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The Journal of Immunology, 1998, 160: 1173-1180.
Copyright © 1998 by The American Association of Immunologists

Migration of Lymphoid Cells from Vaginal Epithelium to Iliac Lymph Nodes in Relation to Vaginal Infection by Herpes Simplex Virus Type 21

Nicholas J. C. King*, Earl L. Parr2,{dagger} and Margaret B. Parr{dagger}

* Department of Pathology, University of Sydney, Sydney, Australia; and {dagger} Department of Anatomy, Southern Illinois University, Carbondale, IL 62901


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether lymphocytes and Langerhans cells in vaginal epithelium are migratory, we stained mouse vaginal epithelium, including its lymphoid cells, by intraluminal administration of H33342, a fluorescent, vital dye. Stromal staining was superficial, and no free dye reached the iliac lymph nodes. The numbers and phenotypes of H33342-stained cells that migrated from the vagina to the iliac lymph nodes during the next 48 h were determined in four groups: normal mice, mice infected intravaginally with wild-type herpes simplex virus type 2 (HSV-2), mice that were immune to vaginal HSV-2 infection, and immune mice that received vaginal challenge with HSV-2. H33342-stained cells migrated from the vaginal epithelium to the iliac lymph nodes in all groups and were mainly Thy-1.2+ cells and B220+ cells. The number of migrating Thy-1.2+ cells was similar to the sum of CD4+ and CD8+ cells in all groups and was not significantly different from the number of CD44+ cells, suggesting that most of the migrating T cells were memory cells. B lymphocytes comprised 31, 32, 43, and 68% of the migrating cells in the four groups, respectively. We found no evidence that Langerhans cells or macrophages were migrating. Thus, most MHC class II+ cells in all groups were accounted for by B cells, and migrating cells did not express B7.1 or F4/80 or exhibit indented nuclei or dendritic processes. We suggest that the migrating T cells and B cells probably belonged to a pool of lymphocytes that recirculates from blood to tissues and back to the lymph nodes via their afferent lymphatics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vaginal infection of mice with attenuated HSV-23 elicits strong immunity against subsequent vaginal infection with wild-type HSV-2 (1, 2, 3, 4). Information about the immune mechanisms that protect the vagina against challenge infection in this convenient animal model should provide insight into the requirements for effective vaccination against viral sexually transmitted diseases in women. Studies in this model using neutralization of virus by secreted Abs, passive transfer of immune IgG, and removal of vaginal secretions from immune mice before challenge have indicated that Abs in the vaginal secretions of immune mice, especially IgG viral Ab, play a major role in protection (5, 6). A potential involvement of cell-mediated immunity in the model was first suggested by the observation that adoptive transfer of lymphocytes from the iliac lymph nodes of immune mice protected naive mice against vaginal challenge (7). Recently, we found that in vivo administration of mAbs to immune mice effectively eliminated T cells within 7 days without any effect on vaginal Ab titers. Twenty-four hours after vaginal challenge with wild-type virus, the T cell-depleted mice had significantly greater infection of the epithelium than did intact immune mice (4). These observations indicate that immune T cells act rapidly to protect the vaginal epithelium against challenge infection, and they emphasize the need for information about the origins and possible migratory behavior of the T cells in the most superficial region of the vaginal mucosa.

Thymus-derived lymphocytes are present in the vaginal epithelium of normal humans, mice, and other species along with other kinds of leukocytes, including neutrophils and Langerhans cells. Information about the phenotypes of the cells and their distribution, TCR repertoire, and functional activity has been obtained by immunostaining of tissue sections (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) and by studies of isolated cells in suspension after enzymatic dissociation of the tissues (19, 20, 21, 22). It is likely that most of the T cells in the vaginal epithelium are derived from the blood, since the normal mouse vagina and most specimens of human vagina have few, if any, primary or secondary lymphoid nodules where local lymphocyte proliferation might occur (18). However, little is known about the fate of T cells after they enter the vaginal epithelium. Do they continue migrating outward into the lumen with the neutrophils, remain in the epithelium in readiness for immune function, or reverse course and exit the vagina via stromal lymphatic vessels? The vaginal Langerhans cells are probably also derived from the blood, and since epidermal Langerhans cells in skin have been shown to migrate to the draining lymph nodes after contact sensitization, it has been suggested that vaginal Langerhans cells may also migrate to the draining lymph nodes (23, 24). A demonstration of this migration would be of interest.

