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The innate immune response is a key barrier against pathogenic microorganisms such as human immunodeficiency virus type 1 (HIV-1). Because HIV-1 is rarely transmitted orally, we hypothesized that oral epithelial cells participate in the innate immune defense against this virus. We further hypothesized that secretory leukocyte protease inhibitor (SLPI), a 12-kDa mucosal antiviral protein, is a component of the host immune response to this virus. Here we demonstrated constitutive expression and production of SLPI in immortalized human oral keratinocytes. Brief exposure of cells to HIV-1 BaL and HXB2 significantly increased SLPI mRNA and protein production compared to that in mock-exposed cells (P < 0.01), as evaluated by real-time quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. HIV-1-mediated stimulation of SLPI occurred at the transcriptional level, was dose and time dependent, was elicited by heat-inactivated and infectious viruses, and did not depend on cellular infection. Experiments with purified retroviral proteins showed that the stimulatory effect was induced specifically by external envelope glycoproteins from HIV-1 and simian immunodeficiency virus. SLPI responsiveness to HIV-1 was also observed in an unrelated oral epithelial cell line and in normal (nonimmortalized) human oral epithelial cells isolated from healthy uninfected gingival tissues. In this first report of SLPI regulation by HIV-1, we show that the expression and production of the antimicrobial and anti-inflammatory protein can be stimulated in oral epithelial cells by the virus through interactions with gp120 in the absence of direct infection. These findings indicate that SLPI is a component of the oral mucosal response to HIV-1.
Oral tissues are naturally resistant to infection with human immunodeficiency virus type 1 (HIV-1), unlike genital and anorectal tissues (7). The mechanisms contributing to the protective effect in the oral cavity are incompletely understood. The innate immune response is a key defense against HIV-1, particularly at mucosal surfaces (27). Mucosal epithelium participates in this response by providing a physical barrier to infection and producing anti-HIV-1 peptides and proteins that serve as key effector molecules. Oral antimicrobial factors having anti-HIV-1 activity at physiologic concentrations include secretory leukocyte protease inhibitor (SLPI), human beta defensins (hBDs), salivary agglutinin, thrombospondin 1, and mucins (33, 42, 49). Knowledge gained by studying the oral innate defense against this virus will shed light on new strategies for reducing transmission at genital and anorectal sites, where most infections occur in vivo.
SLPI, a member of the trappin gene family that includes elafin (45), is a 107-kDa protein produced and secreted primarily from epithelial cells lining mucosal surfaces (12) and skin (52), neutrophils (44), and lipopolysaccharide-stimulated macrophages (22). Originally described for its activity against serine proteases (e.g., leukocyte elastase and cathepsin G) (53), SLPI participates in the mucosal defense by reducing inflammation (16); suppressing matrix metalloproteinase production and activity (61); blocking the in vitro growth of selected bacteria (17), fungi (54), and non-HIV-1 viruses (5); promoting fertility (39); and enhancing wound healing in skin (4, 62).
SLPI anti-HIV-1 activity was first described by McNeely et al. (33) in studies demonstrating that saliva-derived and recombinant forms of SLPI protected human monocyte-derived macrophages and CD4+ T cells against infection with HIV-1 BaL. Inhibition was dose, pH, and temperature dependent; occurred at physiological concentrations in saliva (1 to 10 μg/ml); and targeted viral entry through high-affinity interactions with a cell surface molecule (33, 34, 58) recently identified as annexin II (30). Reports of no or inconsistent inhibition by two research groups (25, 55) prompted a reexamination of SLPI antiviral activity. SLPI inhibition in vitro was subsequently confirmed by several groups (19, 48, 51), including one group that initially reported negative findings (47) with various cell culture models and HIV-1 isolates having different subtypes and chemokine coreceptor usage patterns. Additional evidence for SLPI antiviral activity came from African studies of perinatal HIV-1 transmission in which high transmission rates were associated with low levels (below 0.1 μg/ml, the MIC required in vitro) (33) of vaginal SLPI in infected mothers (40) and salivary SLPI in breast-fed babies (11). Thus, SLPI does indeed possess anti-HIV-1 properties and likely plays a key role in protecting mucosal surfaces against viral infection.
