In Situ Analysis of the Evolution of the Primary Immune Response in Murine Chlamydia trachomatis Genital Tract Infection

RESULTS

In situ analysis of lineage-specific cells in genital tract tissue during the course of C. trachomatis infection.At various times during the course of chlamydial genital tract infection, animals were sacrificed and the entire genital tract (vagina, uterine horns, and oviducts) was removed and analyzed by immunohistochemistry for cell surface markers that define specific cell lineages. Cells of the myeloid lineage express cell surface molecules recognized by anti-CD11b, a phenotype shared by macrophages/monocytes and polymorphonuclear neutrophils (PMN) (21). Anti-Ly6G reacts with a surface molecule on mature granulocytes (8). Anti-CD3e and anti-CD45R (B220) were used to identify T cells and B cells, respectively.

The course of chlamydial genital tract infection is shown in Fig.1. Tissues were harvested from infected mice at various times following infection and correspond to different stages of infection. For example, tissues harvested at days 3 and 7 represent a time when infection is progressing and specific immune responses are developing (22), whereas tissues harvested at days 21 and 28 are representative of a resolving infection and those harvested at days 42 and 70 are representative of a resolved infection.

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Fig. 1.

Time course of chlamydial genital tract infection. Mice were infected with 100 50% infectious doses of C. trachomatis strain MoPn. The course of infection was monitored by swabbing the vaginal vault at selected times and enumerating IFU by isolation onto HeLa cell monolayers. Inclusions were visualized by indirect immunofluorescence using monoclonal antibody Mo-33b and fluorescein-labeled goat anti-mouse IgG. Data are presented as mean IFU ± standard error of the mean for nine mice.

The cellular profiles of vaginal tissues from control mice and from mice on day 3 of infection are shown in Fig.2. Cells with the Ly6G and CD11b cell surface phenotype predominate and correspond to the PMN infiltrate that has been reported previously (22). Numerous CD11b- and Ly6G-positive cells were found in the luminal exudate, the epithelial layer, and the lamina propria (Fig. 2E and F). Cells of the B-cell lineage (CD45R) were rare (Fig. 2H), and a few T cells (CD3e⁺ cells) (Fig. 2G) were found in the mucosal epithelium and submucosa. As infection progressed, cells began to infiltrate and accumulate in the uterine horns (Fig.3). Ly6G- and CD11b-positive cells were numerous by day 7 postinfection (Fig. 3E and F) and localized to the uterine lumen, mucosal epithelium, and lamina propria. As the infection resolved (3 to 4 weeks postinfection), the PMN population decreased (Fig. 3Q and R). T cells (CD3e⁺ cells) and B cells (CD45R⁺ cells) were rarely observed in uterine tissues until about day 7 postinfection, at which time they contributed significantly to the inflammatory infiltrate (Fig. 3G and H). T cells and B cells localized primarily to the lamina propria but were also occasionally observed within the mucosal epithelium. CD3⁺ T cells were the predominant cell type during and following the resolution of infection (days 14 to 70).

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Fig. 2.

Immunohistochemical staining of CD11b, Ly6G, CD3e, and CD45R in vaginal tissue. Vaginal tissues were collected from uninfected mice (A to D) and from chlamydia-infected mice at 3 days post-infectious challenge (E to H) and stained with anti-CD11b (A and E), anti-Ly6G (B and F), anti-CD3e (C and G), or anti-CD45R (D and H). Magnification, ×300. Representative tissues from four or five mice at each time point are shown.

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Fig. 3.

Immunohistochemical staining of myeloid and lymphoid cell populations in uterine tissue. At weekly intervals uterine horns were harvested from chlamydia-infected mice and stained for CD11b, Ly6G, CD3e, or CD45R cell surface antigens. (A to D) Noninfected mice; (E to H) 7 days postinfection; (I to L) 14 days postinfection; (M to P) 21 days postinfection; (Q to T and U to X) 28 days postinfection. Anti-CD11b (A, E, I, M, Q, and U), anti-Ly6G (B, F, J, N, R, and V), anti-CD3e (C, G, K, O, S, and W), and anti-CD45R (D, H, L, P, T, and X) were used. Magnifications, ×300 (A to T) and ×60 (U to X). Representative tissues from four or five mice at each time point are shown.

An interesting feature of the T-cell inflammatory response in the uterus, and to some extent in the vagina, was the development of perivascular T-cell clusters. Clusters became apparent by day 21 postinfection (Fig. 3O) and were observed as late as 70 days postinfection (data not shown). B-cell clusters were less evident, but anti-CD11b also stained clusters of cells. Low-power magnification of immunostained tissues (Fig. 3U to X) clearly demonstrated the clustering of T cells throughout the uterine horns.

