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Immunology |
Department of Obstetrics,3 Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
CONRAD,4 Department of Obstetrics and Gynecology,
Department of Pathology,5 Eastern Virginia Medical School, Norfolk, Virginia 23507
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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cytokines, immunology, sperm, toxicology, vagina
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unfortunately, some commercially available spermicidal microbicides, including the popular nonoxynol-9 (N-9), were proven less efficacious in clinical trials than it had been hoped according to their in vitro potency against HIV-1 and other sexually transmitted disease (STD) pathogens [4–8]. This discrepancy may be due to a mucosal inflammatory reaction induced by N-9, which exerts a facilitating effect on HIV-1 transmission. In support of this hypothesis, strong clinical evidence shows that both ulcerative and nonulcerative inflammatory STDs increase the risk of HIV-1 infection approximately 3–5 times [9, 10]. Pro-inflammatory chemokines and cytokines play a key role in recruiting and activating CD4+ cells to the mucosal port of entry for HIV, and some cytokines can amplify viral replication in infected cells [11–15]. Despite their FDA-approved status, recent studies on N-9 products showed inflammatory lesions in the genital tract mucosa after repeated and frequent use [5, 16–19]. Furthermore, only three consecutive applications of N-9 were sufficient to trigger cytokine and chemokine release and a dramatic influx of activated macrophages in the cervicovaginal secretions of healthy volunteers [8].
In the present study, we characterize the pro-inflammatory potentials of four surface-active agents that are currently on the market as spermicides or under consideration as vaginal microbicides. Taking this information together, it is clear that existing models to understand, predict, and evaluate vaginal mucosal toxicity induced by spermicides/ microbicides are inappropriate, especially in regard to the identification of subtle or subclinical inflammatory reactions, which as indicated above, are crucial to these compounds' impact on HIV transmission. We focused this study on nonoxynol-9 (nonionic), benzalkonium chloride (cationic), sodium dodecyl sulfate (anionic), and sorbitan monolaurate (nonionic) because they possess different electrochemical charges that condition different membrane-associating properties. In addition to studying their antisperm and anti-HIV activities, we characterized their proinflammatory effects using an in vitro culture system based on immortalized human vaginal cells [20] and an in vivo model consisting of single or repeated intravaginal applications in rabbits. The rabbit model was chosen for its proven high sensitivity to chemical insult and the long history of use for evaluating mucosal toxicity of vaginal products [1, 21]. In its original version, however, vaginal irritation is assessed after a 10-day compound administration by one-time anatomical observation and histologic evaluation of edema, congestion, epithelial erosion, and leukocyte cell infiltrate [3, 21]. Our model uses short-term applications and monitors the release of inflammatory mediators in daily cervicovaginal lavages. We place the emphasis on the value of interleukin (IL)-1, IL-6, and IL-8 as markers of mucosal toxicity because they represent three families of cytokines with different roles in inflammatory tissue damage and HIV-1 pathogenesis. IL-1 and IL-6 induce HIV-1 expression via NF-kB mediated HIV-1 long terminal repeat (LTR) activation, while IL-8 triggers the recruitment of HIV-1-susceptible cells to the inflammation site and may also stimulate HIV-1 replication in T cells and macrophages [12, 22– 27]. All three of these cytokines are expressed constitutively at low levels by cervicovaginal keratinocytes and are significantly induced via NF-kB mediated signal transduction pathways by pro-inflammatory stimuli and common sexually transmitted pathogens [24, 28–31].
Our results show that surface-active agents with different electrochemical charges and membrane-associating properties differ in their virucidal, spermicidal, and mucosal inflammatory properties. The assessment of IL-1, IL-6, and IL-8 release induced in vitro and in the rabbit vagina links cellular and tissue markers of inflammation and provides a more sensitive and dynamic measure of the proinflammatory potential of spermicides and microbicides, a determining element of their safety and ultimate impact on HIV transmission.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Benzalkonium chloride (BZK), SDS, and sorbitan monolaurate (SML) were purchased from Sigma Co. (St. Louis, MO). Nonoxynol-9 (N-9) was a kind gift from Personal Products Company (Skillman, NJ).