Little is known of SLPI function and regulation in oral tissues. Most studies have been conducted with cells and tissues derived from the lungs (1-3, 21, 32, 43, 44, 57), the reproductive tract (10, 39, 60), and skin (4, 52, 62). These studies have revealed that the human SLPI gene is nonpolymorphic (23) and is expressed in a tissue- and mucosa-specific manner (3). Regulation of the antiprotease activity occurs primarily at the transcriptional level, although gene expression can also be modulated posttranscriptionally (3, 32). Treatment of cells with the proinflammatory mediators interleukin 1β and tumor necrosis factor alpha (24, 43, 50, 52), phorbol ester (32), neutrophil elastase (1, 57), corticosteroids (2), and progesterone (24) increased SLPI mRNA and protein production. SLPI expression was also induced by lipopolysaccharide and lipoteichoic acid in macrophages (22) and by insulin-like growth factor I and transforming growth factor alpha in skin keratinocytes (52).
Studies of SLPI expression in oral tissues have been limited to the major and minor salivary glands, where SLPI mRNA and protein production is localized to serous and ductal epithelial cells (12, 37, 59). In salivary glands, inhibitor production is anatomically distinct from areas of active HIV-1 replication in the stroma (59). SLPI expression in other tissues of the oral cavity has not been reported.
Because HIV-1 transmission rarely occurs through the oral route, we hypothesized that oral keratinocytes participate in the innate immune response to the virus by stimulating local SLPI production and secretion. In this study, we used reverse transcription (RT)-PCR and immunohistochemical analyses to demonstrate constitutive expression of SLPI in oral epithelial cells. Real-time quantitative RT-PCR (QRT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that SLPI mRNA production and protein production were increased 100-fold and 3-fold, respectively, after brief contact of oral epithelial cells with HIV-1 compared to controls. The stimulatory effect occurred in a dose- and time-dependent manner, was controlled at the transcriptional level, did not require cellular infection, and was mediated through interactions with viral external envelope glycoprotein gp120. The effect was observed in two immortalized oral keratinocyte cell lines and in normal (nonimmortalized) oral keratinocytes. HIV-1-mediated regulation of SLPI represents a novel protective mechanism in the oral cavity and suggests unique strategies for reducing HIV-1 infection at nonoral mucosal sites.
(This work was presented by D. C. Shugars and N. K. Jana in part at the 82nd General Section of the International Association of Dental Research, Honolulu, Hawaii, 10 to 13 March 2004 [abstr. 3854].)
The human oral epithelial cell line GMSM-K (a generous gift from V. A. Murrah and E. P. Gilchrist, University of North Carolina School of Dentistry) was derived from normal oral buccal mucosa of a stillborn fetus and immortalized with simian virus 40 T antigen (14). GMSM-K cells exhibit differentiation markers characteristic of normal keratinocytes, including the expression of low- and high-molecular-weight keratins, squamous cell-specific keratin 14, and epithelial cell-specific antigen Ber-EP4. As determined by immunoblotting and immunohistochemical analysis, GMSM-K cells no longer express the T antigen (14). Cells were grown in keratinocyte serum-free medium (SFM) with supplements (epidermal growth factor, bovine pituitary extract, and 1% penicillin-streptomycin) (Invitrogen, Carlsbad, CA) (growth medium).
Normal human gingival epithelial (HGE) cells were isolated from uninflamed gingival tissues overlying partially impacted third molars (26). Tissue was obtained from non-HIV-1-infected healthy donors according to procedures recommended by the University of North Carolina Institutional Review Board. Briefly, excised tissues were rinsed twice in phosphate-buffered saline (PBS) containing 1% penicillin-streptomycin and 1 mg/ml amphotericin B (all from Invitrogen) (PBS+), cut into small blocks (2 mm by 2 mm), and incubated overnight at 4°C with dipase (6 mg/ml; Becton Dickinson, Franklin Lakes, NJ) in PBS+. Single-cell suspensions were generated by trypsinizing the epithelial layer and plating in T-25 flasks containing growth medium further supplemented with hydrocortisone, bovine insulin, gentamicin sulfate, amphotericin B, and 0.15 mM CaCl2 (Invitrogen) (growth+ medium). Cells were grown to near confluence, passaged once, and cryopreserved (5 × 105 cells/vial) by a standard procedure. Frozen HGE cells were thawed and cultured in growth+ medium without further passage prior to use. HGE cultures contained primarily (>98%) oral epithelial cells based on morphology determined by light microscopy.
