal subjects studied here, the mean value for total ₁PI was 36.0 ± 13.9 µM and ranged from 18 to 53 µM between the 5th and 95th percentiles (Fig. 1). The mean ₂M concentration in the present study was 3.36 ± 1.82 µM and ranged from 1.26 to 5.57 µM between the 5th and 95th percentiles. Values for ₁PI and ₂M determined here are consistent with previous studies (5, 12).

View larger version (37K): [in this window] [in a new window]   FIG. 1.   1PI and 2M concentrations in serum by clinical category of HIV disease. (A) Values were not normally distributed. Category medians are represented by bars and are statistically compared in Table 1. (B) HIV RNA was found to be correlated with total 1PI in asymptomatic patients (P < 0.04) but not in other clinical categories. HIV RNA was not related to CD4 levels in any clinical category. (C) There was no relationship between 2M and 1PI concentrations in asymptomatic and symptomatic pre-AIDS patients; however, in AIDS patients, 2M was correlated with both active 1PI (r2 = 0.73, P < 6 × 106) and inactive 1PI (r2 = 0.45, P < 0.04). (D) Schematic representation of CD4 (.....), HIV RNA (), active 1PI (----), and total 1PI (----) concentrations in this patient population.

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View this table: [in this window] [in a new window]   TABLE 2.   Sensitivities and specificities of 1PI, CD4, and HIV RNA as prognostic indicators for transition between clinical categories in HIV diseasea

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Anti-₁PI in HIV disease. Since autoantibodies recognizing multiple cytokines and cellular proteins are known to occur in HIV disease (14), the potential for an autoimmune process in producing decreased active ₁PI was examined. Antibodies recognizing immobilized ₁PI were not discernible in serum from this patient population by traditional ELISA (data not shown). However, it was found that ₁PI-anti-₁PI immune complexes were detectable by capturing them with immobilized anti-₁PI and detecting them with anti-human IgG. Control sera from 17 normal subjects and 1 subject with inherited ₁PI deficiency were found to have background values of 11.2 ± 8.4 milliabsorbance at 405 nm (mA₄₀₅)/min and to range between 0.9 and 26.0. In contrast, 56% of asymptomatic, 57% of symptomatic pre-AIDS, and 31% of AIDS patients exceeded the mean control value by 2 standard deviations (Table 3). The high concentration of ₁PI in serum suggested that the appearance of anti-₁PI would rapidly produce immune complexes, precluding detection by this method. It was further considered that immune complex formation might obscure ₁PI epitopes, preventing capture using anti-₁PI and resulting in underestimation. We have previously shown in insulin-dependent diabetes mellitus that insulin-anti-insulin immune complexes are obscured in this manner (28). We determined that acidification of serum allows dissociation of immune complexes and that neutralization of acidified serum in the presence of competing radiolabeled ligand allows the detection of insulin-anti-insulin immune complexes. Using a modification of this approach, acidification and neutralization of sera in the presence of immobilized competing anti-₁PI allowed detection of ₁PI-anti-₁PI immune complexes in an additional 33% of asymptomatic patients, an additional 7% of symptomatic pre-AIDS, and an additional 14% of AIDS patients. Background values (mA₄₀₅ per minute) in normal control sera were not influenced by acidification and neutralization. Overall, 89% of asymptomatic, 64% of symptomatic pre-AIDS, and 44% of AIDS patients had detectable anti-₁PI IgG, and this decline in anti-₁PI IgG is consistent with the decline in specific antibody response during AIDS. An increased anti-₁PI level was correlated with decreased active ₁PI in the asymptomatic category of HIV disease (P < 0.05). Increased anti-₁PI was correlated with increased inactive ₁PI in symptomatic (P < 0.03) and AIDS patients (P < 0.03), and this suggests that ₁PI autoimmune antibodies are responsible in part for acquired ₁PI deficiency. In patients with detectable anti-₁PI IgG immune complexes, the odds were 3.6:1 that active ₁PI would be deficient (P < 0.01). Further, in AIDS patients, the mean reactivity (mA₄₀₅ per minute) was significantly lower than in the other clinical categories (P < 0.002, Fig. 2), and this result is consistent with the decline in detectable anti-₁PI. The correlation between ₂M and active and inactive ₁PI in AIDS suggests that infection-related dysregulated proteolysis also contributes to the deficiency in ₁PI detected.

View this table: [in this window] [in a new window]   TABLE 3.   Circulating 1PI-specific IgG immune complexes in HIV disease

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Autoantibodies reactive with 1PI in HIV disease. (A) Autoantibodies reactive with 1PI were detected by ELISA. Bars represent mA405 per minute and are statistically compared in Table 3. The mean reactivity was significantly lower in AIDS patients than in asymptomatic (P < 0.0005) or symptomatic pre-AIDS (P < 0.02) patients. (B) A monoclonal antibody with specificity for gp120 epitopes near the fusion domain also recognized 1PI () but not CD4 (). Binding of anti-gp120 to 1PI was inhibited in the presence of 8 µM competing gp120 (). A monoclonal antibody with specificity for gp120 epitopes near the V3 loop failed to recognize 1PI (). (C) Alignment of amino acid sequences for human 1PI and the epitope recognized by anti-HIV gp120 near the fusion domain revealed significant homology. Boxed residues are identical or conservatively substituted. The immunizing peptide for monoclonal anti-gp120 antibody is underlined. The hydrophobic 1PI pentapeptide is depicted in boldface. The proteinase reactive site of 1PI is Met-358. HIV gp120 and gp41 are produced by cleavage near Lys-517 and Arg-518 to reveal the fusion domain.