Modified Sander-Cramer Assay
Semen samples were collected with informed consent from normal donors by masturbation, allowed to liquefy, and assessed for motility. The protocol was approved by the Eastern Virginia Medical School IRB. Serial twofold dilutions in 0.9% NaCl of the test compound were incubated with semen aliquots for 30 sec. Motility was evaluated under the microscope during the first incubation and after dilution in buffer and further incubation for 1 h. Only those dilutions that immobilize 100% of the spermatozoa pass the test [3]. Results were expressed as minimum effective concentrations (ED100).
Double-End Test
Compound solutions were incubated with microcolumns of cervical mucus (CM) (Penetrak; Biochem Immuno System, Norwell, MA) for 1 h, after which the columns were sealed, opened by the other end, and reimmersed in semen. Sperm migrate through the CM until they find a bioactive concentration of the compound [3]. Results were expressed as percent penetration of control (saline-treated) sperm.
Sperm Motility and Viability Tests
Compound twofold serial dilutions were incubated with semen aliquots for 30 sec, after which the mixtures were overdiluted with nutrient medium (termination of compound's effect). Sperm were pelleted down, resuspended in fresh medium, and incubated at 37°C, 5% CO2 for 30 min. Progressive motility was assessed under the microscope. Results were expressed as percentage of compound-treated motile sperm as compared with control (saline-treated) sperm. To verify membrane integrity, sperm were further incubated in a hypo-osmotic buffer for 30 min (hypo-osmotic swelling test, HOST), and the response of their tails was evaluated microscopically (coiled tails indicated conserved membrane integrity). Not less than 200 cells were counted [32].
Computer-Assisted Sperm Motion Analysis
Compound concentrations that immobilize 50% of the sperm (ED50) were incubated with semen in the same way as above (sperm motility assay). This time, however, a Hamilton-Thorne Motion analyzer (IVOS v. 10.4; Hamilton Thorne Biosciences, Beverly, MA) was used to determine sperm motion parameters such as velocity (VSL and VCL), head displacement (ALH) and linearity of trajectory (LIN and STR). Data were normalized against saline control [32].
Cell-Free HIV Inactivation Assay
HIV-1 RF, obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, MD), was incubated with half-log dilutions of compounds for 2 min. Contact time was terminated by 10-fold serial dilutions and virus-compound mixtures were added to MT-2 cells. After 7 days in culture, virus-induced cytopathic effect (syncytium formation) and cytotoxicity (no virus) were evaluated microscopically. Reed-Muench calculations were employed to obtain 50% tissue culture infectious doses (TCID50s) and log reduction in virus titers [33].
Cytotoxicity to Human Vaginal Cells
HPV16/E6E7-immortalized human vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cell lines [20] were grown until confluence in 96-well tissue-culture plates and incubated with two-fold dilutions of test compounds or plain culture medium for 30 min, 6 and 24 h. The 24 h exposure experiments were repeated with 3rd and 5th passage of primary human ectocervical epithelial cells (CrEC-Ec) obtained from Clonetics (BioWhitaker Inc., San Diego, CA). The nonradioactive Cell Titer 96 MTT assay (Promega, Madison, WI) was performed to assess viability based on mitochondrial enzyme function as previously described [8]. This assay has been shown to discriminate well the irritating potential of surfactants on the skin [8, 34]. Following removal of the test compounds, cells were incubated for 4 h with tetrazolium salt (15 µl MTT/ well). Subsequently, 100 µl of DMSO solution was added overnight to each well to dissolve the blue formazan produced by viable cells. All incubation steps were performed at 37°C in a 5% CO2 incubator. Absorbencies were read with Dynatech MRX ELISA reader (Dynex Technologies, Chantilly, VA) at 540 nm and a reference wavelength of 620 nm.