In the murine model of chlamydial genital tract infection, the oviducts are the primary site for inflammatory damage, which subsequently results in infertility. The cellular infiltrate of the oviducts following chlamydial infection was evaluated and found to be similar to that of uterine tissues (data not shown). Briefly, as infection ascended into the oviducts (approximately day 7 postinfection), PMN predominated in the luminal exudate and in mucosal and submucosal tissues. T cells and B cells accumulated as the course of infection progressed. Unlike that of uterine tissues, however, the cellular infiltrate of oviducts diminished greatly, and only a few scattered T cells were observed following the resolution of infection. Thus, few inflammatory cells remained in oviductal tissue, but as reported previously (22), tubal ectasia, loss of ciliated columnar epithelial cells, and hydrosalpinx were frequently observed.

In situ analysis of CD4 and CD8 T-cell subsets.Both CD4⁺ and CD8⁺ T cells have been implicated in the protective immune response to C. trachomatis genital tract infection (11, 22, 34-36). At 3 days following infection, CD4⁺ and CD8⁺ T cells were observed in the vaginal mucosal epithelium and lamina propria (Fig.4C and D). By 7 days postinfection, the uterine mucosal epithelium and lamina propria contained CD4⁺ and CD8⁺ T cells (Fig.5). CD4⁺-T-cell clusters were evident by 14 to 21 days postinfection (Fig. 5C and D). Clusters were observed throughout the uterine horns, and, in general, clusters of CD4⁺ T cells were more numerous and were comprised of more cells than CD8⁺-T-cell clusters (Fig. 5F and L).

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Fig. 4.

Immunohistochemical staining of CD4⁺- or CD8⁺-T-cell subsets in vaginal tissues from noninfected mice (A and B) and chlamydia-infected mice (C and D) at 3 days postinfection, stained with either anti-CD4 (A and C) or anti-CD8 (B and D). Magnification, ×300. Representative tissues from four or five mice at each time point are shown.

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Fig. 5.

Immunohistochemical staining of either CD4⁺- or CD8⁺-T-cell subsets in uterine tissue from noninfected or chlamydia-infected mice. (A to F) Staining with anti-CD4; (G to L) staining with anti-CD8. (A and G) Noninfected; (B and H) 7 days postinfection; (C and I) 14 days postinfection; (D and J) 21 days postinfection; (E, K, F, and L) 28 days postinfection. Magnifications, ×300 (A to K) and ×60 (F and L). Representative tissues from four or five mice at each time point are shown.

The frequencies of cell populations in genital tract tissue during the course of infection are depicted in Fig.6. Myeloid-lineage cells (CD11b) and CD3⁺ T cells were the predominant cell types in early infection (day 7), but T cells, and particularly CD4⁺ T cells, were the predominant cell type during the resolution (days 14 to 21) and following the resolution (days 28 to 70) of infection. The kinetics of B-cell infiltration into infected genital tract tissue was similar to that of T cells, but the number of B cells never approached the level of T cells. The inflammatory infiltrate of the genital tract tissues diminished as infection resolved, but appreciable numbers of T cells remained localized to uterine tissue for at least 70 days postinfection.

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Fig. 6.

Characterization of the local cellular inflammatory response following genital tract infection with C. trachomatis. Uterine horns were harvested at various times postinfection, processed, stained, and enumerated as described in Materials and Methods. Bars represent the mean inflammatory score ± standard deviation in uterine tissues from groups of four or five mice at each time point.

Cellular characteristics of the vagina and uterus following the resolution of genital tract infection.Previous studies have shown that the adaptive immune responses which develop during the course of primary chlamydial infection confer a considerable degree of protection from a secondary infectious challenge (22). The protective response is characterized by the shedding of fewer chlamydiae and a much shortened course of infection. To determine the characteristics of the inflammatory cell response in genital tract tissue at a time when infection had resolved and animals demonstrated a level of immunity to reinfection, we examined vaginal and uterine tissues for various cell populations at 42 days following primary infection (2 weeks past the first culture-negative time point).