Methods to study lymphoid cell migration in vivo are limited, especially in small animals such as mice and in specific tissue compartments such as vaginal epithelium. Several studies have used injection of a vital dye such as FITC directly into thymus tissue (25, 26), Peyer’s patches (27), or the blood vessels supplying a limited part of the small intestine (28). Such techniques minimize alterations to lymphoid cells that might occur during in vitro labeling, but they are not widely applicable because of anatomical considerations (29). Vital staining of lymphoid cells in vitro with the DNA-binding, fluorescent dye, H33342 (bisbenzimide), for studies of their migration in vivo after i.v. injection was introduced by Brenan and Parish (30) and was used by Austyn et al. (31) to study in vivo migration of splenic dendritic cells. Use of this vital dye for direct staining of lymphoid cells in vivo has not, to our knowledge, been reported previously.

In the present study we stained the mouse vaginal epithelium, including its lymphoid cells, by intraluminal administration of a 15-min pulse of H33342. We then monitored the subsequent migration of fluorescent lymphoid cells to other regions of the vagina and to the iliac lymph nodes, which receive the lymph that drains from the vagina. We present evidence that there is a lively traffic of lymphoid cells from the superficial vaginal mucosa to the deeper stroma and the iliac nodes. We report the numbers and phenotypes of the cells that migrated within 48 h to the lymph nodes in four groups of mice: 1) normal mice, 2) mice that were acutely infected with wild-type HSV-2 intravaginally, 3) mice that were immune to vaginal HSV-2 infection because of prior vaginal immunization with attenuated virus, and 4) immune mice that were challenged by intravaginal (ivag) inoculation of wild-type HSV-2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, hormone treatment, and virus

Female, BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used when they were 60 to 200 days old. They were housed in the Southern Illinois University vivarium and used in compliance with all institutional and federal guidelines. Susceptibility to vaginal HSV-2 infection was induced by s.c. injection of 0.10 µg of estradiol benzoate (Sigma Chemical Co., St. Louis, MO) in peanut oil, followed 1 day later by 2.0 mg of Depo-Provera (Upjohn Co., Kalamazoo, MI; E/DP treatment). Stocks of attenuated HSV-2 (TK-HSV-2) lacking a functional gene for thymidine kinase and wild-type HSV-2 strain 333 (TK+HSV-2) were provided by Dr. Mark McDermott, McMaster University (Hamilton, Canada). Virus infection or challenge was initiated by ivag inoculation of 20 µl of virus, 5 days after treatment with Depo-Provera.

HSV-2 infection and H33342 staining

Four groups of animals were treated as follows before vaginal staining with the fluorescent vital dye, H33342 (bisbenzimide, Calbiochem, La Jolla, CA): a normal group that was treated with E/DP 5 days previously, an infected group that received wild-type virus (4.0 x 107 pfu/ml) ivag 40 h previously, an immune group that received ivag infection with the attenuated virus (2.0 x 106 pfu/ml) 6 wk previously and an additional treatment with E/DP 5 days previously, and an immune/challenged group consisting of immune mice that received ivag inoculation of wild-type virus (4.0 x 107 plaque-forming units/ml) 24 h previously. The rationale for dye inoculation 24 h after virus challenge in immune mice was that histologic studies (2) revealed substantial lymphocyte infiltration into the vagina and that T cell-mediated immune protection against challenge was already evident at this time (4). In contrast, the same histologic studies revealed little or no lymphocyte infiltration into the vagina 24 h after virus inoculation in normal mice. Therefore, to give lymphocytes of normal mice additional time to respond to vaginal infection, we inoculated dye 40 h after virus in the infected group.

The vaginas of these normal, infected, immune and immune/challenged mice were stained by ivag inoculation of 40 µl of H33342 (50 µg/ml in HBSS). Before inoculation of dye the mice were anesthetized with tribromoethanol (Sigma), and after inoculation they were inclined head downward so that the dye remained in place in the vagina for 15 min, followed by two washes with 50 µl of PBS. The mice were killed 48 h later, and the vagina and iliac lymph nodes, which receive lymph from the vagina, were taken for examination. The schedule of virus infection and H33342 staining in the four mouse groups is summarized in Figure 1Go.



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  		FIGURE 1. Schedule of wild-type HSV-2 (TK+HSV-2) infection and H33342 staining in normal, infected, immune, and immune/challenged groups.