Cytokine Determination
Cytokine levels were measured in cell culture supernatant by ELISA using commercially available human cytokine kits (R&D system, Minneapolis, MN). For these experiments immortalized and primary epithelial cell cultures were grown until confluence in 96 or 24 well plates and treated for 24 h in duplicates or triplicates with respectively 0.1 ml or 1 ml compound supplemented medium/well. MTT assay was performed on each plate after removal of culture supernatants to adjust cytokine concentrations by percentage of viable cells. IL-1, IL-8 and IL-6 were also quantified in rabbit vaginal washings using cross-reactive kits (human IL-1ß from Genzyme, Cambridge, MA; human IL-8 and rat IL-6 from R&D Systems, Minneapolis, MN). Optical densities were read using a Multilabel Microplate Counter Victor 2 (Perkin Elmer Life Sciences, Boston, MA) and WorkOut Version 1.5 Wallac Software (DAZDAQ Ltd., Brighton, UK). Cytokine concentrations were calculated by quadratic regression analysis based on logarithmically transformed optical densities. Interference of compounds with cytokine ELISA was ruled out by spiking various compound concentrations with cytokine standards provided by manufacturer.
Rabbit Experiments
Adult (3–4 kg) nulliparous non-pregnant New Zealand white female rabbits were employed in this study. Rabbits were dosed intravaginally with 1 ml of active formulation (2% N-9, SDS, BZK or SML in carboxymethylcellulose [CMC]), vehicle control (CMC), or normal saline (0.9% NaCl) using a 12-cm flexible catheter (polyethylene tubing, Fisher Scientific, catalog # 22–204222, Hampton, NH) introduced up to its 8-cm mark. The same catheter was used to collect the saline lavage (5 ml). Two different protocols were designed for parallel assessment of histopathological effects (RVI scores) and proinflammatory cytokine release. In the first protocol the rabbits received a single 1-ml dose of 2% compound or saline, and cervicovaginal lavages (CVLs) were collected before (baseline) and 24 h after the dose administration. Thirty-six vaginal lavage specimens were obtained from three rabbits per group. In the second protocol the rabbits were dosed on three consecutive days, and CVLs were collected at 24 h intervals before each treatment and on two consecutive days after the last application. A total of 75 CVLs were collected from 3 rabbits per treatment group.
CVLs were then centrifuged to separate the soluble supernatant from cell debris and frozen until used in ELISA for cytokine determination. Rabbits were killed with a combination of Acepromazine and Euthasol after the last lavage. Their vaginas were surgically excised and medially opened. After macroscopic observations were recorded, three pieces of tissue taking the entire thickness of the wall were cut at the upper, mid, and lower portions of the abdominal vagina and processed for histopathology. Scores were based on Eckstein et al. [35].
All experimentation with live animals was carried out within the provisions of the Animal Welfare Act (Public Law 99–198), the National Research Council (NRC), Public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS), the "Guide for the Care and Use of Laboratory Animals" (1996), the Health Research Extension Act of 1985 Public Law 99–158 (11/20/86), and United States Department of Agriculture (USDA) regulations. The work was performed under an EVMS Institutional Animal Care and Use Committee (IACUC)-approved protocol and with further approval by the Standing Committee on Animals of the Harvard Medical School Office for Research Subject Protection.
