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Blood, 15 December 2003, Vol. 102, No. 13, pp. 4479-4486. Prepublished online as a Blood First Edition Paper on August 21, 2003; DOI 10.1182/blood-2003-05-1635.
IMMUNOBIOLOGY HIV-1 preferentially binds receptors copatched with cell-surface elastaseFrom the Laboratory of Cellular Physiology and Immunology, The Rockefeller University and Population Council, New York, NY, and the Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill.
Human leukocyte elastase (HLE) interacts with HIV-1 glycoprotein (gp)41, suggesting a nonenzymatic receptor function for HLE in the context of HIV-1. HLE is found localized to the cell surface, but not granules in HIV permissive clones, and to granules, but not the cell surface of HIV nonpermissive clones. Inducing cell-surface HLE expression on HLE null, HIV nonpermissive clones permits HIV infectivity. HIV binding and infectivity diminish in proportion to HLE RNA subtraction. HIV binding and infectivity show dose dependence for the natural HLE ligand  1 proteinase inhibitor (1antitrypsin, 1PI). Chemokines prevent, whereas 1PI promotes, copatching of HLE with the canonical HIV receptors. Recent demonstration that decreased viral RNA is significantly correlated with decreased circulating 1PI in HIV seropositive individuals is consistent with a model in which HLE and 1PI can serve as HIV coreceptor and cofactor, respectively, and potentially participate in the pathophysiology of HIV disease progression. (Blood. 2003;102:4479-4486)
HIV-1 entry and fusion are thought to involve an initial interaction between the envelope glycoprotein, gp120, and cell-surface CD4. This interaction exposes a site within gp120 that then interacts with a coreceptor (ie, CCR5 or CXCR4), inducing a conformational change within the gp41 portion of the viral envelope; this set of events results in insertion of the fusion domain of gp41 into the cell membrane. Cell-surface expression of specific chemokine receptors and CD4 is necessary, but not sufficient, to confer HIV-1 permissivity.1,2 Cellular resistance to HIV-1 infectivity can be due to fusion/entry failure, suggesting this differentiation-associated restriction is due to a positive factor or negative factor. Comparison of HIV-1 nonpermissive and permissive U937 subclones revealed relatively equivalent cell-surface CD4 and CXCR4 and a lack of CCR5.1 A notable difference was that nonpermissive, but not permissive, subclones expressed detectable granule-associated proteinases, cathepsin G (CatG) and human leukocyte elastase (HLE).3 These same proteinases are known to be cell-surface-associated in certain situations and to bind HIV-1 envelope proteins,4,5 but in association with the lipid bilayer, enzymatic activity and antigen detection are absent or compromised.6 This suggests that plasma membrane-associated proteinases may exhibit nonenzymatic receptor functions and that location, rather than gene expression, might impact HIV-1 permissivity.
Traditionally, the proteinase activity of HLE has been characterized in aqueous environments, and cell-surface lipids are known to negatively influence its catalytic activity6 further supporting a nonenzymatic function for plasma membrane HLE.7 In fact, the primary actions of cell-surface HLE and CatG involve adhesion, chemotaxis, and stem-cell mobilization.8-10 Granule-associated HLE rapidly translocates to the cell surface in response to many agonists including the bacterial endotoxin lipopolysaccharide (LPS),11 suggesting these activation signals rapidly mobilize HLE to the cell surface in the absence of protein synthesis. The precise domains in HLE that allow its association with the plasma membrane are not completely known; however, the
In HIV-1-seropositive patients, viral load is correlated with circulating levels of
Cells and reagents