The human H9 T-cell line was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (31). H9 cells were grown in RPMI 1640 medium with 2 mM l-glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum.
OKF6/TERT-1 cells, hTERT-immortalized human oral epithelial cells (8), were obtained from J. G. Rheinwald (Harvard Institutes of Medicine, Boston, MA). The cells were propagated in growth+ medium further supplemented with 0.4 mM calcium chloride.
The following reagents were obtained through the AIDS Research and Reference Reagent Program: HIV-1 BaL (13) and recombinant HIV-1 proteins LAV p55 Gag, Rev (38), NL4-3 integrase (F185H/C280S; from Robert Craigie), HXB2 reverse transcriptase p51 (46), simian immunodeficiency virus (SIV) mac239 gp130 (18), and BaL gp120. HIV-1 HXB2 was purchased from Advanced Biotechnologies (Columbia, MD). Recombinant gp120 from HIV-1 MN and IIIB was obtained from ImmunoDiagnostics (Woburn, MA). All recombinant proteins were generated in bacteria and purified according to standard protocols (13, 18, 38, 46). Cell-free virus stocks were prepared and titers were determined with phytohemagglutinin-stimulated human peripheral blood mononuclear cells (PBMCs) by standard procedures (9).
Total RNA (2 μg) was purified from cell lysates (RNeasy; Qiagen, Valencia, CA) and subjected to RT-PCR amplification (Titan One Tube RT-PCR system; Roche, Branchburg, NJ) in 35 cycles with published PCR primers specific for SLPI (50) or the β-actin housekeeping gene (Promega, Madison, WI). Controls included reactions lacking RT or template RNA. RT-PCR products were separated by 2% agarose gel electrophoresis, visualized by ethidium bromide staining, and verified by sequencing.
Unstimulated GMSM-K cells were cytospun onto ProbeOnPlus glass microscopic slides (Fisher Scientific, Pittsburgh, PA), air dried, fixed with methanol-acetone, permeabilized with PBS containing 0.05% Triton X-100 (Sigma-Aldrich, St. Louis, MO), and blocked in 15-min room-temperature incubations sequentially with peroxide (3% in PBS; Sigma-Aldrich) avidin (Vector, Burlingame, CA), biotin (Vector), and Power Block (BioGenex, San Ramon, CA). Cells were incubated for 1 h at 37°C with goat anti-human SLPI polyclonal antibody (R & D Systems, Minneapolis, MN) diluted 1:10 in common antibody diluent (BioGenex), washed four times with automation buffer (Biomeda, Foster City, CA), and incubated for 20 min at 37°C with biotinylated donkey anti-goat secondary antibody diluted 1:2,500 in PBS containing 0.05% Tween 20 (Jackson ImmunoResearch, West Grove, PA). Cells were washed with automation buffer and treated with ABC Elite reagents and NovaRed peroxidase substrate (both from Vector) to identify SLPI-producing cells. Cell preparations were immunostained with the secondary antibody only (no primary antibody) to assess background staining.
In triplicate, cells (105 cells/well in a 24-well tissue culture dish) were plated in growth medium. When confluence reached 80 to 90%, the cells were washed with PBS and incubated with 1 ml of SFM for 3 h to minimize the potential effects of epidermal growth factor and pituitary extract on SLPI expression. After the medium was removed, cell-free virus (multiplicity of infection [MOI] of 0.1 in 200 μl SFM) or purified recombinant protein (200 ng/200 μl SFM) was added to the wells and incubated for 30 min at 37°C. Because the virus stocks and recombinant proteins were diluted a minimum of 1:30 in SFM to obtain the desired concentrations, mock cultures were treated with SFM only (200 μl/well). After the incubation period, growth medium (1 ml) was added, and the cells were incubated for 4 days without a medium change. Every 24 h, an aliquot of culture supernatant was removed and analyzed for SLPI protein content by an ELISA (R & D Systems). At 96 h, the cells were washed and either centrifuged onto glass slides for immunocytochemical evaluation or harvested in RNAlater (Ambion, Austin, TX) for RT-PCR-based analyses. Cell viability was monitored by trypan blue dye exclusion at the end of each experiment. Results were verified in at least two independent experiments for each treatment condition.
