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Immunology |
Sealy Center for Vaccine Development3
Department of Pediatrics,4 University of Texas Medical Branch-Galveston, Galveston, Texas 77555-0436
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, immunology, toxicology, vagina
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several candidate compounds have been identified that act by inactivating the pathogen directly or blocking infection of susceptible cells. Sulfated polymers represent a broad category of compounds that have been shown to have antimicrobial activities against a range of sexually transmitted pathogens. The naphthalene sulfonate polymer PRO 2000 has been shown to be active in vitro against HIV [2], both in vitro and in vivo against herpes simplex virus (HSV) [3], and has recently entered clinical evaluation. The sulfated polysaccharide lambda carrageenan, a component of red seaweed, has been shown to have antimicrobial properties both in vitro and in vivo [4–6]. Based on clinical exposure [7, 8], lambda carrageenan is considered safe and has been formulated as the active component of the candidate microbicide, Carraguard, which is currently in clinical trial.
There has also been interest in developing surface-active agents as microbicides based primarily on their ability to disrupt lipid membranes or envelopes of bacterial and viral pathogens. Interest focused initially on nonoxynol-9 (N-9), a nonionic detergent used as the spermicidal ingredient in commercial contraceptive gels. While promising results were obtained in preclinical trials [9, 10], efforts to incorporate N-9 in candidate microbicides have diminished due to concerns about the development of cellular disruptions and erosions [11, 12] and infiltration of inflammatory cells [12, 13] following repeated N-9 use. The naturally occurring anionic detergents, bile salts, have been proposed as an alternative to nonionic or cationic detergents and have been shown to inhibit infections with HIV, Neisseria gonorrhoeae, Chlamydia trachomatis, HSV-1, and HSV-2 infections in vitro [14, 15].
Because topical microbicides might potentially be applied on a frequent basis, it is important that these compounds should be safe. CD4+ and CD8+ T lymphocytes are an important component of the immune cells normally found in the female genital tract and function in providing immune protection and maintaining homeostasis. The ability of candidate microbicide compounds to deplete, deregulate, or interfere with the function of resident immune cells in the genital tract could have important consequences on the ability to resist infection and to maintain a healthy vaginal microenvironment. In the present study, we report the effects of sulfated polymer-based and anionic detergent-based microbicides on antigen recognition by primed CD4+ and CD8+ T cells. The results of this study are important for understanding potential detrimental interactions between candidate microbicide compounds and resident vaginal immune cells as well as for the prioritization of candidate microbicides for clinical development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DO11.10 transgenic mice [16] and C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). OT-1 transgenic mice [17] were obtained from Dr. Raphael Hirsch (Children's Hospital Research Foundation, Cincinnati, OH). DO11.10 and OT-1 mice were maintained as breeding colonies at the AALAC-approved facility at the University of Texas Medical Branch. All animals were housed in specific pathogen-free conditions and all experiments were conducted in accordance with the National Research Council publication, Guide for Care and Use of Laboratory Animals.
Candidate Microbicide Compounds
Lambda carrageenan and cholic acid were purchased from Sigma-Aldrich (St. Louis, MO). PRO 2000 was obtained from Dr. Albert Profy (Indevus, Inc., Lexington, MA). All compounds were dissolved in T cell culture media (RPMI 1640, 10% fetal bovine serum [FBS], 1% L-glutamine, 1% penicillin/streptomycin [pen/strep]). The concentrations of candidate microbicide compounds used in the immune assays studies were based on solubility in T cell media and the results of in vitro cytotoxicity testing on spleen lymphocytes.
Cells
Single cell suspensions of lymphocytes were obtained by pushing dissected spleens through a stainless steel screen mesh. EL-4 thymoma cells (H-2b) and A201.11 lymphoma cells (H-2d) were obtained originally from Dr. Vivian Braciale (University of Texas Medical Branch, Galveston, TX). The B6 embryo fibroblast cell line, B6/WT-3, was obtained from Dr. Stephen Jennings (Louisiana State University Health Sciences Center, Shreveport, LA). EL-4 cells were cultured in RPMI 1640 + 10% FBS and 1% pen/strep. B6/WT-3 and A201.11 cells were grown in Dulbecco minimum essential media + 10% FBS + 1% L-glutamine + 1% pen/strep.