U937 subclones were generously provided by the Laboratory of Immunoregulation, National Institute for Allergy and Infectious Diseases, National Institutes of Health (NIH), and maintained using RPMI-1640 containing 10% fetal bovine serum (FBS) determined to have endotoxin levels less than 0.3 endotoxin units (EU)/mL. Cells were harvested during exponential growth and were more than 95% viable as determined by trypan blue exclusion. Endotoxin stimulation was performed by incubation with Escherichia coli strain O26:B6 LPS (Sigma Chemical, St Louis, MO) at a concentration of 5 µg/µL/2 x 105 cells in the presence or absence of 0.5 µg recombinant LPS-binding proteins (LBPs; Xoma, Berkeley, CA) in AIM V (R) serum-free media (Gibco Invitrogen, Carlsbad, CA) for 60 minutes at 37°C in a humidified chamber containing 5% CO2. Cells in AIM V (R) were examined by flow cytometry to exhibit CD4 and CXCR4 levels not different from cells in RPMI-1640, 10% FBS (data not shown). The functionally active concentrations of 2 preparations of Immunocytochemical transmission electron microscopy (TEM) TEM was performed by Wallace Ambrose, Microscopy Laboratory, Dental Research Center, University of North Carolina-Chapel Hill. Grids were stained using rabbit anti-HLE (Biodesign, Kennebunkport, ME) or anti-CatG (Biodesign) at concentrations of 1 mg/mL in 0.01 M phosphate, 0.15 M NaCl, pH 7.2 (PBS). Antibody binding was detected using Protein A polygold (0.23 µg Protein A/mL; Sigma) and photographed using a Philips CM/12 TEM/STEM transmission electron microscope (FEI, Hillsboro, OR). Positive-control grids were stained with rabbit anti-nuclear factor kappa-B (NFkB) p50 (Biodesign) and negative-control grids were stained without primary antibody. Receptor/ligand binding analysis Preparation of solubilized clone 10 membrane extract, the synthetic pentapeptide FLGFL representative of the HIV-1 fusion domain, and receptor/ligand binding analysis was performed as previously described.5 Binding by 125I-U937 clone 10-solubilized membranes to FLGFL was performed in hypertonic phosphate buffer, pH 7.4, 0.5 M NaCl, 0.05% tween-20 (HPBS) containing 2.5% lipid-free bovine serum albumin (BSA) to limit nonspecific ionic binding. The bound and unbound (U) fractions were counted. Specific binding (B) was determined as the difference between binding to coated and uncoated wells. Immunofluorescent receptor staining and flow cytometric analysis Three-parameter flow cytometric analysis was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Cells were washed twice in PBS and resuspended at 1 x 106 cells/mL. Monoclonal antibodies included anti-CXCR4-phycoerythrin (PE) (PharMingen, San Diego, CA), anti-CD4-peridinin chlorophyll-alpha protein (PerCP) (clone SK3; Becton Dickinson), and isotype-matched controls. To detect cell-surface HLE, cells were incubated with polyclonal sheep anti-HLE-fluorescein isothiocyanate (FITC) (Biodesign, Kennebunkport, ME) or negative-control sheep immunoglobulin G (IgG)-FITC (Cappel, Aurora, OH). Alternatively, cell-surface HLE was detected using polyclonal rabbit anti-HLE (Biodesign) or negative-control rabbit IgG (Chemicon, Temecula, CA), and binding was detected using biotinylated antirabbit IgG (Amersham, Amersham, United Kingdom) followed by streptavidin-FITC (PharMingen). To investigate the influence of alternate receptor ligation, whole blood was stained by incubating 100 µL cells with 10 µL of each antibody stepwise for 15 minutes each at 20°C in the order anti-HLE-FITC, anti-CXCR4-PE, anti-CD4-PerCP, or in the order anti-CD4-PerCP, anti-HLE-FITC, anti-CXCR4-PE. Cells were washed in 2 mL PBS between each staining step. After the final wash, the cell pellet was resuspended and fixed in 0.5 mL 1% paraformaldehyde in PBS. For each analysis, at least 10 000 to 30 000 events were acquired. List mode multiparameter data files were analyzed using CellQuest Software (Becton Dickinson). Median fluorescence intensity (MFI) for each coreceptor was detected, and relative fluorescence intensity (RFI) was determined as MFI of coreceptor minus MFI of isotype control. Receptor patching and colocalization
U937 cells (1 x 106 cells/mL) were incubated with various receptor ligands for 60 minutes in humidified 5% CO2 at 37°C. Ligands included polyclonal rabbit anti-HLE (1.4 mg/mL; Biodesign), monoclonal murine anti-HLE (65 µg/mL; Dako, Carpinteria, CA), 1.25 nM
After washing 3 times in HBSS, aliquots (30 µL) were applied to the sample chambers of a cytospin apparatus (ThermoShandon, Pittsburgh, PA), and slides were centrifuged at 850 rpm for 3 minutes. Slides were fixed by application of 50 µL 10% formalin in PBS to the sample chambers of the cytospin apparatus followed by an additional centrifugation at 850 rpm for 5 minutes. Slides were sequentially incubated for 90 minutes at 20°C with a monoclonal antibody having specificity for human To demonstrate receptor copatching induced by HIV, an inoculum of 1 x 106 infectious units of HIV-1 T-tropic strain NL4-3 (HIVNL4-3, a generous contribution from Dr W. Resch, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill) was