MILLIGAN, GREGG N. PhD*; DUDLEY, KRISTEN L. BSc†; BOURNE, NIGEL PhD*; REECE, ALISA†; STANBERRY, LAWRENCE R. MD, PhD*
THE HIGH RATES of sexually transmitted diseases (STDs) detected globally highlight the need for effective strategies to prevent disease transmission. 1 Although immunization to control the spread of STDs would be ideal, protective vaccines are not currently available. Topical microbicides represent a logical alternative approach to preventing horizontal transmission of sexually transmitted pathogens, and the development of vaginally applied microbicides is a high priority. 2 An attractive benefit of this approach is that microbicide use would be a female prerogative, allowing use in situations in which condom use cannot be negotiated.
Nonoxynol-9 (N-9), a nonionic detergent used as a spermicidal ingredient in commercial contraceptive gels, has been considered for use as a topical microbicide. N-9 has been proven effective against a number of sexually transmitted pathogens in vitro, including HIV. 3,4 The microbicidal properties of a surface-acting agent such as N-9 are thought to result from the disruption of lipid membranes or envelopes of bacterial and viral pathogens. Unfortunately, frequent vaginal application of N-9 in humans has been reported to result in disruption of the genital epithelia and erosions, 5,6 the presence of inflammatory cells, 6–8 and alteration of the vaginal flora, including decreased numbers of lactobacilli and increased numbers of aerobic gram-negative bacilli and gram-positive cocci. 9 Although the long-term consequences of these effects are not fully understood, it is possible that any or all of these outcomes might increase the risk of infection with STD pathogens. In fact, in a recent phase 3 clinical trial involving women, use of an N-9-containing gel resulted in more genital lesions and a higher HIV seroconversion rate than use of a placebo gel. 10 There has been similar interest in using the sulfated anionic detergent sodium dodecyl sulfate (SDS) as an active ingredient in candidate microbicides. Vaginal application of SDS has been shown to protect mice against lethal genital infection with herpes simplex virus type 2 (HSV-2). 11 Studies in vitro suggested that primary vaginal keratinocytes were more sensitive to N-9 than to SDS 12; however, the effects of SDS on the vaginal epithelium in vivo have not been characterized.
Ionic polymers are also being explored as alternatives to surface-active vaginal microbicides. These compounds lack detergent activity and may act by blocking pathogen attachment to epithelial cells. The naphthalene sulfonate polymer PRO 2000 has been shown to have antimicrobial properties in vitro 13 and in animal studies 14 and has recently entered clinical trials. The sulfated polymer T-PSS has likewise been shown to prevent infection with HSV-1, HSV-2, Chlamydia trachomatis, and Neisseria gonorrhoeae in vitro and has shown efficacy against HSV-2 in vivo. 15 T-PSS has also entered clinical trials.
The immune system of the female genital tract is not fully understood but is clearly involved in protection against a variety of bacterial, fungal, and viral pathogens. Adverse interactions between candidate microbicides and the innate and adaptive immune cells of the genital tract might impact host protection or contribute to tissue pathology. To investigate the interaction between candidate microbicides and innate immune cells in the genital tract, we developed a mouse model in which changes in vaginal leukocyte populations could be identified and measured after vaginal application of a candidate microbicide.
We tested two classes of microbicides with different physicochemical properties and modes of action for their ability to induce inflammatory responses in the vaginal epithelium. We found that a single intravaginal administration of SDS, N-9, or N-9-containing gels could alter the number and composition of vaginal leukocytes. Significantly higher numbers of inflammatory cells, including macrophages, were detected in the vaginal lumen by 4 hours after N-9 treatment. In contrast, vaginal application of the ionic polymers PRO 2000 and T-PSS did not result in a significant increase in vaginal leukocytes. The results of this study demonstrate the utility of this model in screening for local inflammatory responses in the vaginal epithelium following application of candidate microbicides. This model may be useful as part of a screening program for prioritizing candidate microbicides before clinical studies in humans.
Female Swiss Webster mice weighing 18 to 21 g were obtained (Harlan Sprague–Dawley, Indianapolis, IN) and housed under pathogen-free conditions. Animal studies were approved by the Institutional Animal Care and Use Committee.