Considerable numbers of inflammatory cells remain localized to the vaginal and uterine tissues following the resolution of primary infection (Fig. 7). At 42 days following infection, moderate numbers of CD11b⁺ cells and CD3⁺ T cells were detected in the vagina. CD45R⁺ B cells were also detected but were less numerous than T cells. B cells and T cells were found to localize to both the lamina propria and mucosal epithelium. CD4⁺-T-cell clusters were evident, and CD8⁺ T cells were often observed to be localized to the mucosal epithelium. The uterus displayed a similar inflammatory picture; T cells predominated, and only scattered CD45R⁺ B cells and Ly6G⁺ PMN were observed. CD4⁺ T cells were scattered throughout the lamina propria and within clusters, whereas the less numerous CD8⁺ T cells were observed within the mucosal epithelium and sparsely scattered throughout the lamina propria. Although single CD11b⁺ cells were present in the uterine tissue at this time, a more dominant feature was the diffuse CD11b staining of regions that appeared to be near perivascular clusters of lymphocytes.

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Fig. 7.

Immunohistochemical staining of inflammatory cells in the vagina (A to F) and uterine horns (G to L) following the resolution of chlamydial genital tract infection (day 42 postinfection). Anti-CD11b (A and G), anti-Ly6G (B and H), anti-CD45R (C and I), anti-CD3e (D and J), anti-CD4 (E and K), and anti-CD8 (F and L) were used. Magnification, ×300. Representative tissues from four or five mice are shown.

Detection of cytokine-producing cells in the genital tracts of infected mice.To determine the kinetics of cytokine production and the location of cytokine-producing cells during genital tract infection, vaginal and uterine tissues were analyzed for cells producing specific cytokines at various times throughout the course of infection (Fig. 8; Table1). Cells producing IL-12 or TNF-α were detected in vaginal tissues by day 3 postinfection (Fig. 8); however, neither IL-4- nor IL-10-producing cells were detected at that time. At 7 days postinfection, cytokine-producing cells were evident in uterine tissue (Table 1). The individual variation in cytokine-expressing cells was large, but as a whole IL-4- and IL-10-producing cells outnumbered IL-12- and TNF-α-producing cells throughout the course of infection. Considerable variability in the number of IL-12- and TNF-α-producing cells also occurred during the course of infection and was not significantly different at any time point analyzed. Similar variability in the TNF-α response has been reported previously (6). In contrast, the number of IL-4- and IL-10-producing cells was greatest at 3 weeks postinfection, a time when infection is nearing resolution, and declined thereafter. However, even at 70 days postinfection the number of cytokine-producing cells in genital tract tissues was greater than that in naive mice.

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Fig. 8.

Immunohistochemical staining of cytokine-producing cells in the vaginal (A to H) and uterine (I to P) tissues of chlamydia-infected mice. Tissues were harvested, processed and stained as described in Materials and Methods. (A to D) Vagina, noninfected; (E to H) vagina 3 days postinfection; (I to L) uterine horn, noninfected; (M to P) uterine horn 28 days postinfection. Anti-TNF-α (A, E, I, and M), anti-IL-12 (B, F, J, and N), anti-IL-4 (C, G, K, and O), and anti-IL-10 (D, H, L, and P) were used. Representative tissues from four or five mice at each time point are shown. Magnification, ×600.

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Table 1.

Enumeration of cytokine-producing cells following primaryC. trachomatis genital tract infection^a

Tissues were also probed for IL-1β, IL-2, IL-5, IL-6, and IFN-γ, but we were unable to convincingly demonstrate the production of those cytokines using immunohistochemical staining. Those data do not necessarily imply that the cytokines were not produced, but may instead simply reflect the limitations of the detection methology. Our enumeration of IFN-γ-producing cells in genital tract tissues of infected mice was also confounded by the observation that immunoaffinity-purified anti-IFN-γ stained chlamydial inclusions quite intensely. The reason for the cross-reactivity of anti-IFN-γ with chlamydial inclusions is not understood, but because of that reactivity we were unable to confidently enumerate IFN-γ-producing cells.

DISCUSSION

Previous studies of the primary immune response to C. trachomatis genital tract infection have utilized methods that disrupt the genital tract tissue to enumerate infiltrating cell populations or to determine the pattern of cytokine production by infiltrating cells (25, 27, 41). The in vitro production of cytokines by antigen-stimulated splenic lymphocytes has also been used to define the systemic cellular immune response following primary chlamydial genital tract infection (27, 37-39). In recent studies, the molecules that traffic lymphocytes to genital tract tissue following chlamydial infection have been defined using in situ immunohistochemical analysis (15, 28). In the present study, we have extended those findings by analyzing the development of the local cellular inflammatory response using in situ immunohistochemical staining for cell surface antigens and for the production of intracellular cytokines. Our intent was to identify and characterize changes in the local inflammatory and cytokine responses induced during the course of chlamydial genital tract infection.