incubated with 1.5 x 107 cells in 1 mL RPMI-1640 containing 10% FBS at 37°C in humidified 5% CO2. Aliquots of 250 µL were removed at 15-minute intervals for 90 minutes. Cells were washed 3 times in fresh medium and fixed using 1% paraformaldehyde. Fixed cells were sequentially incubated for 60 minutes at 20°C with rabbit polyclonal anti-HLE (1 mg/mL; Biodesign), biotinylated donkey antirabbit immunoglobulin (100 µg/mL; Amersham, Arlington Heights, IL), FITC-conjugated streptavidin (50 µg/mL; Becton Dickinson), monoclonal anti-gp120 (approximate epitope V3 loop, 10 µg/mL; Research Diagnostics, Flanders, NJ), biotinylated sheep antimouse immunoglobulin (100 µg/mL; Amersham), gold-conjugated streptavidin (1/50; Nanoprobes, Yaphank, NY), and either PerCP-conjugated monoclonal anti-CD4 (clone SK3; Becton Dickinson) or Per-CP-conjugated CXCR4 (Becton Dickinson). Bound anti-gp120 was detected using silver enhancement as recommended by the manufacturer (LI Silver; Nanoprobes). Antibody controls were purified rabbit IgG (1 mg/mL; Chemicon) and murine IgG (10 µg/mL; Jackson Laboratories). Cells were washed once between each staining step. Slides were examined by epi-illumination UV microscopy on a Zeiss Axioskop (Thornwood, NY) equipped with filters for excitation/emmission wavelengths (nm) 546/590 and 450/520 to 490/520. Optical sections of intact cells were examined by confocal laser scanning microscopy (CLSM) performed by Dr C. R. Bagnell Jr, Confocal Microscopy Facility, Department of Pathology and Laboratory Medicine, UNC-Chapel Hill. Digital images were artificially colorized using Adobe Photoshop software (San Jose, CA). Antisense oligomers
Morpholino antisense oligomers (Gene-Tools, Corvallis, OR) were 22- or 25-mers, targeted respectively to the HLE start codon (5'-CCGAGGGTCATGGTGGGGCTGGG) or to the 3' splice site of HLE exon 4 (5'-CGACCCGTTGAGCTGTGGCGGTGGG). Anti- HIV-1 infectivity
To demonstrate the influence of To demonstrate the influence of antisense oligonucleotides, antisense-treated clone 10 cells were enumerated and resuspended at 5 x 105 cells/200 µL/well in 96-well tissue-culture plates in RPMI-1640 containing 10% FBS. HIVNL4-3 (4.9 x 105 IU/mL MAGI cells) was incubated at a concentration of 1 x 105 IU/well for 2 hours at 37°C in a humidified chamber containing 5% CO2. Cells were washed free of virus and resuspended in RPMI-1640 containing 10% FBS. Cells were harvested at 48 hours and analyzed by flow cytometry for cell-surface expression of HLE and by polymerase chain reaction (PCR) for the presence of HIV-1 minus-strand, strong-stop DNA. Enumeration of viable cells in suspension at 48 hours revealed 1.7 x 105 cells/well to 3.2 x 105 cells/well that were 99% to 100% viable. HIV-1 DNA PCR
An equivalent number of cells from each treatment were harvested from tissue culture and washed once in PBS. Lysis, PCR amplification, and
Different subcellular compartments occupied by HLE in HIV-1 permissive and nonpermissive U937 subclones
Immunocytochemical TEM examination of U937 subclones revealed HLE associated with the plasma membrane in HIV-1 permissive clone 10 (Figure 1A). Plasma membrane-associated HLE was never seen in unstimulated HIV-1 nonpermissive clone 17 (data not shown). Instead, HLE was found in clusters in clone 17 associated with intracytoplasmic, electron-dense, irregular-shaped bodies resembling granules (Figure 1B). Localization of HLE to an internal compartment in clone 17 was confirmed by CLSM (data not shown). Intracytoplasmic clusters of HLE were never seen in permissive clone 10. Positive-control protein CatG was found to localize to the same structures as HLE in clones 10 and 17, whereas NF
The endotoxin LPS, a glycolipid found on Gram-negative bacteria, produces multiple effects on cells including release of granule-associated HLE, which becomes bound to the plasma membrane.11 Granule release is mediated by the interaction of LPS with 2 different signaling receptors including CD14 and Analysis of HIV-1 coreceptors by flow cytometry in the presence or absence of LPS stimulation was compared using 3-color fluorescence. Because HLE density was expected to be low or not detectable, HIV-1 permissive clone 10 was examined with or without anti-CD4 as a staining control. Surprisingly, HLE was greatly increased when clone 10 was interacted with antibodies specific for CD4 first and specific for HLE secondarily; HLE was undetectable when the order of addition was reversed (Figure 