Microbicide Preparations and Treatment
N-9 (Sigma–Aldrich, St. Louis, MO) and SDS (BioRad, Hercules, CA) detergents were both diluted in phosphate-buffered saline (PBS) before use. A gel preparation containing 4% N-9 (Conceptrol; Ortho Pharmaceutical Corporation, Raritan, NJ) was obtained from a local pharmacy. A formulated gel containing 4% PRO 2000 and a control gel of comparable viscosity were obtained from Albert Profy (Interneuron, Inc., Cambridge, MA). A gel formulation containing 10% T-PSS and a control gel of comparable viscosity, manufactured under Good Manufacturing Procedure conditions (Advanced Care Products, North Brunswick, NJ) also were obtained. The reproductive cycle was synchronized by treatment with medroxyprogesterone acetate (Upjohn, Kalamazoo, MI) as described previously. 16 Progesterone-conditioned mice were treated by vaginal application of 15 μl of a microbicide gel or solution in PBS. Microbicide-containing gels and solutions were applied intravaginally with use of a model M25Gilson Microman positive displacement pipette with a CP25 capillary-piston (Gilson Medical Electronics, Villiers-le-Bel, France). This method has been utilized previously for application of viscous gels in small animal models. 14
Quantification of Vaginal Leukocytes
Leukocytes were harvested from the vaginal vault at 2, 4, 8, 24, or 48 hours after compound application by two consecutive washes with 75 μl Hanks balanced salt solution plus 5% newborn calf serum. Newborn calf serum was included in the wash fluid to maintain cells and prevent cell clumping before subsequent manipulation. Lavage fluid was applied to and withdrawn from the vagina with a Rainin air displacement pipette (Rainin, Emeryville, CA) and standard pipet tips. Viable cells from individual mice were quantified by hemocytometer counts with the viable stain trypan blue as diluent. Vaginal lavage cells from individual mice were spun onto glass slides at 500 rpm for 10 minutes with a Shandon cytospin 3 (Shandon Life Sciences International [Europe] Limited, Astmoor, Runcorn, England). Differential cell counts were obtained by staining cell preparations from individual mice with the Hema 3 stain kit (Fisher Scientific, Inc., Pittsburgh, PA). Macrophage and neutrophil counts were derived from the total viable cell count by the following formula:
Total viable cell count × % macrophage or neutrophil count (from differential staining) = total number of macrophages or neutrophils.
Flow Cytometric Analysis
Groups of 30 mice were treated intravaginally with Conceptrol or PBS. After 8 hours, leukocytes were harvested by vaginal lavage and pooled. Viable cells were obtained by centrifugation of lavage fluid over Lympholyte M (Cedarlane Laboratories, Ontario, Canada), washed three times with Hanks balanced salt solution plus 5% newborn calf serum, and incubated with purified rat IgG (20-μg/ml final concentration) to block Fc receptors. Cells were stained with a recombinant phycoerythrin (rPE)–conjugated monoclonal antibody specific for the murine macrophage–specific marker F4/80 (Serotec, Inc., Raleigh, NC) or an rPE-conjugated rat IgG2B isotype control antibody (Becton Dickinson–PharMingen, San Diego, CA). Cells were fixed in 1% formaldehyde before analysis on a Becton Dickinson FACSCalibur with Cell Quest software.
Virucidal Efficacy of N-9-Containing Gel In Vivo
Groups of 15 progesterone-treated mice were treated by instillation of 15 μl of Conceptrol either 4 hours or 20 seconds before inoculation or were control-treated with PBS 20 seconds before inoculation with 104 pfu of HSV-2 strain 186. This dose of virus routinely infects greater than 90% of the mice inoculated and has been used as a challenge dose in previous efficacy studies of candidate microbicides in a murine model. 14 Vaginal swab specimens were obtained on day 2 and stored frozen at −70 °C until titration on Vero cell monolayers as described previously. 16 Animals were scored as infected if virus was detected in day 2 vaginal swabs. Mice were evaluated daily through day 21 postinoculation for evidence of symptomatic infection, as described previously. 14 Symptoms included hair loss and erythema around the perineum, chronic urinary incontinence, hind-limb paralysis, and mortality.
Data were analyzed by t test, one-way analysis of variance with the Bonferroni correction for multiple groups, or two-tailed Fisher exact test as appropriate.