MTT Assay for Cytotoxicity
Cell viability was assessed using a modification of the colorimetric assay described by Carmichael et al. [18]. Briefly, spleen cells from C57BL6/J mice were added to sterile U-bottom, 96-well plates in 200 µl of T cell media or T cell media containing various concentrations of microbicide compound. Cells were incubated at 37°C for 48 h, then centrifuged at 250 x g for 2 min. The supernatant was aspirated and 100 µl of 1 mg/ml (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) (MTT, Sigma-Aldrich) dissolved in phenol red-free RPMI was added to each well. Plates were incubated for an additional 3 h at 37°C and centrifuged before aspiration of supernatant and addition of 120 µl/well of isopropanol. The plates were agitated for 10 min at ambient temperature before transfer of 100 µl of supernatant to an ELISA plate. The optical density (OD560) was measured on a Thermo Max microplate reader (Molecular Devices, Sunnyvale, CA). Four-hour MTT assays to test PRO 2000 for cytotoxic effects on A201.11 antigen-presenting cells were performed in an identical manner.
Assay of Antigen Presentation
DO11.10 or OT-1 bulk cultures were prepared by culturing 108 splenocytes from transgenic mice with a final concentration of 100 µg/ml OVA protein (for DO11.10 cultures; Sigma-Aldrich) or 1.0 µM OVA257–264 peptide (SIINFEKL; for OT-1 cultures) in T cell media. Assays to screen candidate microbicides for effects on T cell-antigen presenting cell (APC) interactions were performed in flat-bottom, 96-well plates. A201.11 or EL-4 cells were used as APC and were treated with 50 µg/ml mitomycin C (Sigma-Aldrich) and washed thoroughly before addition to culture plates at 105 cells/well. Responder T cells were obtained from bulk DO11.10 or OT-1 spleen cell cultures 4–5 days after initial antigen stimulation, washed, and added to cultures at 3 x 105 cells/well. Candidate microbicide compounds and ovalbumin (OVA) antigen were added to duplicate wells over a range of concentrations and plates were incubated at 37°C for 48 h. Supernatants were harvested and plated in serial dilution on anti-IFN-
-coated plates for quantification of IFN-.
For short-term exposure studies on APC function, mitomycin C-treated APCs, antigen, and various concentrations of candidate microbicide compounds were incubated in 96-well plates for 4 h at 37°C, then washed thoroughly before addition of antigen-specific T cells. Plates were incubated at 37°C for 48 h before collection of supernatants for quantification of IFN-. Studies of effects of short-term microbicide exposure on T cell function were performed by incubating various concentrations of candidate microbicides and responder T cells for 4 h before washing and addition of APC and antigen. Forty-eight hour supernatants were collected for quantification of IFN-.
Quantification of IFN-
IFN- present in culture supernatants was quantified using a specific enzyme-linked immunosorbent assay as described previously [19]. Briefly, assay plates were coated with anti-IFN- (R4-6A2; BD-Pharmingen, San Diego, CA) and blocked with 2.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) before addition of serial dilutions of recombinant IFN- standard and experimental culture supernatants. Plates were developed by the addition of biotinylated anti-IFN- (XMG 1.2; BD-Pharmingen), streptavidin-peroxidase (Sigma-Aldrich), and citrate buffer containing o-phenylenediamine dihydrochloride and hydrogen peroxide (Sigma-Aldrich). The optical density at 490 nm (OD490) was determined on a Thermo Max microplate reader (Molecular Devices). To determine if the presence of candidate microbicides interfered with the ELISA assay, compounds were added over a range of doses to culture media containing recombinant mouse IFN-. Serial dilutions of sample were plated on anti-IFN--coated ELISA plates and developed as described previously. There were no significant differences in OD490 readings between wells containing candidate microbicides and control wells, indicating that the presence of the microbicide compounds did not interfere with detection of IFN- by this assay.
Intracellular Cytokine Staining
Unless otherwise noted, all antibodies and reagents were obtained from BD PharMingen. B6/WT-3 cells (2 x 105 cells/well) were seeded into 24-well plates and cultured overnight. OT-1 T cells from Day 5 cultures were added at 1 x 106 cells/well to B6/WT-3 cultures in the presence or absence of 1.0 mg/ml PRO 2000. T cells were stimulated for 4 h with SIINFEKL peptide (1 µM final concentration) or 100 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 5 µM ionomycin (Sigma-Aldrich). Brefeldin A (Golgiplug; BD PharMingen) was added for the last 3.5 h of culture. Cells were harvested, incubated with rat anti-mouse CD16/CD32 (2.4G2) to block FcR, and then surface stained with FITC-conjugated anti-Vß5.1, 5.2 (MR9-4,) or mouse IgG1 isotype control. Cells were permeabilized using a Cytofix/Cytoperm Plus kit (BD PharMingen) and stained with PE-conjugated anti-IFN- (XMG1.2) or Rat IgG1 isotype control. Assays with DO11.10 T cells were performed in a similar fashion using A201.11 cells as antigen-presenting cells. Following stimulation with the OVA323–339 peptide (ISQAVHAAHAEINEAGR) at 1 µM final concentration, cells were stained with PE-conjugated anti-DO11.10 clonotypic TCR antibody (KJ1-26) or mouse IgG2a isotype control (Caltag Laboratories, Burlingame, CA) and FITC-conjugated anti-IFN-. Samples were acquired on a Becton Dickinson FACS Scan flow cytometer at the University of Texas Medical Branch Flow Cytometry Core Facility. Data were analyzed using Flow Jo software (Tree Star, Inc., Ashland, OR).