 
Lymph node cell preparation and labeling

Animals were killed, and the iliac lymph nodes were removed. Cells were isolated from the nodes by gently forcing them through a 70-µm cell strainer (Falcon, no. 2350 Becton Dickinson, Lincoln Park, NJ) into 3.0 ml of HBSS containing 2% FBS. Cells from each mouse were counted in a hemocytometer to determine the total numbers isolated on each occasion. They were washed by a single centrifugation at 200 x g for 5 min and divided into five samples. Each sample was incubated in a primary rat Ab against one of a panel of mouse leukocyte surface markers at 0°C for 60 min. Cells in each sample were then washed by a single centrifugation through a bed of FBS and resuspended in FITC-conjugated secondary Ab at 0°C for an additional 60 min. At the end of this time, cells were washed once through FBS and resuspended in 50 µl of HBSS for counting. Cells were placed in a hemocytometer and examined in an Olympus fluorescence microscope (Olympus Corp., Lake Purchase, NY) equipped with filters for examination of H33342 and FITC fluorescence without spectral overlap. The numbers of blue fluorescent cells and double-stained cells were counted in the same fields of view. Between three and six sets of counts were made for each leukocyte marker, and each set came from a different mouse. Data were averaged, and an unpaired analysis of variance (ANOVA) was used to compare data from three or four mouse groups. A two-tailed unpaired t test was used to determine statistical significance between pairs of groups.

Preparation of vaginas and lymph nodes

All vaginas used in the present study were fixed in 2% paraformaldehyde in PBS for 2 h. Lymph nodes that were sectioned for study of the localization of H33342 within the node were similarly fixed. The fixed tissues were washed twice in 10% sucrose in PBS over 16 h before freezing and cryosectioning. Sections were examined with an Olympus fluorescence microscope as described above.

Antibodies

To identify cells migrating to the lymph nodes from the vaginal mucosa, Abs to the following leukocyte surface markers were used at 10 µg/ml: Thy-1.2 (clone 30-H.12, Boehringer Mannheim, Indianapolis, IN), B220 (clone RA3-6B2, Boehringer Mannheim), CD8 (clone 53-6.7, Becton Dickinson, Mountain View, CA), CD4 (clone GK-1.5, Becton Dickinson), MHC class II (clone M5-114, Boehringer Mannheim), CD44 (clone IM7, PharMingen, San Diego, CA), F4/80 (Serotec, Indianapolis, IN), CD80/B7.1 (clone 1G10, PharMingen), and LGL-1 (clone 4D11, Dr. Llewellyn Mason, National Institutes of Health, Bethesda, MD). In addition, IgG2a and IgG2b control primary Abs, isotypic with the specific primary Abs, were used at the same concentrations to assess nonspecific labeling. The secondary Ab was affinity-purified, FITC-conjugated mouse F(ab')2 anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Staining of vaginal mucosa

The dose and time for in vivo staining of the superficial vaginal mucosa with H33342 were determined in preliminary experiments. The protocol that was finally adopted for these studies was ivag inoculation of 40 µl of H33342 at 50 µg/ml, followed by washout 15 min later. Figure 2Go shows fluorescent staining of the normal vagina by this method. The vaginal epithelium shows bright, full thickness staining everywhere except in a few crypts. The vaginal stroma shows faint staining of a few fibroblast nuclei immediately beneath the epithelium and bright staining of a few cells deeper in the stroma that were surrounded by unstained cells. Double staining revealed that the brightly stained cells surrounded by unstained cells in the stroma were usually CD45+, suggesting that they were lymphoid cells that were stained in the epithelium before they migrated to the stroma during the subsequent 48 h. This interpretation is consistent with the fact that such cells were not observed in the stroma when vaginas were examined 1 h after H33342 staining.



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  		FIGURE 2. Fluorescent staining of normal mouse vagina 48 h after intraluminal inoculation of 40 µl of H33342 at 50 µg/ml for 15 min. The vaginal epithelium consists of a superficial layer of mucus-secreting cells overlying several layers of smaller basal cells. It shows bright, full thickness staining everywhere except in a few crypts. Staining of the stroma is limited to a few regions adjacent to the epithelium and is less bright (large arrow). A few brightly stained cells (small arrows) are present deep in the stroma surrounded by unstained cells. L, vaginal lumen. Magnification, x87.