To determine if genital exposure to SDS or N-9 would induce inflammatory effects as detected by the alteration of the vaginal leukocyte population, groups of Swiss Webster mice were treated intravaginally with 0.25%, 1.0%, or 4% (w/v) SDS or N-9 solutions in PBS. Vaginal leukocytes were harvested by lavage 8 hours after treatment, and viable and differential counts were obtained. A single treatment with either detergent resulted in a dose-dependent increase in the number of viable leukocytes in the vaginal vault (Table 1). The increase was significant in mice treated with either 4% SDS or 4% N-9 (P < 0.05 in comparison with PBS-treated controls). The number of macrophages was significantly greater in mice treated with 4% SDS than in PBS-treated controls (P < 0.01) and accounted for approximately 7% of the vaginal leukocyte population. A more striking result was obtained after treatment with N-9. Macrophages accounted for greater than 42% of vaginal leukocytes in mice treated with 4% N-9, compared with 2% to 3% in PBS-treated mice (P < 0.05). Treatment with either 1% or 0.25% N-9 resulted in a smaller increase in macrophage numbers.
A commercially available spermicidal gel containing 4% N-9 (Conceptrol) was tested to determine if treatment with N-9 in this form resulted in the induction of a similar inflammatory response. Because a placebo gel with similar formulation and viscosity to the commercial preparation was not available, PBS was used as a control in these experiments. A single treatment with Conceptrol resulted in an approximately 10-fold increase (P < 0.0001) in viable leukocytes in the vaginal lumen in comparison with the count in PBS-treated controls (Table 1). Conceptrol treatment resulted in a significant increase in the number of both neutrophils and macrophages present in the vaginal lumen fluid at 8 hours after treatment (P < 0.0001). Macrophages represented less than 2% of the vaginal leukocytes present in control-treated mice, whereas they constituted approximately 50% of the inflammatory cells in Conceptrol-treated mice.
The entry of macrophages into the vaginal lumen was confirmed by flow cytometric analysis. Vaginal lavage cells collected 8 hours after treatment were stained to detect expression of the murine macrophage marker F4/80. In agreement with differential counts, approximately 50% of the total vaginal leukocytes from Conceptrol-treated mice expressed F4/80, versus less than 2% of lavage cells from PBS-treated mice (Figure 1).
Time Course of Macrophage Entry Into the Vagina Following Treatment With an N-9-Containing Gel
The time course of leukocyte entry into the vagina following treatment with Conceptrol was determined. As shown in Figure 2A, the total number of viable leukocytes in Conceptrol-treated mice began to rise by 4 hours after treatment and was significantly higher than in PBS-treated controls at 8 hours after application (P < 0.001). Greater than 95% of the leukocytes present in vaginal lavage fluid of PBS-treated mice at all time points were neutrophils, as determined by differential staining. Neutrophil counts were variable among individual mice, and mean counts were not significantly different between Conceptrol-treated and control-treated mice at any time point in this experiment (Figure 2B). Approximately 97% of the leukocytes present in the vagina at 2 hours after treatment with Conceptrol were neutrophils. However, a significant increase in the number of macrophages was detected by 4 hours (P < 0.01), and macrophage infiltration continued to increase through 8 hours after treatment (P < 0.01) (Figure 2C).
The resolution of leukocyte infiltration was examined in a separate experiment. The total number of viable vaginal leukocytes in Conceptrol-treated mice was significantly higher than in PBS-treated mice at all time points (P < 0.01) and did not change significantly between 8 and 48 hours after treatment (Figure 3A). However, the cellular composition of vaginal leukocytes evolved during this time. The number of neutrophils present in vaginal lavage fluid of Conceptrol-treated mice increased (P < 0.05) between 8 and 48 hours after treatment (Figure 3B). In contrast, the total number of vaginal macrophages began decreasing 24 hours after treatment and was not significantly different from that in PBS-treated controls at 48 hours after treatment (Figure 3C).
Virucidal Activity of Conceptrol In Vivo
To determine if residual Conceptrol was still virucidal at the time macrophages began entering the vagina (4 hours;Figure 2C), mice were treated with Conceptrol either 4 hours before or 20 seconds before intravaginal inoculation of HSV-2. As shown in Table 2, all control-treated mice were infected and demonstrated clinical symptoms following intravaginal inoculation with HSV-2. By contrast, 67% of the mice treated with Conceptrol 20 seconds before virus inoculation were protected against infection (P < 0.001 in comparison with PBS-treated controls). However, mice inoculated with HSV-2 4 hours after treatment with Conceptrol were not protected against infection, and only 7% (1/15 mice) were protected against disease symptoms.