Flow Cytometric Analysis
Flow cytometric analysis was performed using a modification of the procedure described previously [13]. The following antibodies were obtained from BD PharMingen: PE-conjugated anti-mouse CD3 molecular complex (17A2), anti-CD18 (C71/16), anti-CD8
(53-6.7), anti-mouse I-A/I-E (M5/114.15.2), Rat IgG1, Rat IgG2a, Rat IgG2b, and FITC-conjugated anti Vß5.1,5.2 TCR (MR9-4), Rat IgG1, anti-CD54 (3E2), and Armenian Hamster IgG1. OT-1 T lymphocytes were harvested from cultures on Day 5 after peptide stimulation and viable cells were obtained by centrifugation over Histopaque 1083 cushions. T cells were incubated for 4 h at 37°C in the presence or absence of 1.0 mg/ml PRO 2000, and all subsequent blocking and staining steps were performed at 37°C in the continued presence or absence of 1.0 mg/ml PRO 2000. FcR were blocked by incubation with rat anti-mouse CD16/CD32 (2.4G2; BD PharMingen), stained with the indicated antibody conjugates, then washed and fixed with 1% formaldehyde in PBS. A201.11 cells were obtained from log-phase cultures and were treated with PRO 2000 and stained in the same fashion.
Statistics
Data were analyzed by one-way analysis of variance with the Bonferroni correction for multiple groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To test for cytotoxic effects, normal splenic lymphocytes were cultured for 48 h with candidate microbicide compounds over a range of doses and assessed for viability by MTT assay. No toxicity was observed in cultures of spleen cells containing the sulfated polymers PRO 2000 or lambda carrageenan. As shown in Figure 1A, splenocytes tolerated the sulfated polymers at doses ranging from 0.0002 to 2.0 mg/ml. Similar results were obtained by testing the ability of splenocytes to exclude Trypan Blue in the presence of PRO 2000 (data not shown). The bile salt, cholic acid, exhibited toxicity at 0.5–1.0 mg/ml (P < 0.001) but was not toxic or only minimally so at concentrations up to 0.25 mg/ ml (Fig. 1B).
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Detection of Interference with Antigen Recognition by Specific T Lymphocytes
Candidate microbicides were screened for detrimental effects on antigen recognition by antigen-primed T cells. EL-4 thymoma cells were used as APCs and were cultured with OVA-specific OT-1 T cells for 48 h in the presence of the OVA257–264 peptide and various concentrations of the candidate microbicides lambda carrageenan, cholic acid, or PRO 2000. Recognition of the OVA peptide was assessed by quantifying the antigen-specific production of IFN- by OT-1 T cells. As shown in Figure 2A, lambda carrageenan did not interfere with recognition of the OVA peptide over a range of doses between 0.008 and 1.0 mg/ml. The presence of cholic acid in doses between 0.25 and 0.5 mg/ml interfered (P < 0.05) with OVA peptide recognition at high antigen dose (10 µM) but was not inhibitory at lower antigen concentrations (Fig. 2B). As shown in Figure 2C, recognition of OVA over the entire range of peptide concentrations (0.1–10 µM) was significantly inhibited by the presence of 1.0–2.0 mg/ml PRO 2000 compared with the untreated control (media) group (P < 0.001). Recognition of low doses (0.1 µM) of OVA peptide by OT-1 T cells was inhibited in the presence of 0.5 mg/ml PRO 2000 whereas recognition of higher antigen concentrations (10 µM) was not inhibited compared with the untreated control group.