SLPI mRNA in cell lysates was quantitated by real-time RT-PCR with TaqMan technology (Roche). TaqMan-specific SLPI forward (5′-GCTGTGGAAGGCTCTGGAAAGT-3′) and reverse (5′-GCATTTGATGCCACAAGTGTCA-3′) primers and a 5′-VIC (6-carboxyrhodamine)/3′-minor groove binding-labeled SLPI probe (5′-AGAAACCTGAGTGCCAGAGT-3′) were designed by using Primer Express computer software (Applied Biosystems) to span across introns and prevent amplification of contaminating genomic DNA. TaqMan-specific primers and a 6-carboxyfluorescein-labeled probe for β-actin were obtained from Applied Biosystems. Briefly, first-strand cDNA was generated from purified RNA with RT (SuperScript II reverse transcriptase; Invitrogen) and the TaqMan-specific SLPI reverse primer. The cDNA was amplified by real-time PCR with an ABI prism 7000 PCR and detection instrument (Applied Biosystems, Foster City, CA). Each 50-μl reaction mixture contained 10 ng of cDNA, 25 μl of 2× TaqMan universal PCR master mix with UNG AmpErase (final concentrations of 3 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate, and 1.25 U of Amplitaq Gold), 200 nM each probe, and 300 nM each primer (all from Applied Biosystems). After a 2-min incubation at 50°C and a 10-min incubation at 95°C, reaction mixtures were subjected to 40 amplification cycles of denaturation (95°C for 15 s) and annealing/extension (60°C for 1 min). Data were acquired during the annealing/extension phase and analyzed by using the SDS program (Applied Biosystems).
The concentrations of SLPI primers and probe in the PCR mixtures were optimized and validated by using serial dilutions of total RNA from unstimulated GMSM-K cells. A control PCR containing the SLPI primers (but excluding the probe) were run under identical conditions, and the products were evaluated by 2% agarose gel electrophoresis to confirm the amplification of a single 60-bp DNA fragment (data not shown). The fold change in SLPI expression over time was determined by comparing the quantity of SLPI RNA to that of β-actin RNA (internal control) for each reaction.
SLPI concentrations (μg/ml) were expressed as the mean ± standard error (SE) and analyzed with regard to treatment conditions by using Statview statistical analysis software (Abacus Concepts). Differences in test conditions were evaluated by using the unpaired Student t test (significance was set at a P value of <0.05).
The first cells that HIV-1 likely encounters in the mouth are epithelial cells lining intraoral mucosal surfaces. To date, studies of SLPI gene expression in the oral cavity have been limited to salivary glands (37, 59). To determine whether oral epithelial cells also express the antiprotease, we analyzed SLPI expression and production in transformed GMSM-K oral keratinocytes and normal human gingival cells established from healthy gingival tissues of non-HIV-1-infected donors. As shown in Fig. Fig.1A1A (top gel), the SLPI gene was constitutively expressed in unstimulated GMSM-K and HGE cells but not in the negative control H9 T cells. RT-PCR specificity was verified in reactions lacking RT or RNA and by sequencing the SLPI product generated from GMSM-K cells. Amplification of the housekeeping β-actin gene confirmed the RNA integrity in the reactions (Fig. (Fig.1A,1A, bottom gel). Immunohistochemical staining with an anti-SLPI antibody demonstrated a punctate cytoplasmic pattern of SLPI production consistent with protein secretion (Fig. (Fig.1B).1B). As determined by an ELISA, SLPI levels in the medium of unstimulated triplicate cultures of GMSM-K cells were (without a medium change) 63.4 ± 3.8 pg/ml at 24 h, 139.3 ± 0.9 pg/ml at 48 h, 427.0 ± 11.8 pg/ml at 72 h, and 1,216.1 ± 17.7 pg/ml at 96 h. SLPI production in the cells was confirmed by immunoblotting (data not shown).