The influx of hematopoietic cells into the vagina at day 3 of infection, and into the uterus at day 7 of infection, correlated with a large increase in cells of the myeloid lineage (CD11b⁺cells). Although we did not distinguish unequivocally between PMN and macrophages, Ly6G is a cell surface antigen found predominantly on PMN (8), and previous studies using hematoxylin and eosin staining of infected genital tract tissue have indicated that the early inflammatory cellular infiltrate is comprised primarily of PMN (22). The mere presence of PMN at the site of infection is not sufficient to resolve infection, however. For example, mice deficient in T-cell responses do not resolve infection even though a marked PMN inflammatory response is evident (22), and in vivo depletion of PMN with anti-Ly6G only slightly prolongs the course of chlamydial infection (2). It is not known if that modest effect on chlamydial shedding induced by PMN depletion results from decreased killing of chlamydiae by PMN or from the loss of mediators produced by PMN that direct lymphoid cells to the site of infection. PMN also comprised the predominant cell type in the vaginal and uterine luminal exudates, and cells of the lymphoid lineage were rarely observed in the lumens. Thus, the analysis of vaginal washes for specific cytokines might not necessarily reflect the predominant cytokines produced during infection but instead may provide information only on those cytokines released by cells of the exudate (PMN) or on mediators released by epithelial cells.

Changes in the cellular composition of infected genital tract tissues were determined throughout the course of infection. In addition to measuring the myeloid cells, the influx of T and B cells was monitored. By 7 days postinfection, cells of the lymphoid lineage had increased significantly in vaginal and uterine tissues. The delay in lymphoid cell expansion is not surprising, since signals from myeloid cells are necessary for their activation. The accumulation of CD3e⁺cells (T cells) and CD45R/B220⁺ cells (B cells) into genital tract tissue was very rapid and thus was unlikely to result from the in situ expansion of antigen-specific cells. Instead, the increased number of lymphocytes probably resulted from the influx of non-antigen-specific cells. T cells outnumbered B cells throughout the course of infection, and CD4⁺ T cells were more numerous than CD8⁺ cells at all times evaluated. Consistent with the findings of others (28), T cells were predominantly T-cell receptor αβ positive, and T-cell receptor γδ-positive cells were only rarely observed (data not shown).

CD45R/B220 and CD19 are B-cell lineage differentiation antigens expressed on the surface of B lymphocytes from the pro-B-cell through the mature B-cell stage (10, 19). Both CD45R/B220 and CD19 have been used as pan-B-cell markers, although CD19 expression is reported to be more restricted to the B-cell lineage (18). Neither CD45R nor CD19 is found on plasma cells. In our initial studies we attempted to use anti-CD19 antibody to identify cells of the B-lymphocyte lineage. Although we found that splenic B cells were visualized using anti-CD19 antibody, only a few weakly staining cells were observed in genital tract tissues from infected mice (data not shown). However, numerous cells were easily visualized when anti-CD45R/B220 was used as a marker for B cells. We have interpreted the CD45R/B220⁺ cells as representing cells of the B-lymphocyte lineage, even though we were unable to confirm those results using anti-CD19. Those disparate results may simply reflect differences in the utility of the antibodies in immunohistochemistry and may not have occurred if a different detection methodology, such as fluorescence-activated cell sorting was used for these studies. However, because splenic B cells stained with anti-CD19 antibody under identical experimental conditions, our results may indicate the presence of a CD45R⁺ CD19⁻ cell population in chlamydia-infected genital tract tissues. Natural killer (NK) cells do not express CD19 or other B-cell markers but have been reported to express CD45R (33). The CD45R-positive cells in genital tract tissue may therefore represent a population of NK cells. Our attempts to stain genital tract tissues for NK cells using other NK cell markers (anti-NK1.1 or anti-asialo-GM1) were unsuccessful. Alternatively, the CD45R/B220-staining cells may represent a population of activated T cells (42). At this time we know only that a population of CD45R/B220⁺ cells infiltrate genital tract tissue following chlamydial infection and that few cells expressing the B-cell lineage CD19 cell surface antigen are present. If this CD45R⁺ population of cells does represent an NK cell or activated T-cell population, then quite possibly those cells might contribute to the resolution of intracellular chlamydial infection. Additional studies using other methods to separate cell populations (e.g., fluorescence-activated cell sorting) will be needed to address that question.