1D). The explanation for differences in detection of surface HLE on unfixed cells in the presence or absence of anti-CD4 is not known, but evidence suggests a potential role for membrane remodeling22 or autodegradation.23 In contrast, the order of coreceptor ligation had negligible influence on the fluorescence intensity of CD4 or CXCR4. LPS stimulation of HIV-1 nonpermissive clone 17 was found to have little or no influence on CD4 or CXCR4 density; however, HLE expression was found to increase. Further, as with clone 10, the order of receptor ligation influenced detection of HLE, but not detection of CD4 or CXCR4. The relatively identical HLE expression induced by LPS with or without LBP is interpreted to result from factors in the serum-containing tissue-culture medium. Results suggest a dynamic functional association between membrane-associated HLE and the HIV-1 coreceptor CD4. HLE binding to the HIV-1 fusion domain
Association constants for soluble proteins are conventionally measured in 0.15 M NaCl, pH 7.2, to accommodate comparisons between proteins competing for the same soluble ligand and yield values in the range of 106 M-1. To optimize binding of nonsoluble proteins, association constants may be determined by modifying salt, pH, or temperature. Although a constant determined in 0.5 M NaCl at 0°C is not directly comparable to a constant determined in 0.15 M NaCl at 25°C, each constant is valid and definitive for the conditions under which affinity is measured. We have previously found that hypertonic saline (0.5 M NaCl) can be used to limit nonspecific ionic interactions between cell-surface receptors and the hydrophobic pentapeptide FLGFL, representative of the HIV-1 fusion domain.5 Using these conditions for Scatchard analysis, we demonstrated the existence of a single receptor (Kassoc = 1 x 103 M-1) on CEM T-lymphoblastoid cells for FLGFL. By competitive inhibition, the receptor was identified as HLE (HLE IC100 = 8.0 mM,
In support of this hypothesis, HLE visualized by CLSM using a monoclonal antibody was found to patch in response to soluble FLGFL or
Immunolocalization of receptors on cells sequentially incubated with
Enhanced HIV-1 infectivity in the presence of exogenous
It has previously been shown that HIV-1 gp120 induces colocalization of CD4 and CXCR4.28 That
Since HLE is not constitutively expressed on the cell surface of clone 17, it was considered that LPS-induced cell-surface expression of HLE might confer permissivity. Clone 17 was cultured in serum-free medium and stimulated with LPS in the presence or absence of LBP prior to addition of HIV. Cells were subsequently infected in the presence or absence of physiologic concentrations of
To examine the possibility that patching might occur during HIV-1 entry, clone 10 was incubated at 37°C with infectious HIV-1NL4-3, and cells were fixed and examined at various time points by CLSM for colocalization of HIV-1 and coreceptors. HIV-1 was detected using a monoclonal antibody with specificity for epitopes proximate to the V3 loop (Research Diagnostics). This antibody has recently been shown not to recognize Requirement for cell-surface HLE during HIV-1 infection
The data presented thus far suggest the participation of HLE as a receptor during HIV-1 capture and infection. Linking HIV-1 entry with the novel expression of CD4 and chemokine receptor cDNA in cells lacking their expression has established this method as the gold standard for identifying HIV-1 receptors. On the other hand, silencing gene expression has proven to be an equally powerful tool for demonstrating dependence of HIV-1 entry on CD4 expression.30 Since novel expression of HLE cDNA is technically not possible at this time, the importance of HLE during HIV-1 entry and infectivity was examined by inhibiting HLE expression. Morpholino antisense oligonucleotides complementary to HLE mRNA and pre-mRNA were introduced into HIV-1 permissive clone 10 and cultured for 48 hours. Antisense oligonucleotides complementary to
Oligonucleotide-treated cells were incubated with HIV-1NL4-3, washed free of virus, and cultured for 48 hours. The presence of minus-strand, strong-stop DNA, the earliest RT product following HIV-1 entry, was determined by PCR amplification of cell lysates using R/U5 primer pairs.21 As was found with HLE expression, minus-strand, strong-stop DNA was diminished in cells transfected with antisense oligonucleotides complementary to the HLE start codon or the 3' splice site of exon 4 (Figure 4B). In contrast, this DNA was easily detected in cells transfected with antisense oligonucleotide complementary to