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A similar assay was performed using antigen-primed CD4+ T cells (DO11.10). The presence of PRO 2000 at 1.0 mg/ml completely inhibited (to background IFN- levels) recognition of the OVA323–339 peptide (ISQAVHAAHAEINEAGR) (Fig. 3A) or native OVA protein (Fig. 3B) at all antigen doses tested (P < 0.001). Addition of PRO 2000 at 0.2 mg/ml significantly inhibited recognition at all antigen concentrations tested (P < 0.001), whereas concentrations of PRO 2000 from 0.0016 to 0.040 mg/ml did not inhibit recognition of APCs cultured with either OVA peptide or native OVA protein.
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As an alternative approach to detecting inhibitory effects of PRO 2000 on antigen recognition, antigen-specific T cells were cultured with APC and antigenic peptide in the presence or absence of PRO 2000 and IFN- production was assessed after 4 h by intracellular cytokine staining. As shown in Figure 4, stimulation of CD4+ DO11.10 T cells with antigenic peptide resulted in the production of IFN- in the absence, but not in the presence, of 1.0 mg/ml PRO 2000. Similarly, the recognition of SIINFEKL peptide by CD8+ OT-1 T cells was prevented in the presence of 1.0 mg/ml PRO 2000 (Fig. 5). Importantly, OT-1 T cells nonspecifically stimulated with PMA and ionomycin in the presence of PRO 2000 produced IFN-, suggesting that failure to produce IFN- following exposure to antigenic peptide in the presence of PRO 2000 was not due to toxic effects on T cells. Decreased expression of the T cell receptor in these experiments most likely reflects previously described downmodulation of the TCR following recognition of peptide/MHC complexes [20, 21].
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Antigen Recognition by T Lymphocytes Is Inhibited Only in the Continued Presence of PRO 2000
Exposure of A201.11 APC to 4.0 mg/ml PRO 2000 for 4 h resulted in toxicity as measured by MTT assay (Fig. 1C). However, doses of PRO 2000 from 2.0 to 0.25 mg/ml were not toxic. Similar results were obtained by testing the ability of A201.11 cells to exclude Trypan Blue in the presence of 2.0 or 1.0 mg/ml PRO 2000 (data not shown). Thus, short-term exposure of APC to these concentrations of PRO 2000 did not result in toxicity. To test if PRO 2000 inhibited antigen uptake and processing, A201.11 antigen-presenting cells were simultaneously exposed to PRO 2000 and OVA protein for 4 h, then repetitively washed before addition of DO11.10 T cells. As shown in Figure 6A, DO11.10 cells produced IFN- in response to exposure to either PRO 2000 treated or untreated, antigen-pulsed APC. These results suggest that uptake and processing of native OVA were not inhibited by the presence of PRO 2000 at concentrations up to 2.0 mg/ml.
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To determine if transient exposure of T cells to PRO 2000 would result in long-lasting effects on the ability to recognize and respond to peptide/MHC complexes on antigen-presenting cells, DO11.10 T cells were cultured with PRO 2000 for 4 h, then washed thoroughly before addition of antigen and A201.11 antigen-presenting cells. As shown in Figure 6B, 4-h exposure of T cells to PRO 2000 at concentrations between 0.5 and 2.0 mg/ml did not result in inhibition of antigen recognition by DO11.10 T cells, suggesting there were no long-term detrimental effects on T cells.
Diminished Detection of Surface Proteins Involved in Antigen Recognition Events in the Presence of PRO 2000
PRO 2000 is highly sulfated and may form salt linkages or ion pairs with oppositely charged molecules on the APC and T cell surfaces. We determined if proteins critical for TCR signaling or stabilization of T cell-APC interactions were masked in the presence of PRO 2000 using a flow cytometric approach. Purified OT-1 T cells were incubated for 4 h with either T cell media alone or media containing 1.0 mg/ml PRO 2000. The ability of fluorochrome-labeled-specific antibodies to bind to and detect CD3, CD8, CD18 (integrin ß2chain of LFA-1), or the TCR (Vß5.1) in the continued presence of PRO 2000 was then assessed. As shown in Figure 7, A–C, staining for CD3, CD8, and CD18 was inhibited in the presence of PRO 2000 as demonstrated by a decrease in the mean fluorescence intensity of staining. Interestingly, staining of the TCR was only minimally inhibited by PRO 2000 (Fig. 7D). Similar results were obtained using DO11.10 T cells (data not shown). Studies were also performed to determine effects on A201.11 antigen-presenting cells. Binding of specific antibody to CD54 (ICAM-1, Fig. 7E), but not MHC class II proteins (Fig. 7F), was partially inhibited in the presence of 1.0 mg/ml PRO 2000.