To evaluate whether exposure to HIV-1 affected SLPI mRNA and protein production in oral epithelial cells, GMSM-K cells were incubated with R5 (CCR5 utilizing) HIV-1 BaL, X4 (CXCR4 utilizing) HIV-1 HXB2, or SFM only (mock) for 30 min at 37°C and then cultured for 4 days without a medium change. Because the virus stocks (generated in phytohemagglutinin-stimulated PBMCs) were diluted at least 1:30 in SFM to obtain the desired MOI, control cells were treated with SFM rather than supernatant from stimulated PBMCs. At 24-h intervals, culture supernatants were collected and analyzed for secreted SLPI by an ELISA. As shown in Fig. Fig.2,2, SLPI production significantly increased threefold at 48, 72, and 96 h after treatment in virus-exposed cells compared to mock-exposed cells (P < 0.05). Cells treated with the R5 and X4 viruses showed equivalent SLPI levels (P > 0.05), suggesting that the stimulatory effect was independent of chemokine coreceptor usage. The effect was not due to a stimulation of cellular proliferation by HIV-1, as virus-treated and mock cultures exhibited similar growth kinetics (data not shown). Cell viability remained greater than 95% throughout the culture period. No cellular infection was detected under these experimental conditions, as assessed with a PCR-based assay to detect integrated proviral DNA and viral p24 quantitation of culture supernatants (unpublished data).
SLPI mRNA expression in oral keratinocytes in response to HIV-1 was examined by real-time QRT-PCR. Here, GMSM-K cells were incubated with BaL for various durations prior to RNA isolation, RT, and quantitative PCR amplification. Change in SLPI expression over time was measured by comparing the fold change in SLPI cDNA relative to β-actin cDNA (internal control) for each lysate. Compared to mock-treated cells, SLPI mRNA level rapidly increased 50-fold at 1 h after virus addition, peaked at a 100-fold level at 2 h after virus addition, and was subsequently maintained at an approximately 60-fold-higher level for the remainder of the observation period (Fig. (Fig.3).3). These results indicate that virus-cell contact leads to a rapid and sustained increase in SLPI expression and that the stimulatory effect occurs at the transcriptional level.
To further characterize the effect, the SLPI stimulation assay was repeated under various conditions. In one experiment, GMSM-K cells were incubated with various concentrations of BaL (serial dilutions; MOIs from 1.0 to 0.0075), infectious BaL (MOI, 0.1), BaL heat inactivated for 1 h at 56°C (MOI, 0.1), or SFM only for 30 min at 37°C prior to the addition of growth medium. For clarity, results from the dose-response conditions are presented in Fig. Fig.4A,4A, while results from the infectious/heat-inactivated conditions are presented in Fig. Fig.4B.4B. The mock treatment data are reproduced in Fig. Fig.4B4B for comparison. Significant dose-dependent elevations in SLPI secretion were first detected at 24 h after virus exposure in cells treated with MOIs of 0.0075, 0.03, 0.1, and 1.0 compared to controls (Fig. (Fig.4A)4A) (P < 0.05). At 48, 72, and 96 h after exposure, all virus-treated cultures exhibited significantly higher SLPI levels than controls (P < 0.05). Similar results were observed in cells exposed to serial dilutions of HIV-1 HXB2 (data not shown). As shown in Fig. Fig.4B,4B, SLPI production was similar in cells treated with infectious or heat-inactivated virus, indicating that infectious virus is not essential for SLPI stimulation. This finding suggests that a component of the virus particle is responsible for enhanced SLPI secretion.