Cell-surface HLE ligation by 1PI, anti-HLE, and HIV-1 fusion peptide was shown here to induce copatching of HLE, CD4, and chemokine receptors. A model is proposed in which HLE ligation induces clustering of HIV-1 receptors, and that this dense focus of receptors increases the odds of the dense focus of HIV-1 envelope proteins to interact with remaining free receptors. Ligand-occupied receptors thereby produce a tangible increase in proximal receptors rather than producing steric interference and decreased accessibility of HIV-1 receptors. Consistent with this model, SDF-1 was found to prevent receptor clustering, and this suggests that chemokines may suppress HIV-1 infectivity by decreasing HIV-1 receptor clusters rather than by directly blocking receptors as previously believed.15 Increased circulating 1PI is correlated with increased viral load in the HIV-1 population, suggesting the hypothesis that 1PI might facilitate HIV-1 binding and infectivity. Here we have shown that RT activity produced by HIV-1 infectivity is dependent on 1PI in a dose-dependent manner, that 1PI facilitates copatching of HLE with the canonical HIV-1 receptors, that live HIV-1 preferentially binds copatched receptors, and that binding of virus to copatched receptors does not occur in the absence of 1PI. We have further demonstrated that translocation of HLE from granules to the cell surface conveys HIV-1 permissivity to nonpermissive cells and that inhibiting HLE mRNA proportionally diminishes detection of the earliest RT product, minus-strand, strong stop DNA. In support of these findings, previous studies have demonstrated that in contrast to permissive clones, nonpermissive clones are unable to efficiently form complexes between gp120, CD4, and CXCR4.31
The critical parameters determining the kinetics of HIV-1 infectivity have been defined as virus concentration, number of virions produced by one cell, and the time for one complete cycle of infection.32 In a homogeneous cell population, the time to peak RT activity has been shown to be directly related to virus concentration. We found that time to peak RT activity produced by clone 10 was diminished by 2 days for each logarithmic dilution of HIV-1NL4-3, and these results are consistent with previous kinetic studies.32 However, clone 10 produced negligible RT activity when cells were infected in serum-free medium lacking
The dose-dependent manner in which Evidence that HIV-1 gp120 binds CD4 includes the demonstration that binding is reversible and saturable,33 that introducing CD4 cDNA expression in CD4 null cells results in a corresponding increase in HIV-1 infectivity,34 and that silencing CD4 mRNA expression blocks HIV-1 infectivity.30 Since HLE may reside either on the cell surface or in granules, and since HLE localization is dependent neither on DNA, mRNA, nor protein structure, but on whether the cell is producing granules, an as-yet-undefined process, introduction of HLE mRNA into HLE-negative cells is not a good test of its receptor function. The true test is to identify cells lacking HLE on the cell surface; test whether these cells are not susceptible to HIV-1 entry in the presence of CD4, CCR5, and CXCR4; and determine whether coexpression specifically of HLE on the cell surface now rescues HIV-1 entry into these cells. Reversible, saturable binding of the HIV-1 fusion domain to HLE has been shown here and elsewhere.5 We have demonstrated here that inhibiting HLE mRNA expression blocks HIV-1 infectivity and that favoring HLE association with the plasma membrane favors HIV-1 infectivity. Clark's receptor occupancy model24 states that in addition to reversible, saturable binding of the ligand to the receptor, binding must produce a biologic response proportional to the number of receptors bound. Evidence presented suggests that suppression of HLE expression proportionally suppresses the earliest HIV-1 RT product, and this supports a requirement for HLE during HIV-1 binding and the earliest steps of infection consistent with a model of HIV-1 entry that requires HLE as a fusion receptor. We have previously found that in vitro viral infectivity outcome correlates with HLE, but not CD4, CXCR4, or CCR5,13 and this suggests HLE is a rate-limiting receptor for viral entry potentially by a mechanism involving the phenomenon of receptor copatching.