Vaginal Application of Sulfated Polymers
To determine if application of candidate microbicides containing sulfated polymers as active agents would also result in a vaginal inflammatory response, groups of mice were treated with either PRO 2000 or T-PSS. Concentrations and volumes of PRO 2000 and T-PSS used were based on efficacy studies in mice 14 (N. Bourne, submitted for publication). As shown in Table 3, a single treatment of mice with PRO 2000 did not result in increased numbers of vaginal leukocytes and did not alter the composition of the leukocyte population in comparison with PBS or control gel. An elevated vaginal leukocyte count was observed in mice treated with T-PSS in comparison with PBS-treated or control gel–treated mice. However, this increase did not reach significance. Additionally, the composition of vaginal leukocytes did not change in these mice in comparison with PBS-treated controls. The majority of vaginal leukocytes were neutrophils (98%) in T-PSS-treated mice in comparison with Conceptrol-treated mice (45.7% neutrophils, 54.3% macrophages).
Mice were treated daily with PRO 2000 or T-PSS for 1 week to determine if repeated exposure to these agents would result in an inflammatory response. The number of viable leukocytes in PRO 2000 and control gel–treated mice was slightly higher than but not significantly different from that in PBS-treated mice (Table 4). Similarly, a 7-day treatment regimen with T-PSS resulted in an approximately twofold increase in total leukocytes in comparison with PBS-treated mice. Again, this difference was not significant. Similar to the results obtained after a single exposure to sulfated polymers, the leukocyte population of T-PSS- and PRO 2000–treated mice was composed almost entirely of neutrophils, and this mirrored the results obtained following PBS or control gel treatment.
The female genital tract is populated with innate immune cells such as macrophages, neutrophils, and Langerhans cells. In addition, effector T lymphocytes are present in cervical and vaginal tissue, 17–19 and IgG and secretory IgA antibodies can be readily detected in cervicovaginal secretions. 20 The role of the genital immune system is not completely understood but likely includes immune surveillance, maintenance of normal vaginal flora, and protection against sexually transmitted pathogens. The interaction of potential microbicide compounds with the immune cells responsible for innate and acquired immunity in the genital tract needs to be understood to prevent or minimize undesirable consequences resulting from frequent microbicide application. We have developed a murine model in which local inflammatory responses following vaginal application of candidate compounds can be rapidly detected and quantified. This model can be used together with other small animal models of genital infection to screen new compounds for efficacy and safety before preclinical trials with nonhuman primates and clinical trials in humans. The utility of this model is based on the ability to rapidly sample, identify, and quantify changes in the leukocyte population at the vaginal epithelial surface in sufficient numbers of animals to ensure that any detrimental response observed is truly representative within a population.
A naturally occurring population of neutrophils has been observed previously in the vaginal lumen of normal mice 21,22 and was also detected in PBS-treated mice in the current study. This population may be attracted to the vagina by the local production of the murine interleukin-8 homologue protein during the metestrus-2 phase of the murine reproductive cycle. 23 By contrast, macrophages have rarely been detected in the vaginal lumen of normal mice. 21 The use of specific pathogen–free animals with synchronized reproductive cycles in this study allowed the quantification of vaginal cells under defined conditions. It is interesting that some variability was detected in cell counts between individual control-treated animals despite control of these physical and environmental parameters. The reason for this variability in cell number is unclear, but it is important to note that the cellular composition of vaginal leukocytes was remarkably invariable in that neutrophils routinely accounted for greater than 95% of the normal vaginal leukocytes. Variability in the number of resident vaginal immune cells has also been observed in humans and may reflect the presence of subclinical bacterial or viral infection. 19
A single application of the N-9-containing gel Conceptrol resulted in the entry of inflammatory leukocytes into the vagina within 4 hours. A significant proportion of cells were identified as macrophages on the basis of morphologic characteristics and expression of the murine macrophage–specific marker F4/80. The macrophage component of the infiltrate resolved by 48 hours after Conceptrol application. In a rabbit vaginal model, repeated application of N-9 resulted in a similar infiltration of leukocytes into the vaginal submucosa. 24 Cervical biopsy specimens from pigtailed macaques taken after a single application of a gel containing 4% N-9 revealed the presence of an inflammatory infiltrate in the submucosal tissues, comprised predominantly of polymorphonuclear leukocytes. 25 In a human clinical trial, repeated vaginal exposure to N-9 resulted in an infiltration of macrophages and CD8+ T lymphocytes to the superficial aspect of the vaginal lamina propria. 6 The results of the current study with a murine model are similar to those observed following application of N-9 in humans and demonstrate the utility of this model in predicting inflammatory properties of candidate microbicides.