	DISCUSSION

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We unexpectedly found in this study that active ₁PI was deficient in all asymptomatic patients. On the other hand, total ₁PI was elevated in all symptomatic pre-AIDS HIV-positive patients. The strong association between ₁PI and disease progression suggested ₁PI might be central to the pathophysiology of HIV disease. Combined with CD4⁺ levels, determining the viral load (plasma viral RNA) is integral to the recommended guidelines for the clinical management of HIV disease. Discordant values for plasma viral RNA and CD4⁺ cell counts occur in approximately 14% of patients receiving antiretroviral therapy, and this complicates therapeutic decisions. The prognostic value of ₁PI concentration for determining clinical category of HIV disease was equivalent to that of CD4, and this finding supports recent evidence that this self antigen acts as an entry cofactor.

More than 75 codominant alleles of human ₁PI have been identified that are correlated in mucosal secretions and serum (16). In 1,084 plasma samples representing six species of monkeys (Macaca irus, M. mulatta, M. cyclopis, M. nemestrina, M. speciosa, and M. fuscata), five codominant alleles have been identified (3). These genetic differences might serve as a key to understanding the different pathologic outcomes of HIV infection in humans and chimpanzees. Neither antibodies to ₁PI nor deficiency in ₁PI were detected in macaques infected with simian/human immunodeficiency virus (SHIV) (data not shown), suggesting that different pathophysiologic outcomes result during HIV disease in humans and SHIV disease in macaques.

The primary source of circulating ₁PI is thought to be the liver. Synthesis and secretion of ₁PI by lymphocytes, mononuclear phagocytes, cornea, and intestinal Paneth cells in response to interleukin-6, transforming growth factor , granulocyte-macrophage colony-stimulating factor, HLE, and lipopolysaccharide from gram-negative bacteria is under the regulation of NF-B (22). Convincing evidence from ₁PI phenotype-mismatched patients undergoing bone marrow transplantation suggests that the mononuclear phagocyte system does not contribute to the systemic pool (13). However, it can be speculated that ₁PI secreted by the mononuclear phagocyte system might explain the low levels of ₁PI in patients homozygous for the PI_zz phenotype who lack the capacity for hepatocyte secretion of ₁PI. It can be further speculated that ₁PI secreted by cells of the mononuclear phagocyte system might primarily remain tissue associated, creating a localized gradient.

The distribution of human ₁PI phenotypes has been reported to differ between ethnic groups and between groups dichotomized by sexual preference (9). Testosterone can induce a 20-fold increase in ₁PI mRNA, and during pregnancy ₁PI increases by 100% and ₂M increases by 20% (10).

The detection of anti-₁PI IgG in HIV-seropositive patients suggests that autoimmune recognition of ₁PI may be one determinant in producing ₁PI deficiency. This result further suggests that in the presence of competing anti-₁PI immune complexes, the quantitation of total and inactive ₁PI by ELISA in these patients may have been underestimated here. The binding of ₁PI by monoclonal anti-gp120 suggests humoral immunity to gp120 is one potential mechanism for initiating an autoimmune response to ₁PI. Comparative alignment of the antibody-recognized gp120 peptide sequence and the carboxyl-terminal amino acids of ₁PI supports antigenic mimicry. Further, because of the hydrophobic nature of the ₁PI epitope recognized by anti-gp120, these results suggest that neo-epitopes of ₁PI may be exposed during HIV disease. It has been suggested that viral proteins interactive with host proteins can induce conformational changes and the exposure of neo-epitopes, thereby initiating autoimmunity (26), and this suggests a potential mechanism for the initiation of autoimmunity to ₁PI. Antibodies recognizing the V3 loop have been shown to inhibit the activity of II (18), and this suggests that the functional activity of II may also be diminished in the HIV-seropositive population. Deficiency or overexpression of II has not been described in any disease process (4); however, the active derivative of II has been reported to localize to affected tissue in Alzheimer-type dementia (29) and to increase during bacterial infection (24). That morbidity and mortality are documented to result from an imbalance in ₁PI and ₂M ratios (19, 20) suggests that autoimmunity to ₁PI may be cardinal to HIV-associated pathophysiology. The HIV-induced autoimmune recognition of ₁PI raises an important concern regarding vaccine development. It is not known at this time whether the initiation of immunity to HIV by vaccination also may initiate autoimmunity to ₁PI. Of considerable interest is that immune recognition of multiple HIV proteins can be detected in HIV-exposed seronegative individuals; however, neither IgA nor IgG with specificity for gp120 could be detected in these individuals (15). The evidence from this continuing seronegative population for the potential to coexist with HIV suggests, therefore, that protection against AIDS in HIV-infected individuals might arise by preventing autoimmunity to ₁PI.

Results presented elsewhere suggest that ₁PI deficiency, a condition which exists in the asymptomatic category of HIV disease, may promote increased cellular sensitivity to HIV infectivity (8a). On the other hand, the increasing concentrations of ₁PI which occur during bacterial infection may promote increased viral load, conditions known to occur during the symptomatic category of HIV disease. The correlation between HIV RNA and total ₁PI in the asymptomatic category supports the hypothesis that changing concentrations of circulating ₁PI in response to HIV proteins and in response to attendant infections during HIV disease directly impact HIV pathophysiology. Evidence that plasma membrane-associated HLE has been previously shown to interact with gp120 (17) and recent evidence that neither cell lines nor peripheral blood cells infected in vitro produce detectable RT activity in the absence of ₁PI (8a) support the participation of these proteins during HIV disease.