 
The staining pattern shown in Figure 2Go is representative of that seen in 14 normal mice, six immune mice, and six immune/challenged mice that were killed 48 h after H33342 staining in the vagina. Staining of these groups was therefore reproducible, confined to the superficial mucosa, and mainly limited to the epithelial layer. Normal mice that were infected with HSV-2 and stained with H33342 ivag 40 h later had substantial regions of mucosa that lacked epithelium 48 h later due to sloughing of infected cells. Where epithelium remained, it was typically stained. The stroma showed negligible staining everywhere. This is consistent with previous observations (2) that infected vaginal epithelium, as demonstrated by antiviral staining, was still intact 40 h after HSV-2 infection but was lost in some areas during the next 48 h. It is therefore likely that the great majority of the stained cells that migrated from the vagina to the iliac lymph nodes in all four groups, as described below, came from the epithelium.

In addition, localization of H33342 staining in the vagina was the same regardless of whether the mice were killed 1, 24, 48, or 72 h after 15-min exposure to dye. Thus, the initial localization of H33342 was well retained in vivo for at least 72 h. Moreover, this localization of staining was retained during prolonged storage of frozen tissues or dry tissue sections (30 days). However, the dye diffused into surrounding regions within 24 h after sections were mounted with coverslips. Thus, all observations of H33342 in tissue sections were performed immediately after coverslips were mounted.

Migration of stained cells from superficial vaginal mucosa to iliac lymph nodes and axillary lymph nodes

Sections of iliac lymph nodes from normal mice showed a few blue fluorescent nuclei in T cell areas of the node 24, 48, and 72 h after ivag inoculation of H33342, with the maximum number present at 48 h. We observed a few H33342-stained cells in the subcapsular sinuses, consistent with the assumption that the migrating cells reached the lymph nodes via their afferent lymphatics.

No stained cells were detected in lymph node sections from mice that did not receive H33342 ivag, and no direct staining of iliac lymph node cells was observed in the region of the subcapsular sinuses due to free dye in the afferent lymphatics until the concentration of H33342 inoculated into the vagina was increased at least 10-fold. Figure 3Goa shows an iliac lymph node cell suspension from a normal mouse that received H33342 ivag 48 h earlier. The cells are in a hemocytometer and are simultaneously illuminated from above with 350 nm light to excite H33342 fluorescence (cell at arrow) and from below with a tungsten lamp to show the nonfluorescent cells in the background. No blue fluorescent cells were observed in iliac lymph node cell suspensions unless the mice received H33342 ivag. We conclude that some lymphoid cells that became stained while in the H33342-stained superficial vaginal mucosa later migrated to the iliac lymph nodes during the following 48 h. Examination of axillary lymph nodes from three mice revealed that small numbers of H33342-stained cells (~0.02% of the total lymph node cells) were present in these nodes as well.



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  		FIGURE 3. Iliac lymph node cell suspension from a mouse that received H33342 ivag 48 h earlier. The cell suspension was also stained with FITC to detect Thy-1.2. a shows an H33342-stained cell (arrow) and unstained cells in the background. The stained nucleus is similar in size and shape to the nuclei of small lymphocytes. b shows FITC-stained cells and unstained cells in the background. Comparison of the two micrographs shows that the H33342-stained (migrating) cell is Thy-1.2+ (arrow). Magnification for both aand b, x325.

 
Number and phenotype of cells migrating from vagina to iliac lymph nodes

Cell suspensions from the iliac lymph nodes of mice that received H33342 ivag 48 h earlier were stained with rat mAbs to mouse leukocyte markers, followed by FITC-conjugated secondary Ab. The cell suspensions were placed in a hemocytometer and viewed by fluorescence microscopy, as shown in Figure 3Go. Total numbers of lymph node cells as well as total H33342-stained cells were counted with illumination as in Figure 3Goa, after which the percentage of H33342-stained cells that exhibited surface membrane FITC staining of the leukocyte marker was determined by comparing the blue and green fluorescent cells (Fig. 3Go, a and b). Attempts to obtain this quantitation by flow cytometry were not consistently successful, probably due to the low frequency (0.2–0.5%) of H33342-positive cells in the lymph node suspensions.