The authors wish to gratefully acknowledge D. Irlbeck, F. DiMeo, and Drs A. S. Fauci, H. Moriuchi, M. Moriuchi, M. Pope, W. Resch, E. Miller, M. Gonzales-Gronow, S. V. Pizzo, and R. R. Arnold for reagents and technical assistance; and Drs O. J. Cohen, R. W. Doms, H. Moriuchi, R. R. Arnold, H. Patel, and M. Pope for critically reviewing the manuscript. Special thanks to Drs J. D. Folds, K. A. Jacobson, and H. H. Fudenberg for advice and encouragement.
Submitted May 21, 2003; accepted August 19, 2003.
Prepublished online as Blood First Edition Paper, August 21, 2003; DOI 10.1182/blood-2003-05-1635.
Supported by grants from the Center for AIDS Research, University Research Council of the University of North Carolina-Chapel Hill, and The Rockefeller Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Cynthia L. Bristow, Laboratory of Cellular Physiology and Immunology, The Rockefeller University and Population Council, New York, NY 10021; e-mail: bristoc{at}mail.rockefeller.edu.
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Top
Abstract
Introduction
-globin oligonucleotides were used as negative controls as described previously.
32P-labeled R/U5 primer pairs for HIV-1 minus-strand, strong-stop DNA were as previously described.
B was found to localize to the cytoplasm and mitochondria in both clones equivalently (data not shown). That HLE was found to reside in granule-like structures in nonpermissive cells, but on the cell surface in permissive cells suggested these clones had become arrested at disparate stages of differentiation. 
). Alternatively, cells were first interacted with anti-HLE and second with anti-CD4 and anti-CXCR4 (
). HLE was detected on the surface of nonpermissive clone 17 after stimulating with LBP and LPS to induce granule release. CD4, CXCR4, and HLE expression on clone 17 in the presence of LBP was indistinguishable from medium alone (data not shown). Relative fluorescence intensity (RFI) were calculated as receptor MFI minus isotype control MFI. Flow cytometric analysis was performed on 10 000 to 30 000 events in 4 separate experiments. Data represent one analysis.
) with 10 mM HIV-1 fusion peptide (FLGFL) and incubated with 125I-U937 clone 10 solubilized cytoplasmic membrane proteins. Hypertonic saline (0.5 M NaCl) was used to limit nonspecific ionic interactions between solubilized membranes and the hydrophobic peptide. Maximum bound cpm/well at saturation was 1.3 x 105 (r2 = 0.94), Binding to uncoated wells was 9.0 ± 0.3 x 103 cpm. Specific binding to fusion peptide is represented as the difference between coated and uncoated wells, and binding to uncoated wells is depicted for comparison. (B) Scatchard analysis revealed a linear character (r2 = 0.81) to the interactions and association constant 1 x 103 M-1 in hypertonic saline. Interaction of cell-surface HLE with FLGFL (C) or 
), or 30 µM (
) active 
). Infectivity experiments were confirmed independently by the Laboratory of Immunoregulation, NIAID, NIH. A representative set of results is depicted. Infectivity outcome was determined in duplicate by measuring RT activity of cell-free supernatants. Mean values are depicted. (D) RT activity produced by clones 10 and 17 infected simultaneously in the presence (closed symbols), but not in the absence (open symbols), of 
), 3' splice site of HLE exon 4 (
), or