To determine the minimum stimulation period required for the effect, the stimulation assay was repeated using GMSM-K cells incubated with infectious BaL for 0, 10, 20, or 30 min and then washed to remove unbound virus prior to medium addition. Culture supernatants were collected 96 h later and assayed for SLPI content. An increase in SLPI secretion was detected after virus exposures of 10 min or longer compared to controls (Fig. (Fig.5A).5A). To determine whether shorter periods of virus contact can also induce SLPI production, the assay was repeated using cells incubated with virus for 0, 1, or 5 min prior to virus removal. Incubation of cells with BaL for 5 min but not 1 min led to a significant increase in SLPI stimulation compared to the control (P < 0.05). In experiments not shown, exposure of cells to virus for up to 2 h prior to medium addition did not further enhance SLPI secretion compared to cells treated with virus for 30 min. Thus, only a brief (5-min) exposure of HIV-1 is needed to induce SLPI secretion from oral keratinocytes. Also, continuous exposure to virus is not required for the effect.
The component of the HIV-1 particle likely to elicit the effect is the external envelope glycoprotein gp120, since this protein is located on the exterior of the viral envelope and provides initial contact with target cells. Other viral proteins, however, might be responsible for the effect. To test this possibility, GMSM-K cells were incubated for 30 min with either SFM alone or a panel of purified recombinant (bacterially expressed) HIV-1 proteins that included BaL gp120, Rev, Gag, integrase, and RT. SLPI secretion was measured at 24-h intervals as described above. Exposure of cells to recombinant gp120 from BaL significantly increased SLPI secretion compared to mock-treated cultures and cultures treated with the other viral proteins (P < 0.01). The level of SLPI stimulation with BaL gp120 was similar to levels seen using intact infectious and heat-inactivated BaL (compare Fig. Fig.6A6A with Fig. Fig.2,2, 4A and B, and and5A),5A), indicating that gp120 alone was likely responsible for the effect. The nonenvelope viral proteins as well as human soluble CD4 (data not shown) failed to stimulate SLPI secretion.
To assess whether SLPI stimulation is a property shared by retroviruses, the SLPI stimulation experiment was repeated with recombinant gp120 from HIV-1 MN, IIIB, and BaL and gp130 from SIV mac239 as inducers (Fig. (Fig.6B).6B). All of the HIV-1-derived gp120 proteins as well as the SIV gp130 protein stimulated SLPI production to similar levels.
The SLPI stimulation experiments described above utilized an oral epithelial cell line originally immortalized with simian virus 40 T antigen but no longer expresses the oncogene (14). To determine whether SLPI regulation by HIV-1 also occurred in other keratinocytes, we repeated the assay with two cell sources: oral epithelial OKF6/TERT-1 cells immortalized by the human TERT catalytic telomerase subunit (8) and normal HGE cells established from healthy gingival tissues of a non-HIV-1-infected donor. The cultures were exposed to infectious BaL or HXB2 (MOI, 0.1), recombinant BaL gp120 (200 ng/well), or SFM alone for 30 min prior to the addition of growth medium. As shown in Fig. 7A and B, the infectious viruses and recombinant gp120 induced SLPI secretion in OFK6/TERT-1 (Fig. (Fig.7A)7A) and normal HGE (Fig. (Fig.7B)7B) cultures. Similar results were obtained with normal HGE cells from another uninfected healthy donor (data not shown).
The innate immune defense appears to be particularly adept at protecting the oral cavity against HIV-1 infection, as evidenced by the paucity of HIV-1 transmission through the oral route compared to genital and anorectal routes. Here, we demonstrate that SLPI, an antiviral mucosal protein, is constitutively expressed in immortalized and normal oral keratinocytes. Brief exposure of cells to HIV-1 led to a significant increase in SLPI mRNA and protein secretion that was dose and time dependent, occurred rapidly after virus contact, did not require productive cellular infection, and was elicited specifically by the external envelope glycoproteins of HIV-1 and SIV strains. In this first in-depth characterization of SLPI regulation by a virus, our findings suggest that SLPI stimulation by HIV-1 represents a previously unrecognized mechanism that contributes to the oral host immune defense against this virus.
SLPI has been recognized as an important component of the innate mucosal immunity to microbial pathogens. Previous studies have confirmed its anti-inflammatory, antimicrobial, and wound-healing properties and its role in the maintaining the integrity of mucosal surfaces by inactivating destructive serine proteases during inflammation (reviewed in reference 16). The natural abundance of SLPI in oral secretions (33) has led to the speculation that the antiprotease is a major inhibitor of HIV-1 in saliva. Here, we show that oral keratinocytes are a previously unrecognized source of the inhibitor.