Four groups of mice were prepared as described in Materials and Methods, i.e., normal, infected, immune, and immune/challenged. Figure 4Go shows the total number of cells and the number of H33342-stained cells in iliac lymph nodes in each group. The number of lymph node cells increased markedly after ivag inoculation of HSV-2. Thus, infected and immune/challenged groups showed 5-fold (p < 0.0001) and 2-fold (p = 0.0105) increases compared with normal and immune groups, respectively. This is consistent with the increased size of the lymph nodes in the infected and immune/challenged groups, as observed grossly upon dissection. Similarly, the numbers of H33342-stained cells migrating from the superficial vaginal mucosa to the iliac lymph nodes increased markedly after HSV-2 inoculation. Thus, the infected group showed an increase of approximately 5-fold (p < 0.0001) compared with the normal group, while the immune/challenged group showed an increase of approximately 2-fold (p = 0.0047) compared with the immune group. Interestingly, the number of migrating cells was 3.5-fold greater (p = 0.0027) in the immune group than in the normal group, i.e., in the absence of virus inoculation. The percentage of H33342-stained cells in the lymph nodes was similar in normal and infected mice, as was the percentage of such cells in immune and immune/challenged mice. However, the values in the latter two groups were approximately 2.5-fold higher than those in the former groups.



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  		FIGURE 4. The total number of iliac lymph node cells and the number of H33342-stained (migrating) cells in the iliac nodes were counted in each mouse. The numbers of mice in the four experimental groups were: 13 (normal), six (infected), seven (immune), and six (immune/challenged).

 
Lymph node cells in each group were labeled for selected leukocyte surface markers. Isotype control Ab labeling was <0.1% in all cases. Figure 5Go, a and b, shows the results for migrating T cells (Thy-1.2) and B cells (B220), respectively. The percentage of the migrating cell population that expressed Thy-1.2 was similar in normal and acutely infected mice. This was true also for B220-positive cells. However, in immune mice the percentage of migrating cells that expressed Thy-1.2 was reduced, while the percentage that expressed B220 was increased from 32 to 43%. When immune mice were challenged with virus, the percentage of Thy-1.2+ cells in the migrating population was unchanged, but the percentage of B220+ cells was further increased from 43 to 68% (p = 0.0183). Thy-1.2+ cells and B220+ cells together accounted for 74% of the migrating cells in normal mice and for 99% of the migrating cells in immune/challenged mice.



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  		FIGURE 5. The percentage of H33342-stained (migrating) cells that expressed Thy-1.2 and the number of Thy-1.2+ migrating cells in iliac lymph nodes of the four mouse groups (a) and similar data for B220 (b). The data represent the means of three to six mice per group ± SEM. The percentage of migrating cells that expressed Thy-1.2 was significantly decreased in immune mice compared with those in normal and infected mice (p = 0.047, by three-group ANOVA; p = 0.011, by two-tailed t test; immune vs normal). The percentage that expressed B220 was correspondingly increased, although this change was not statistically significant. The percentage of migrating cells that expressed B220 was further increased from 42% in immune mice to 63% in immune/challenged mice (p = 0.0183, by two-tailed t test). The numbers of migrating T and B cells in the four experimental groups were significantly different (Thy-1.2, p = 0.014; B220, p < 0.0001; by four-group ANOVAs). The numbers of migrating T and B cells were relatively small in normal mice, increased in infected mice (Thy-1.2, p = 0.013; B220, p < 0.0001; by two-tailed t tests), reduced in immune mice compared with those in infected mice but not to the levels in normal mice (Thy-1.2, p = 0.090; B220, p = 0.024; by two-tailed t tests), and increased in immune/challenged mice compared with those in immune mice (Thy-1.2, p = 0.11; B220, p = 0.0035; by two-tailed t tests). While not all differences were statistically significant, the trend of the data is clear.

 
The numbers of migrating T cells and B cells were markedly increased in the infected group compared with those in the normal group (Fig. 5Go, a and b). These numbers were reduced in immune mice, but not back to the levels in normal mice, and then increased markedly after immune mice were challenged ivag with virus (immune/challenged group). In particular, the number of migrating B cells in immune/challenged mice was about 4-fold greater than that in immune mice and was >21-fold greater than that in normal mice.

The large increase in the number of B cells migrating from the superficial vaginal mucosa to the iliac lymph nodes in immune/challenged mice compared with that in normal mice suggested that the number of B cells in the vaginal epithelium of these mice might also be increased. This was confirmed by counting the number of B220-positive cells per unit length of vaginal epithelium, which increased from 0.27 ± 0.04 in normal mice to 4.0 ± 1.0 (p < 0.001) in immune/challenged mice, a 15-fold increase (Fig. 6Go).