Vaginal application of SDS, another detergent being considered for use in topical microbicides, also resulted in an inflammatory response with a significant macrophage component. In this study, the inflammatory response to SDS was not as prominent as the response to N-9. This finding is consistent with in vitro data suggesting SDS might be tolerated better than N-9 by vaginal keratinocytes. 12 However, the results from the current study demonstrate that SDS also elicits an inflammatory response after vaginal application in mice.
Evidence of efficacy of N-9 against sexually transmitted pathogens has been demonstrated in animal models, 26,27 usually by application just before exposure to the pathogen. In this study, the microbicidal activity of N-9 was lost if applied 4 hours before exposure to HSV-2. It must be noted that the macrophage infiltrate in this study was first detected 4 hours after application, at a time when N-9 was no longer virucidal. Recent studies of vaginal transmission of simian immunodeficiency virus (SIV) have identified dendritic cells, macrophages, and CD4+ T cells as the earliest cell types supporting SIV infection. 28,29 Given the potential for misuse of microbicides, specifically the failure to reapply microbicide before initiation of further coitus, it is possible that the presence of infiltrating macrophages might increase susceptibility to HIV infection by increasing the number of targets for HIV infection during a period when the microbicide is no longer active. In the current study, vaginal application of N-9 did not result in an infiltration of CD3+ T lymphocytes into the vaginal lumen, as determined by differential staining and flow cytometry (data not shown). However, the influence of microbicide application on cells of dendritic lineage was not determined. Given the potential role of these cells in vaginal transmission of HIV, future studies to examine the effect of topical microbicides on vaginal dendritic cell populations are clearly warranted.
Detergents such as N-9 or SDS act by disruption of lipid membranes, whereas the topical microbicides PRO 2000 and T-PSS act primarily by blocking infection of epithelial cells. Previous studies showed that a single application of PRO 2000 was sufficient to provide significant protection against HSV-2 infection in a mouse model. 14 In the current study, no inflammatory infiltrate was detected following vaginal application of the naphthalene sulfate polymer PRO 2000. Although not statistically significant, increased numbers of neutrophils were detected in the genital tracts of T-PSS-treated mice. In contrast to the result of treatment with SDS, N-9, or N-9-containing gels, elevated numbers of macrophages were not detected in the vaginal lumen of T-PSS-treated mice, even after repeated application. Furthermore, entire sheets of vaginal epithelia were detected in vaginal lavage fluid from N-9-treated mice 2 hours after treatment, whereas the cellular constituency of vaginal lavage fluid from PRO 2000–treated mice was not different from that of PBS-treated mice (data not shown). Taken together, these results suggest that vaginal application of sulfated polymers in humans may not result in disruption of vaginal and cervical epithelia or in local inflammation, including infiltration of macrophages.
The presence of genital ulcers has been associated with an increased risk of infection by HIV and other sexually transmitted pathogens. 30,31 The repeated vaginal application of N-9 in humans has been shown to irritate or disrupt vaginal and cervical epithelia, 5,32 induce an inflammatory response, 6 and influence the maintenance of peroxide-producing lactobacilli normally present in the vagina. 9,33 The long-term consequence of these adverse effects on the female genital tract is not well understood. Given the potential for nonspecific tissue damage following release of degradative enzymes and oxygen or nitrogen metabolites by activated inflammatory cells, it will be important to understand the interaction between topical microbicides and the innate immune system and possible modification of this interaction by reproductive hormones and preexisting infections or inflammation. While it may be possible to formulate N-9 in such a way as to reduce its toxic properties, it is also logical to develop effective, noninflammatory microbicides. In this regard, the sulfated polymers T-PSS and PRO 2000 appear superior to surface-active agents such as N-9 or SDS.
In practice, microbicidal compounds would most likely be applied frequently over a period of months or years. Therefore, in addition to providing protection against a broad range of bacterial and viral pathogens, frequent application of these compounds should not result in adverse effects that might ultimately predispose the user to other infections. The murine model employed in this study should prove useful for rapid screening of candidate microbicides for inflammatory effects and therefore aid in prioritizing development of candidate microbicides.
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