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surface-active agents such as ionic and nonionic detergents may directly inactivate pathogens by disrupting membranes or envelopes and thus have been proposed as candidate topical microbicides. However, there are safety concerns involved with the use of these compounds. Although available as an ingredient of over-the-counter spermicides, repeated use of N-9-containing gels has been shown to result in epithelium irritation and erosions [11, 12, 28], increases in the incidence of urinary tract infections [28], and increased acquisition rates of sexually transmitted diseases, including HIV [29]. The anionic detergent, cholic acid, and derivative compounds have been suggested as alternative active ingredients for topical microbicides [14]. In the current study, we found that cholic acid was toxic for lymphocytes at concentrations above 0.250 mg/ml. Similar levels of cholic acid toxicity for human peripheral blood lymphocytes have previously been reported [30]. The results of viability testing on immune cell populations raise concerns about possible toxic effects of cholic acid on resident immune cells in the genital tract. For example, long-term or repeated exposure to cytotoxic compounds might result in depletion of antigen-specific lymphocyte populations in the genital tract. Given the variable effects of different formulation components on other important parameters such as sperm motility and alteration of normal vaginal flora [31], it is possible that formulation of cholic acid with an appropriate carrier might abrogate or reduce the cytotoxic effects. In the current study, cholic acid interfered with antigen recognition by OVA-specific T lymphocytes only at the highest antigen concentration. The reason for this inhibition is unclear but may involve increased sensitivity of OT-1 T cells activated with high antigen doses to toxic effects of cholic acid.
Sulfated polymers represent another category of compounds that are being considered as candidate microbicides based on the ability to inhibit infection of enveloped viruses such as HIV in vitro and HSV both in vitro and in vivo [2–6]. The mechanism of action of these highly charged compounds is thought to result from the formation of salt linkages or ion pairs between oppositely charged molecules on the pathogen or host cell surface and the sulfated polymer [32]. Thus, critical proteins on either the target cells or the pathogen may be bound, preventing contact between the host and pathogen. We hypothesized that important cell-cell interactions such as T cell recognition of infected cells or recognition of antigen presented by resident APC may also be prevented or inhibited by nonspecific coating of host cells with these compounds. Recognition of some, but not all, APC and T cell surface proteins by specific antibody was inhibited in the presence of PRO 2000, suggesting some proteins involved in antigen recognition events might have been less accessible for critical protein-protein interactions. The reduction in accessibility of proteins involved in T cell signaling (e.g., CD3, CD8) and stabilization of T cell-APC interaction (e.g., LFA-1, ICAM-1) may have been responsible, in part, for the diminished recognition of antigen in the presence of PRO 2000. In addition to these nonspecific blocking effects, it is possible that cellular interactions may have also been inhibited by charge repulsion of T cells and APC coated with the highly charged PRO 2000 molecule. In further support of this hypothesis, Perotti et al. [33] presented evidence that similar interference with cell-cell interactions may be involved in the sulfated polymer-mediated inhibition of macrophage migration from the genital tract to draining lymph nodes. In initial screening, the presence of the naphthalene sulfonate polymer, PRO 2000, inhibited T cell-APC interactions. Importantly, transient exposure of T cells to PRO 2000 did not result in residual impairment of function in these cells. Additionally, the presence of PRO 2000 did not inhibit uptake and processing of antigenic protein by APC. These results suggest that antigen recognition would not be impaired in vivo once metabolic clearance of the compound occurred. That is, there was no evidence for long-term loss of function in populations of primed T cells after exposure to PRO 2000.
No impairment of T cell-APC interaction was detected in the presence of lambda carrageenan, suggesting that the ability to inhibit these immune functions is not a universal characteristic of all sulfated polymers. It should be noted that the inherent viscosity of lambda carrageenan limited the concentrations of compound that could be tested in these in vitro assays. Therefore, it is still possible that higher concentrations of lambda carrageenan, such as those used in the candidate microbicide Carraguard, might inhibit interactions between T cells and APC in vivo.
The in vitro assays used in this study represent a convenient screen for the testing and prioritization of candidate microbicides. It will be important to develop in vivo models to further examine interactions between immune cells and candidate microbicides, including effects on viability and function of B lymphocytes, tissue infiltration, and target cell recognition by natural killer cells, and lymphocyte trafficking and activation mediated by chemokine and cytokine signals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gregg N. Milligan, Sealy Center for Vaccine Development, 301 University Blvd., Galveston, TX 77555-0436. FAX: 409 747 8150; gnmillig{at}utmb.edu
Received: 5 March 2004.
First decision: 3 April 2004.
Accepted: 2 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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complexes after antigenic stimulation. J Exp Med 1997 185:1859-1864
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