A hallmark of both ocular (trachoma) and urogenital chlamydial infections is the development of lymphoid follicles (7, 9, 17, 23, 32, 43). The follicles present in children with active trachoma appear to have germinal centers composed of B cells (7), but in adults with conjuctival scarring, the follicles lack germinal centers and T cells (CD4⁺) are much more numerous than B cells (32). The composition of follicles that develop during chlamydial genital tract infection is less well characterized (9, 13, 17, 23, 43). The mechanism(s) of follicular hyperplasia has not been elucidated, but it may hold important clues to the adaptive immune responses that confer immune protection and/or the pathogenetic immune responses that are thought to contribute to the severe sequelae of chlamydial infection. Although typical lymphoid follicles (i.e., with the architecture of follicles found in primary or secondary lymphoid tissues) were not observed in the genital tract tissues of infected mice, perivascular lymphocyte clusters were easily discernable (Fig. 3, 5, and 7). Clusters were composed predominantly of CD3e⁺ cells and CD4⁺ cells, but much smaller clusters of CD45R⁺ B cells and CD8⁺ T cells were also present. The genitourinary tract, as well as the skin and the pulmonary and gastrointestinal tracts, are the most immunologically active tertiary lymphoid sites. The clusters of T cells that develop during chlamydial genital tract infection are reminiscent of clusters of T cells that accumulate in the dermis following a delayed-type hypersensitivity reaction (30). T-cell clusters were not apparent until about 10 to 14 days following genital tract infection (Fig. 3 and 5), and some clusters remained for as long as 70 days postinfection (data not shown). In a recent study we demonstrated that the level of protective immunity that develops following primary genital tract infection was significantly impaired if animals were treated with doxycycline before the infection resolved (39). The most significant effects were found when mice were treated prior to 10 days postinfection. Perhaps early antibiotic treatment of infected mice ameliorates the development of T-cell clusters. If the clusters consist of memory and/or effector populations of T cells that contribute to the protective immune response in animals that resolve primary infection, then curtailing the formation of T-cell clusters with antichlamydial chemotherapy may significantly impair the development of the long-term protective immune response.

Recently, Su et al. demonstrated that passive immunization of naive mice with chlamydia-pulsed dendritic cells confers a very significant level of protective immunity (38). Protection was equivalent to that of mice which had resolved a primary chlamydial genital tract infection and was greater than that conferred by the adoptive administration of immune T cells (36). Perhaps the activation of postcapillary venules by genital infection enables mice to mobilize chlamydia-pulsed dendritic cells from the circulation and to initiate the perivascular clustering of T cells more readily than mice receiving only immune T cells. Further studies are needed, though, to determine the importance, or lack thereof, of the perivascular T-cell clusters in protective immunity.

Cytokines have been the focus of many recent studies on the adaptive immune response to chlamydial genital tract infection. IL-12 and IFN-γ play crucial roles in antichlamydial immunity, since mice deficient in those cytokines fail to resolve genital tract infection (5, 25). Less is known about local cytokine production during the course of genital infection. However, using reverse transcription-PCR methodologies, IFN-γ, IL-12, IL-10, and TNF-α transcripts have been detected in homogenates of infected genital tract tissues (25, 29, 41). In our present study, IL-12- and TNF-α-producing cells were detected in vaginal tissues by day 3 following infection and in uterine tissues by day 7. The vagina was negative for IL-4- or IL-10-producing cells at 3 days postinfection, but positive cells were numerous in uterine tissues by 14 days following infection and remained elevated even after the infection had resolved. Our results confirm those of other investigators regarding the presence of IL-12, IL-10, and TNF-α and extend the findings by demonstrating the kinetics of appearance and the duration of the responses. Noteworthy is our demonstration of IL-4-producing cells in genital tract tissue of infected mice, compared to results of other investigators who have failed to detect IL-4 transcripts from infected genital tract tissue or IL-4 protein by enzyme-linked immunosorbent assay of supernatants of chlamydia-stimulated T cells (25, 29, 38, 41). Our staining appears to be specific, because we used monoclonal antibodies and we did not experience false-positive reactions due to incompletely blocked endogenous peroxidase activity. Thus, the discrepancy may be due to differences in detection methodologies or in the tissue preparation. Nevertheless, by immunohistochemistry, considerable numbers of IL-4-producing cells contribute to the cellular infiltrate in genital tract tissue of chlamydia-infected mice.

Clearly, the Th1-type T-cell response contributes importantly to the resolution of intracellular infection, and studies generally do not support an essential role for a Th2-type T-cell response. However, we previously reported that antibody-deficient gene knockout mice were more susceptible to reinfection, and we attributed that susceptibility to the lack of antichlamydial antibody (37). Thus, it may be of interest to further investigate the role of IL-4 and IL-10 in the development of Th2-type antichlamydial immune responses and of specific antibody in acquired resistance to chlamydial genital tract infection.