In the time course experiment, SLPI stimulation was observed in GMSM-K cells exposed to virus for as little as 5 min. This time frame has biological relevance in that it mirrors the brief duration that oral epithelial cells are likely to be exposed to virus in vivo during receptive oral sex. Oropharyngeal tissues of infants are likely exposed to virus in infected milk for longer, multiple periods during nursing.
In our experiments, the number of SLPI transcripts rose 50-fold within 1 h after exposure to HIV-1 (Fig. (Fig.3),3), while a significant increase in SLPI protein secretion were first detected 48 h after virus exposure in most experiments (Fig. (Fig.2,2, ,4,4, ,5B,5B, and and7).7). The lag period between inhibitor expression and secretion likely reflects the time needed for the activation of SLPI expression and the translation, intracellular transport, secretion and accumulation of the protein in the culture supernatant. Based on the time lag, it is unlikely that virus-mediated SLPI upregulation protects target cells and tissues against virus acquisition during initial infection. Rather, antiviral inhibitors (including SLPI) already present in salivary fluids likely serve this purpose. When initial protective oral defenses are overwhelmed by a bolus of virus (e.g., during breastfeeding, receptive oral sex, and experimentally infected rhesus macaques; reviewed in reference 49), virus can penetrate the mucosa via transcytosis and possibly direct infection (29, 35), and create an initial focus of viral replication. Over time, subsequent rounds of replication spread the virus through the tissues and draining lymph nodes, and systemic infection occurs. We propose that HIV-1-induced SLPI from oral keratinocytes may help to limit virus spread through tissues rather than prevent initial infection. In addition, distal regions of the oral mucosa may be protected by the elevated SLPI level in oral fluids that ensue after virus stimulation. In support of this latter hypothesis, Lin et al. (28) recently reported higher mean SLPI concentrations in saliva from submandibular/sublingual glands of HIV-1-infected individuals compared to uninfected individuals. The anti-inflammatory property of the inhibitor may also help to control and/or limit viral spread by dampening the inflammatory response and suppressing the activation of susceptible and/or infected cells.
Our data suggest that interactions between HIV-1 gp120 and component(s) of the cellular membrane activate a signaling pathway that ultimately “turns on” SLPI expression. The mechanism of action may include NF-κβ, activated protein 1 (AP-1), and CAAT enhancer binding protein (C/EBP), nuclear transcription factors that activate an array of cytokines and chemokines in response to inflammation, microbial infections, and stress. The human SLPI promoter contains binding sites for NF-κβ, AP-1, and C/EBP (3, 36). HIV-1 binding to cell surface CD4 during infection activates the binding of these transcription factors to the promoters of HIV-1, inflammatory cytokines and chemokines (41), thus creating an inflammatory milieu conducive to viral replication. SLPI appears to be a previously unrecognized cellular gene that is also activated by gp120-CD4 interactions. Given its anti-inflammatory property, SLPI upregulation in virus-exposed keratinocytes may serve as a mechanism to dampen the local inflammatory response to infection, as exogenous SLPI attenuated NF-κβ-dependent inflammatory responses in human endothelial cells and macrophages after atherogenic stimuli (15).
Another possibility is that SLPI stimulation is induced by virus interactions with other cell surface molecules such as Toll-like receptors (TLRs), evolutionarily conserved pattern recognition receptors engaged by specific pathogens on the surfaces of immune and epithelial cells (20). Pathogen-TLR interactions also lead to activated inflammatory gene expression through AP-1 and NF-κβ-mediated pathways. By RT-PCR, we have detected mRNAs for TLRs 1 to 9 in unstimulated GMSM-K cells (unpublished data). Preincubation of GMSM-K cells with antibodies to TLRs 2 and 4 blocked SLPI induction by BaL in GMSM-K cells (unpublished data), suggesting a role for the TLR pathway in the stimulatory effect. Many viruses, including respiratory syncytial virus, measles virus, cytomegalovirus, and influenza virus, modulate host immune responses through TLR activation by viral envelope glycoproteins, core proteins, and replicative nucleic acid intermediates (e.g., single- or double-stranded RNAs) (56). Thus, engagement of TLR-mediated pathways may represent a common mechanism by which cells detect and respond to viruses. Alternatively, interactions between HIV-1 gp120 and a non-CD4 cell surface protein involved in viral entry, such as sulfatide, heparans, galactosyl ceramide (GalCer), and intercellular adhesion molecule 1 (6), may initiate the effect. Future investigations will clarify the mechanism of HIV-1-induced SLPI upregulation and identify the signaling pathway involved in the effect.