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  		FIGURE 6. Histologic section of vagina from immune/challenged group, showing B220+ cells in the epithelium (arrow). L, vaginal lumen. Arrowheads indicate the epithelial basement membrane. Magnification, x350.

 
The percentage of migrating cells that expressed CD4 or CD8 was variable, but the means of the four experimental groups were not significantly different in either case (Fig. 7Go, a and b). However, the numbers of migrating CD4+ and CD8+ cells differed significantly among the groups, being increased in the infected group compared with those in the normal group, reduced in immune mice compared with those in infected mice, and increased in the immune/challenged group compared with those in the immune group. These are the same changes that were observed for total, Thy-1.2+, and B220+ cells in Figure 4Go and in Figure 5Go, a and b, respectively. The small excess of Thy-1.2+ cells compared with the sum of CD4+ and CD8+ cells was probably not due to Thy-1.2-bearing NK cells, since Ab to LGL-1 stained <1% of the total lymph node cells in all groups and stained none of the migrating cells.



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  		FIGURE 7. The percentage of H33342-stained (migrating) cells that expressed CD4 and the number of CD4+ migrating cells in iliac lymph nodes of the four mouse groups (a) and similar data for CD8 (b). The data represent the means of three to six mice per group ± SEM. The percentage of migrating cells that expressed either CD4 or CD8 was variable, but there were no statistically significant differences among the groups in either case (CD4, p = 0.36; CD8, p = 0.084; by four-group ANOVA). The numbers of migrating CD4+ and CD8+ cells, on the other hand, differed significantly among the groups (CD4, p = 0.0008; CD8, p = 0.045; by four-group ANOVAs). The numbers of migrating cells were relatively small in normal mice, increased in infected mice (CD4, p = 0.0002; CD8 p = 0.14; by two-tailed t tests), reduced in immune mice compared with infected mice (CD4, p = 0.011; CD8, p = 0.059; by two-tailed t tests), and increased in immune/challenged mice compared with those in immune mice (CD4, p = 0.31; CD8, p = 0.019; by two-tailed t tests). While not all differences were statistically significant, the trend of the data is clear and is the same as those observed in Figures 4Go and 5Go.

 
The activation status of the vaginal cells migrating to the iliac lymph nodes was assessed by studying the expression of the homing adhesion molecule, CD44. The percentage of migrating cells that expressed CD44 in sufficient quantities to be detectable by fluorescence microscopy was reasonably similar in all groups to the percentage of migrating cells that expressed Thy-1.2, and to the sum of CD4+ and CD8+ T cell populations (Fig. 8Go). Since a CD44high phenotype has been reported to be a marker of mouse memory T cells, these data suggest that many, if not most, of the T cells that migrated from the vagina to the lymph nodes in all groups were memory cells.



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  		FIGURE 8. A comparison of the percentage of H33342-stained (migrating) cells that expressed CD44 to the percentage of migrating cells that expressed Thy-1.2. Also shown is the sum of the cell percentages that expressed either CD4 or CD8.

 
The possibility that Langerhans cells migrate from the vaginal epithelium to the iliac lymph nodes is of considerable interest. We therefore wished to examine the incidence of migrating cells expressing MHC class II. The results indicated that the percentages of MHC-II+ cells were not significantly different from those of B220+ cells in all groups. Since B cells are also MHC class II+, and most of the migrating MHC class II+ cells in all groups could be accounted for by B cells, there was no indication that large numbers of MHC class II+ Langerhans cells or macrophages were migrating. It was therefore of importance to determine whether other possible markers of Langerhans cells, such as B7.1 and F4/80, were detectable in the migrating cell population. We were unable to detect either of these Ags on any of the migrating cells. In addition, the nuclear morphology of migrating cells was always apparent because of H33342 staining, and in all cases we observed the spherical nuclei of small or medium lymphocytes rather than the indented nuclei of Langerhans cells or macrophages. Lastly, no dendritic processes were observed in the surface membranes of MHC class II+ migrating cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To study the migratory behavior of leukocytes in the mouse vaginal epithelium, we stained the epithelial layer and a few cells in the most superficial aspect of the underlying stroma by intraluminal application of a 15-min pulse of the fluorescent vital stain, H33342 (bisbenzimide). We then observed 24 to 72 h later that stained lymphoid cells were present in the T cell areas of the iliac lymph nodes and in the subcapsular sinuses that receive the afferent lymph. These results directly indicate that lymphoid cells migrate from the vaginal epithelium and/or closely adjacent stroma to the iliac lymph nodes in mice.