Statistical Analysis
Area under the curve (AUC) analysis of logarithmically transformed cytokine concentrations in rabbit CVLs was used to assess cumulative compound effects over time in the multiple compound administration protocol. Cytokine values, which were obtained from CVLs after each compound administration, were normalized by subtracting baseline values for each rabbit and log 10 transformed for the AUC analysis. The Pearson r (linear regression) analysis was used to assess correlation between the AUC values and RVI scores. Cytokine concentrations in compound treated cell cultures were normalized per 106 viable cells and when appropriate expressed as a percentage of the control value to allow analysis of multiple experiments. Statistically significant differences (P < 0.05) from baseline and between treatment groups were assessed by analysis of variance (Dunnett multiple comparisons test) or by Bonferroni t-test. Wilcoxon rank test was used to assess differences between ED50 effects on motion parameters. All statistical calculations were performed using Prism version 3.0 (GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonoxynol-9 and sorbitan monolaurate (N-9 and SML, nonionic), benzalkonium chloride (BZK, cationic) and sodium dodecyl sulfate (SDS, anionic) demonstrated significantly different sperm-immobilizing and cytocidal properties. Among them, N-9 was the most potent spermicidal compound followed by BZK and SDS (Table 1). Corrected by molarity, the differences in minimum effective concentration (ED100) were even greater. SML was incapable of completely immobilizing 100% of spermatozoa, even at the highest dose tested (10 mg/ml). At 1%, after diffusing in cervical mucus, N-9, BZK, and SDS showed similar sperm immobilizing activity. Therefore, they were equipotent in blocking sperm penetration in cervical mucus. The very high correlation (r = 0.99, P < 0.0001) between sperm motility and viability dose-responses for all four compounds indicated that the initial cause of sperm immobilization is membrane disruption (Fig. 1). Comparing the dose-response curves, N-9 was slightly more potent than BZK (although not statistically significantly), while both compounds were significantly more spermicidal than SDS and SML; furthermore, SDS was more potent than SML (P = 0.0005 for ED50's compared by ANOVA). Tested at concentrations that immobilized 50% of spermatozoa (ED50), N-9 and BZK induced a significant decrease in sperm velocity, a sensitive marker of initial sperm membrane compromise (Table 2).
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Anti-HIV Virucidal Activity
Assessment of virucidal activity against HIV-1 produced the same compound ranking as the spermicidal testing described above, N-9 being the most active and SML being the least active of the four surfactants evaluated (Table 3). The compounds were incubated with HIV-1 RF, a lymphocytotropic HIV-1 strain, for a short period of time, and then the virus was incubated with MT-2 lymphoblastic cells for 6 days to allow for the formation of virus-induced syncytia. At 0.1%, N-9 and BZK reduced viral titers by >4.8 and 3.6 logs, respectively.
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In Vitro Epithelial Cytotoxicity and Secretion of Pro-inflammatory Cytokines
To study the mechanisms of chemically induced vaginal inflammation, we used immortalized human vaginal and cervical epithelial cell lines that closely resemble the characteristics of their normal tissues of origin and respond to a variety of pro-inflammatory stimuli similarly to primary cells [8, 20, 28, 30, 31]. Our previous investigations using this model system showed that subtoxic doses of N-9 cause IL-1-mediated NF-
B activation and cytokine expression [8].
The epithelial cell cytotoxicity dose responses to the four surface active agents tested in this study after 30-min, 6-h, and 24-h exposure times invariably ranked BZK as the most and SML as the least cytotoxic compounds, regardless of cell line (Fig. 2A). In contrast with spermicidal and virucidal rank orders, N-9 ranked second in cytotoxicity, followed closely by SDS. The addition of 1% human serum albumin to the keratinocyte serum-free medium attenuated the cytotoxic effects, but did not change the cytotoxicity rank order (Fig. 2B).
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Cytokine responses in vitro were obtained for each compound at a series of doses covering a range from 0 to 100% cytotoxicity (TCTD0–TCTD100). The extracellular release of prepackaged cytokines was determined after 30 min of exposure time. As expected by their membrane-perturbing properties, all compounds caused a rapid extracellular release of prepackaged IL-1, which correlated with cell death, measured at 30 min by MTT mitochondrial function assay (Fig. 3, A and B). IL-6 or IL-8 could not be detected in 30-min-culture supernatants, showing that no measurable amounts of these cytokines are stored in the cytoplasm of vaginal and cervical epithelial cells.
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Compound exposures were then extended to 6 and 24 h, starting with the highest nontoxic dose determined at 30 min (Fig. 3C). Within 6 h of exposure, the IL-1 release correlated with the cytotoxicity profile of the four compounds, with BZK being the most potent inducer and SML the least potent inducer of IL-1. SML, however, showed a delayed pattern of IL-1 increase at 24 h despite the fact that it remained nontoxic at this concentration. The 24 h exposure time revealed peaks of IL-6 and IL-8 secretion induced around TCTD50's (Fig. 3, D and E).