Our finding of virus-mediated induction of SLPI mirrors a recent report (42) describing HIV-1 induction of hBD-2 and hBD-3, cationic antimicrobial peptides also produced in epithelial cells. In that study, normal oral epithelial cells exposed to X4 and R5 viruses expressed higher levels of hBD-2 and hBD-3 (but not hBD-1) mRNA compared to controls. The active peptides were specific for X4 viruses, had little effect against R5 viruses, and blocked viral replication through two mechanisms: direct binding to virions and down-modulation of cell surface CXCR4 expression. In contrast, SLPI antiviral activity involves a cellular rather than a viral target (30) and is active against both R5 and X4 viruses (33, 48, 51), although activity is diminished slightly with HIV-1 isolates having broad coreceptor usage patterns (e.g., CCR5, CXCR4, and either CCR2 or CCR3) (51). Thus, in its role as “gatekeeper” of the body, the oral cavity has evolved complementary strategies involving at least two distinct antimicrobial molecules in its quest to protect the body against HIV-1.
Under our experimental conditions, no productive HIV-1 infection was detected in oral keratinocytes, as evaluated by p24 secretion or PCR for newly generated proviral DNA (unpublished data). This result is in agreement with that of Quiñones-Mateu et al. (42), who found no productive infection in normal oral keratinocytes inoculated with either X4 or R5 viruses. The findings, however, contrast with those of Liu et al. (29), who showed infection of oral keratinocytes using high doses of X4 and R5/X4 viruses but not an R5 virus through a CD4-independent mechanism that may involve GalCer and/or CXCR4 as cellular receptors. In another study, Moore et al. (35) showed infection of oral keratinocytes with cell-free R5 viruses but not X4 viruses. The discrepancies may be due to variations in experimental conditions, such as the use of Polybrene during virus exposure by Liu et al. (29) and differences in virus dosages and/or levels of cell surface GalCer, CCR5, and/or CXCR4 expression in the cell models.
It is not known whether HIV-1 stimulates SLPI production at nonoral mucosal sites and what effects enhanced inhibitor production has on the susceptibility of mucosal tissues to virus infection. Oral and genital mucosae differ in several aspects that may alter their susceptibility to this virus, including the architecture of the epithelium, composition and viscosity of the fluids bathing the sites, and local immune responses. In addition, tissue-specific differences in SLPI regulation by HIV-1 may contribute to tissue susceptibility, as treatment with tumor growth factor β1 suppressed SLPI mRNA levels in respiratory epithelial cells (21) but had the opposite effect in endometrial epithelial cells (50). Characterization of the SLPI response to HIV-1 in nonoral mucosal tissues will shed light on this important issue.
In summary, our study reveals that oral epithelial cells constitutively express SLPI. Furthermore, SLPI expression can be manipulated by HIV-1 through infection-independent interactions initiated at the cell surface. Given the anti-inflammatory and antiviral properties of SLPI, the induction of SLPI in virus-stimulated cells represents a tug-of-war between the virus and the host immune response, as the virus attempts to stimulate the local inflammatory response while the inhibitor tries to dampen the response and/or protect neighboring cells against infection. An imbalance between the opposing responses may dictate whether virus exposure ultimately results in productive infection or protection. It will be important to identify other immune response genes whose expression is modified after contact of virus with the oral epithelium. These studies will lead to a greater understanding of the mechanisms through which the host immune response protects against mucosal HIV-1 infection.
We thank Danny R. Gray for technical assistance and helpful discussions.
This work was supported by grant R21DE15055 from the National Institutes of Health.