While we have no direct evidence that the migrating lymphocytes entered the vagina from the blood, it seems unlikely that they originated locally in the vagina as a result of proliferation there because no lymphoid nodules that could be sites of local lymphocyte proliferation were present in the vaginal mucosa (18), and the migrating cells were almost always small lymphocytes rather than blast cells. Therefore, we suggest that many of the lymphocytes in the vaginal epithelium belong to a recirculating pool of cells that enters the vaginal stroma from the blood, crosses the basement membrane to enter the vaginal epithelium, then returns to the stroma and enters lymphatic vessels that transport them to the iliac nodes and ultimately back to the blood. This suggestion is consistent with the conclusion that lymphocytes recirculate through the small intestine in sheep, where the number of lymphocytes in the lymph leaving the intestine declines rapidly if those cells are not returned to the blood (32). Our observations leave open the possibility that other vaginal leukocytes are sessile cells.

Perhaps the most interesting feature of the observed lymphoid cell migration out of the vaginal mucosa was the large number of migrating B cells. A previous study of lymphocytes isolated from the vagina of normal mice failed to detect B cells and concluded that B cells play little, if any, role in the immune defense of the vagina (21). We agree that the number of B cells in the vaginal epithelium of normal mice is not large, but in the present study we were able to detect these cells by immunostaining of vaginal sections. Moreover, among the migrating cells, B cells accounted for 31 ± 9% of the cells migrating to the iliac nodes in normal mice, 32 ± 4% in infected mice, 43 ± 6% in immune mice, and 68 ± 1% in immune/challenged mice. The number of migrating B cells in immune/challenged mice was 21-fold greater than that in normal mice, and this was correlated with a 15-fold increase in the number of B cells per millimeter of vaginal epithelium in this group. These numbers suggest that B lymphocytes may indeed play a significant role in vaginal immunity. In primary immunity they may be the precursors of cells that produce the protective Abs seen in immune mice (5), while in secondary immunity they may recognize challenge Ag in the vaginal epithelium where it first appears and present it to memory T cells (33) either locally or in the draining lymph nodes.

The failure to detect Langerhans cells in the migrating population was surprising. The mouse vaginal epithelium contains a large population of Langerhans cells (11, 16). Extensive studies of skin have shown that epidermal Langerhans cells migrate into lymphatic vessels, where they are called veiled cells, and then into the paracortical areas of the draining lymph nodes, where they are recognized as dendritic cells (34, 35, 36, 37, 38, 39, 40, 41). Since the vagina is histologically similar to the skin, it has been assumed that vaginal Langerhans cells would also migrate (23, 24). It also seemed likely that migration of vaginal Langerhans cells to the iliac nodes might be increased by HSV-2 in both normal and immune mice, since the number of migrating dendritic cells in draining lymph nodes was markedly increased by skin painting with contact Ags (42, 43). Dendritic cells are readily released from lymph nodes by mechanical dissociation (39, 40, 42), and since we used this method to dissociate the iliac nodes in the present study, it seems unlikely that the failure to detect migrating Langerhans cells was due to failure to recover dendritic cells from the nodes. Both vaginal Langerhans cells and lymph node dendritic cells express MHC class II (16, 44), making this marker a good choice for detection of migrating Langerhans cells. Our observation that most MHC class II+ migrating cells were B cells indicates that at most only a very small proportion of the migrating population could have been Langerhans cells. The presence of small numbers of Langerhans cells in the migrating population might not have been detected by the other markers used in our study. It has not been established that B7.1 is expressed on vaginal Langerhans cells, and moreover, it might be down-regulated by virus infection or, as has been reported for F4/80 (45), during migration of the cells to lymph nodes. Unfortunately, we have not consistently been able to label vaginal Langerhans cells with NLDC-145 (46). We examined the iliac lymph nodes at only one time point, 48 h after H33342 staining, which may not be optimum for detection of migrating Langerhans cells in our model, but in the case of skin painting with contact-sensitizing Ag, the number of dendritic cells in the draining lymph nodes was maximal 48 h after skin painting (47). Although further studies of the migratory behavior of vaginal Langerhans cells are needed, our failure to detect migration to draining lymph nodes in the present study may suggest that they mainly present Ag to T cells in situ in the vaginal epithelium, as previously suggested by the observation that Langerhans cells and T cells in the human cervical and vaginal epithelium were often in direct contact (reviewed in 16 .

