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Recurrent vulvovaginal candidiasis (RVVC) is a significant problem in otherwise healthy women of childbearing age (24, 42, 43). Since no exogenous predisposing factors such as pregnancy, use of oral contraceptives or antibiotics, or diabetes mellitus are known to influence the incidence of RVVC, it has been postulated that some form of immune deficiency or dysfunction is responsible for recurrent episodes (more than three per year) of vaginitis (16, 42, 45). Candida albicans, a commensal organism of the intestinal and reproductive tracts, is the causative agent in most cases of RVVC (42). Since cell-mediated immunity (CMI), through the function of T cells and cytokines and specifically through a Th1-type response, is the predominant host defense mechanisms against C. albicans infections of other mucosal tissues (5, 31, 40, 41), we have been examining CMI-type host defense mechanisms against C. albicans at the vaginal mucosa. Our studies have been both clinical, using women with RVVC (9, 11), and experimental, using an estrogen-dependent murine model of vaginal candidiasis (8, 10, 12-15). To date, our studies suggest that Candida-specific Th1-type CMI in women with RVVC, as well as that induced in the peripheral blood and/or secondary lymphoid tissues (i.e., lymph nodes) of mice as a result of vaginal exposure to C. albicans, does not provide protection against vaginal candidiasis (10, 11, 13-15). On the basis of these observations, we postulated that CMI induced at the vaginal site is important for protection against vaginitis (16). Studies were therefore initiated to examine the presence and phenotype of T cells at the vaginal mucosa of naive mice. Interestingly, we and others have found that vaginal T cells are phenotypically distinct from T cells in the periphery (18-20, 30), supporting the concept of immunological independence or compartmentalization at the vaginal mucosa. Specifically, we have noted in vaginal tissue a higher percentage of T-cell receptor (TCR)-positive cells, low or undetectable levels of CD8⁺ cells, and an atypical expression of the CD4 protein on CD4⁺ T cells under nondenaturing conditions (18). The latter was shown by the inability of GK 1.5 but not 2B6 anti-CD4 antibodies to recognize vaginal CD4⁺ cells when detected by flow cytometry following dual staining with the two antibodies on lymph node cells tested separately or when added to the vaginal-cell preparation. However, under denaturing conditions, as shown by immunohistochemistry, vaginal cells could be recognized by GK 1.5 anti-CD4 antibodies, providing evidence that the vaginal CD4 protein was conformationally distinct from that found on systemic CD4⁺ cells (46). The atypical expression of the CD4 protein on vaginal T cells was extended as well to the mRNA level. Despite the presence of sufficient numbers of vaginal CD4⁺ T cells, vaginal CD4 mRNA could be detected consistently only by using a high-efficiency Taq DNA polymerase in reverse transcription-PCR (RT-PCR) and the CD4 mRNA was absent in a purified population of vaginal cells that atypically expressed the CD4 protein. From these data, we postulated that vaginal CD4⁺ cells express a unique CD4 mRNA and that any detectable CD4 mRNA in such reactions represented low-level systemic cell contamination within the vaginal mucosa (46). Thus, vaginal and systemically derived CD4⁺ T cells can be distinguished at both the protein and molecular levels, providing the means to identify and study each within the vagina under various experimental conditions.

Polymorphonuclear leukocytes (PMN) are an important innate host defense mechanism against C. albicans in the systemic circulation (31, 47) and have significant anti-Candida activity in vitro (7, 29). PMN are often observed in the vagina during an experimental infection in mice, but their presence does not seem to correlate with a reduction in the fungal titers in the vaginas of infected animals, calling into question their role in host defense against C. albicans at that site.

The purpose of this study was to evaluate changes in murine vaginal T-cell populations as well as the effects of the depletion of PMN on primary infection in the presence or absence of pseudoestrus. T-cell populations were also assessed following secondary vaginal challenge where partial protection occurs (10).

Analysis of vaginal T cells during a primary C. albicans vaginal infection. Untreated CBA/J mice (H-2^k, 8 to 10 weeks of age; purchased from the National Cancer Institute, Frederick, Md.) or mice treated with 0.02 mg of estradiol valerate dissolved in sesame oil to induce a state of pseudoestrus were inoculated intravaginally with C. albicans 3153A (5 × 10⁴ blastoconidia) (12, 17). Controls included estrogen-treated animals given phosphate-buffered saline intravaginally. Over a period of 5 weeks, groups of 10 to 15 animals were assessed for their vaginal fungal burden by quantitative culture of vaginal lavage fluid (12), and extracted vaginal lymphocytes (enzymatic digestion) (15) and whole tissue were assessed for T-cell phenotypes by flow cytometry (18), immunohistochemistry (46), or RT-PCR (46). In three separate experiments, the vaginal fungal burden in mice infected in the presence or absence of pseudoestrus was similar to that observed previously (13); i.e., the mice were persistently infected (>5 weeks) with high fungal titers (10⁴ to 10⁵ CFU) under pseudoestrus conditions, while short-lived (<3 weeks) infections with lower fungal titers (10¹ to 10⁴ CFU) occurred in the absence of pseudoestrus. C. albicans was not detected in estrogen-treated uninfected mice (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 1.   RT-PCR of T-cell surface marker mRNA expression during primary C. albicans vaginal infection. Total RNA extracted from vaginal tissue or lymph nodes (three mice per group) was subjected to RT-PCR with primers specific for CD3, CD4, CD8, TCR- constant region, and TCR- constant region of systemically derived T cells, using normal or HE Taq DNA polymerase. GAPDH served as the housekeeping gene. (A) RT-PCR of T-cell surface marker mRNA expression in lymph nodes and vaginal tissue of naive mice. (B) Semiquantitative RT-PCR of CD4, TCR-, TCR-, and CD3 during a primary vaginal C. albicans infection. Results over time are expressed as the ratio of each product in pixel intensities to that for GAPDH. Est., estrogen-treated uninfected mice; Est./Inf., estrogen-treated infected mice; Inf., non-estrogen-treated infected mice; beta (b), -chain; delta (d), -chain. The experiment was repeated twice, and the results shown are representative.