Histopathological Findings and Cytokine Production in the Rabbit Cervicovaginal Mucosa
The histopathological examination of cervicovaginal tissues performed after a single compound application clearly identified BZK as the most irritating compound, revealing multifocal damage of the vaginal mucosa with congestion, edema, inflammatory infiltrate, and epithelial disruption (Table 4 and Fig. 4). BZK consistently induced in all test animals more than a log increase in the IL-1ß and IL-6 levels as compared with placebo controls (P < 0.01; Fig. 5). Although at a lower magnitude, SDS and SML also induced a significant IL-1 release (P < 0.05). N-9-induced IL-1 levels were not significantly different from those in the control group. A significant correlation was established between the posttreatment CVL IL-1ß levels and the cumulative (total) RVI scores obtained in 15 test animals 24 h after a single-dose administration (r2 = 0.6370, P = 0,006; Fig. 6A). None of the compounds caused a significant IL-8 induction at this time (data not shown).
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Three compound applications with a 48 h posttreatment follow-up period revealed additional differences among the four test compounds (Table 5 and Fig. 7). The rabbit vaginal irritation (RVI) ranking repeated the vaginal cytotoxicity rank order established in vitro. While the average RVI scores obtained for SDS and SML showed no significant difference from the ones established after single compound administration, the cumulative RVI scores after multiple applications increased for BZK and N-9. This was mostly due to increased epithelial disruption, compounded by increased vascular damage and infiltrate in the case of N-9. The area under the curve (AUC) for IL-1ß again showed a strong correlation of this cytokine with total RVI scores (Fig. 6B; r2 = 0.7561; P = 0.001). The cumulative inflammatory tissue damage induced by BZK and N-9 could also be predicted by IL-8 levels, which were selectively increased over baseline (P < 0.01) after the second and third compound applications. Furthermore, an early (24 h) and sustained IL-6 release over the treatment period was observed for BZK. SDS and, especially, SML showed no significant IL-8 responses, with a tendency to a late IL-6 response, which possibly indicates the need for a more extended contact with the cervicovaginal mucosa before the epithelial integrity is significantly compromised.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our findings show that the nonionic surfactant N-9 was the most potent compound in terms of spermicidal and virucidal activity, while BZK was the most cytotoxic to the vaginal cells. Cationic surfactants like BZK have been shown to be highly toxic to other cell lines such as Hela and NIH 3T3 [38]. In our study, BZK also proved to be the most potent acute inducer of IL-1 cytokine release, both by cultured vaginal epithelial cells and, in situ, by the rabbit vaginal mucosa. Ultimately, BZK was the agent causing the most severe epithelial disruption and vascular damage to the rabbit vaginal mucosa, even after only a single dose. This correlation supports the value of our in vitro evaluation system to predict the in vivo irritative/inflammatory potential of vaginally applied compounds.
Prepackaged IL-1 cytokines are the main initiator of a proinflammatory cascade in membrane-perturbed skin and vaginal keratinocytes. Through NF-kB transactivation, IL-1 would induce the secretion of other proinflammatory mediators, such as IL-8, IL-6, MIP-1, etc., amplifying the inflammatory response [8, 24]. In accordance with their lower cytotoxicity to vaginal cells, the nonionic surfactants N-9 and SML induced significantly less IL-1 ß than BZK in the rabbit vagina. However, they did induce important amounts of IL-8 and IL-6 at subtoxic doses in vitro, which translated into a requirement for repeated administration (N-9) or longer exposure (SML) to induce significant IL-8 secretion in vivo. This would be in agreement with the absence of major histopathological changes in the rabbit vaginal mucosa after a single application of a 2% dose, reported in this study, as well as the need to administer human volunteers with at least three daily doses of Conceptrol (4% N-9) to observe a significant increase of leukocytes and cytokines in their cervicovaginal lavages [8]. Nonionic surfactants, reportedly the least irritating to human skin [36], may require more prolonged exposures, allowing sufficient time for a cytokine-mediated inflammatory cascade to be in full operation. In our study, N-9 induced significant mucosal inflammation only after multiple applications. Again, its proinflammatory potential was appropriately predicted by the in vitro system and the level of IL-1 in rabbit vaginal fluids.