The number of lymphocytes migrating from the vagina to the iliac lymph nodes in infected mice was 5-fold higher than that in normal mice. Similarly, the number of migrating lymphocytes in immune/challenged mice was 2-fold higher than that in immune mice. In the former case the vagina is certainly inflamed, and in the latter case it is probably inflamed. The observations are thus consistent with the suggestion that lymphocyte migration through tissues is increased by inflammation (48, 49, 50). Perhaps more interesting is the finding that the number of migrating lymphocytes in immune mice, where vaginal infection with attenuated virus occurred 6 wk earlier, was 3.5-fold higher than that in normal mice. We have previously shown that by 7 days after inoculation of attenuated HSV-2 into the vagina the virus can no longer be detected by immunostaining, the vaginal epithelium is healed, and the tissue resumes a histologically normal appearance with no indication of ongoing inflammation (2). It would be of considerable interest to know how long lymphocyte migration remains increased in immune mice because a lingering up-regulation of lymphocyte traffic through a mucosal tissue long after a local infection has been resolved would be consistent with the observation that optimum immune protection at a mucosal surface requires local immunization at that surface (51, 52, 53).

There is cogent evidence that memory T cells are present in the vaginal mucosa of immune mice because MHC class II expression on vaginal epithelial cells was up-regulated more rapidly when immune mice were challenged ivag with virus than when normal mice were infected (2). This is presumably due to rapidly increased synthesis and release of IFN-{gamma} by memory T cells in or near the site where challenge Ag is encountered in the epithelium. Memory T cells in mice have been reported to express the homing adhesion molecule, CD44 (54, 55, 56, 57, 58, 59, 60, 61, 62). In particular, T cell memory is associated with a CD44high population of cells, as defined by flow cytometry, whereas several other cell types are present in a CD44low population. A recent study of cells isolated from the combined uteri and vaginas of mice found that most of the Thy-1.2+, CD4+ cells were CD44high (20). In the present study we observed CD44-stained cells by fluorescence microscopy among the migrating population in all four mouse groups. We assume that these cells correspond to the CD44high cells distinguished by flow cytometry and that the cells without detectable staining by microscopy correspond to the CD44low and negative cells. However, it should be recognized that the distinction between detectable and undetectable by fluorescence microscopy may not correspond precisely to the separation between the CD44high and CD44low/negative populations by flow cytometry. Interestingly, the percentage of migrating cells that expressed detectable CD44 was reasonably similar in all groups to the percentage that expressed Thy-1.2 and also to the sum of the CD4+ and CD8+ percentages. Our data therefore suggest that the T cells that migrated through the superficial vaginal mucosa in all four groups were predominantly of the memory cell phenotype, expressing high levels of CD44. These data are consistent with previous studies in sheep that indicated that most of the T cells that migrated through hindlimb tissues and reached the popliteal lymph nodes via their afferent lymphatics were memory T cells (48, 63, 64). Moreover, migration of memory T cells through the superficial vaginal mucosa is likely to be of functional importance, since recent studies have shown that in vivo depletion of CD8+ T cells from immune mice increased challenge infection of the vaginal epithelium by HSV-2 (4). The role of recirculating T cells in vaginal mucosal immunity and the possibility that such recirculation may be enhanced by local vaginal immunization are questions that merit further attention.


   Footnotes
 
1 This work was supported in part by National Institute of Child Health and Human Development Grant 17337. Back

2 Address correspondence and reprint requests to Dr. Earl L. Parr, Department of Anatomy, Southern Illinois University, Carbondale, IL 62901. Back

3 Abbreviations used in this paper: HSV-2, herpes simplex virus type 2; ivag, intravaginal; E/DP treatment, 0.10 µg of estradiol benzoate in peanut oil, followed 1 day later by 2.0 mg of Depo-Provera; ANOVA, analysis of variance; high, high level; low, low level. Back

Received for publication March 25, 1997. Accepted for publication October 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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