Analysis of vaginal T cells during a secondary C. albicans vaginal infection. A similar analysis of T-cell expression was conducted during a secondary Candida vaginal infection, where partial protection has been observed previously (10). For this, animals were inoculated with 5 × 10⁵ stationary-phase C. albicans blastoconidia in the absence of estrogen. After 4 weeks (following spontaneous resolution of the primary infection), the animals were treated with estrogen as above, and 72 h later, they were inoculated a second time with 5 × 10⁴ C. albicans blastoconidia. In two experiments, a significant reduction in the vaginal fungal burden was observed on both days 4 and 10 after secondary inoculation compared to that in estrogen-treated mice given a primary infection (P < 0.002) (data not shown), consistent with the partial protection reported previously (10). Flow cytometric analysis on extracted vaginal lymphocytes (Table 2), however, showed no changes in the various T-cell subsets between mice with primary and secondary infections through 10 days, including the atypical expression of the CD4 cells as assessed by the two epitope-distinct anti-CD4 antibodies. Similarly, no differences were observed in vaginal CD3, CD4, TCR-, and TCR- cells between mice with primary and secondary infections as assayed by immunohistochemistry or RT-PCR of tissue-derived mRNA (data not shown). Thus, if the local T-cell compartment is responsible for the partial protection against a second vaginal infection, it is doing so without significant changes in cell numbers or phenotype. Furthermore, there was no evidence for systemic cell infiltration in animals with secondary infections based on the lack of increases in GK 1.5⁺ CD4⁺ cells as detected by flow cytometry or assessed by amplification products for CD4 with regular Taq DNA polymerase.

Effect of PMN on C. albicans vaginal infection. A second objective of the study was to assess the role of PMN during a vaginal C. albicans infection. This is an important issue because PMN are often observed in vaginal lavage fluid of infected animals and systemically-derived PMN have considerable anti-Candida activity in vitro (3, 8). However, the presence of PMN is erratic during an experimental vaginal infection and rarely correlates with a reduced vaginal fungal burden. Furthermore, PMN are not normally observed in vaginal smears (KOH) from women with vaginitis (43a). Recently, Black et al. reported a lack of effect of PMN depletion on vaginitis in the presence of estrogen (2). Their interpretation was that PMN may be important in anti-Candida vaginal host defense but that estrogen inhibits the ability of PMN to be deployed into the lumen in large enough numbers to affect C. albicans. To test this hypothesis, antibodies to PMN (anti-Ly-6G antibodies; PharMingen) (100 µg) or isotype control antibodies (rat immunoglobulin G; Zymed Laboratories, San Francisco, Calif.) were injected intraperitoneally (2) 1 day before and 3 days after vaginal inoculation in the presence or absence of pseudoestrus. The vaginal fungal burden in such animals, measured 5 days postinoculation, showed in two experiments that depletion or reduction of PMN (<1% in spleen preparations compared to 8 to 10% in isotype control antibody-treated mice, as detected by Wright's stain) had no effect on the vaginal fungal burden in the presence (1.7 × 10⁴ ± 6.1 × 10³ CFU for anti-PMN-treated mice and 6.4 × 10⁴ ± 2.3 × 10⁴ CFU for isotype control antibody-treated mice) or absence (2.1 × 10³ ± 1.7 × 10³ CFU for anti-PMN-treated mice and 4.4 × 10³ ± 4.6 × 10³ CFU for isotype antibody-treated mice) of pseudoestrus. Thus, despite their presence, PMN do not appear to play a role in host defense against vaginitis, irrespective of the state of estrus. In light of these results, we postulate that the erratic presence of PMN during an infection in the presence or absence of pseudoestrus is due to the normal deployment of PMN during the 2-day diestrus phase of the mouse 4-day menstrual cycle rather than in response to C. albicans. If so, it would not appear that a state of pseudoestrus inhibits this process. Recently, it was reported that the chemokine macrophage inflammatory protein 2 (MIP-2) is involved in the recruitment of PMN during diestrus (44). In support of this, we have observed a similar erratic presence of PMN in lavage fluid of estrogen-treated uninfected mice together with the presence of MIP-2 in the vaginal tissue (unpublished observations).