SDS at 0.05% has been reported to be relatively innocuous to human foreskin xenografts [40]. However, data collected with standard skin irritation tests indicate that anionic surfactants are, generally, the most irritating among this category of agents [39, 41]. The attenuation of the cytotoxicity response to SDS in the presence of albumin observed in our in vitro study agrees with its high affinity to proteins [42, 43] and may explain the discrepancies between the severe skin irritation and the milder mucosal effects observed in the mucous, protein-rich vaginal environment. In our study, 2% SDS had a mild histopathological effect as compared with 2% BZK or 2% N-9. However, it showed proinflammatory potential by inducing IL-6 and IL-8 at subtoxic doses in vitro and by causing a sustained IL-1 elevation in the vaginal lumen in the absence of significant epithelial disruption. These findings predicted SDS's significant inflammatory response, even in the absence of serious mucosal damage. This phenomenon bears particular clinical relevance because it masks a facilitating condition for HIV transmission under the clinical appearance of a normal mucosa.
Less toxic compounds, showing favorable (low) rabbit vaginal irritation scores, may still act as potent inflammatory agents via a cytokine-mediated amplification of pro-inflammatory cascades. This might be one of the reasons underlying the poor dose-discriminating power of the standard rabbit vaginal irritation model. Of greater clinical relevance, these findings could explain why N-9-containing products passed phase I clinical safety trials [44, 45], showing no major macroscopic alteration of the cervicovaginal mucosa, but dramatically failed to prevent HIV transmission [4, 46, 47].
Also interesting, SML induced very high levels of IL-8 in vitro at subtoxic doses, while other nonionics such as polyethylene glycol and propylene glycol (data not shown) did not significantly alter the level of any of the cytokines tested, indicating that cytokine secretion profile depends on the individual characteristics of the compounds. These observations point out the importance of assessing the proinflammatory potential of each one of the ingredients of microbicide/spermicide formulations before final development and clinical testing are pursued.
Comparing the spermicidal/virucidal efficacy and irritation/inflammation potential of the screened compounds, one can make a more rational decision as to what agent should be pursued for further development. For instance, in this case, it is clear that BZK or SML would not be the preferred candidates. BZK was ranked first in all inflammatory tests in vivo, and SML, while not so toxic, showed the lowest specific, i.e., spermicidal and virucidal, activity. Additionally, SML induced ominous IL-6 and IL-8 expression by cervical and vaginal epithelial cells and significant IL-1ß release in vivo, with a tendency to a late IL-6 response. SDS and N-9 had similar spermicidal and virucidal activities and, although SDS caused less epithelial disruption in the rabbit vagina after repeated use, both compounds induced similar proinflammatory cytokine production in vitro and in vivo, suggesting that they could have similar deficiencies as antimicrobial spermicides in real use.
In conclusion, compound-induced cytokine secretion by vaginal epithelial cells in culture and by the rabbit cervicovaginal mucosa provides valuable information about the pro-inflammatory potential of vaginal microbicidal and spermicidal products that cannot be predicted by other preclinical and clinical tests in their current format. This information is extremely important in light of the facilitatory effect of inflammation on HIV infection and the potential use of these compounds to curb sexual transmission of HIV. The combination of in vitro and in vivo indices of immunoinflammatory function may lay the foundation for an improved preclinical testing algorithm as well as a better mechanistic understanding for safe and efficacious vaginal microbicidal contraceptives.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Raina Fichorova, 221 Longwood Avenue, RF468, Boston, MA 02115. FAX: 617 713 3018; rfichorova{at}rics.bwh.harvard.edu
Received: 12 March 2004.
First decision: 7 April 2004.
Accepted: 27 April 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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release and cytotoxicity was established after a 30-min exposure to a broad range of compound concentrations in plain (A) or 1% albumin-supplemented medium (B); (C) time-dependent IL-1
