The expression of adhesion molecules on venules within tissues is necessary for the extravasation of lymphocytes into tissue sites. Therefore, the kinetics and type of adhesion molecules present are factors that regulate the types of leukocytes recruited to tissues during inflammation. We have shown here that the majority of CD4 cells were recruited to the upper GT (UH and OD regions), but not to the lower GT (CV region), in response to MoPn vaginal inoculation. We also observed that the appearance of CD4 cells correlated with the expression of VCAM-1 in the upper tract but not in the lower GT tract. These results indicate that the appearance of VCAM-1 or other adhesion molecules in the upper GT facilitated the recruitment of an increased number of CD4 cells to this site during infection. This was functionally confirmed by the finding that lymphocyte adhesion was not increased until 21 days after infection in the upper tract but was transiently elevated on day 7 in the lower GT. Finally, this adhesion could be blocked by incubating the GT sections with specific antibodies, suggesting the likely participation of these molecules in vivo.
Our studies indicate that a noninfected GT is quiescent with respect to adhesion molecule expression. Therefore, the induction and maintenance of these molecules must be important for cells to access various tissue sites. Unexpectedly, we observed a rapid decrease in VCAM-1 expression in the lower GT but not in the upper tract, suggesting that the regulation of ECAMs differed between the upper and lower regions of the GT. In addition, this decrease in ECAM expression in the lower tract was more remarkable since inflammatory cytokines are known to induce the expression of adhesion molecules (4
). In fact, the level of TNF-α in GT secretions was found to increase from days 5 to 8 after MoPn inoculation but then to diminish by day 10 (10
). Furthermore, not only was the expression of VCAM-1, MAdCAM-1, and ICAM-1 decreased but also the adhesive ability of the endothelium in the lower GT was significantly diminished 14 days after inoculation with MoPn. Thus, the inflammatory response in the lower GT may possibly be down-regulated by an as yet unknown mechanism.
The induction of the ECAMs (E-selectin, VCAM-1, and ICAM-1) is regulated through the transcriptional activation of the associated genes genes via nuclear transcription factor-κB (NF-κB) after its release from the cytoplasmic inhibitor of NF-κB (IκB) by proteolytic degradation of IκB (39
). Pharmacologic agents that inhibit the degradation of IκB have been shown to interfere with the expression of ECAMs in vitro (8
). Nitric oxide (NO) has also been reported to limit the expression of VCAM-1 as well as other ECAMs by roughly 50% in the presence of inflammatory cytokines (12
). This effect also appeared to be mediated by inhibiting NF-κB activation and was reversed in the presence l
-monomethyl-arginine. Interestingly, inducible NO synthase knockout mice had a shortened course of infection with MoPn compared to wild-type control mice (35
). In addition, IL-10 has also been shown to diminish ICAM-1 expression by targeting NF-κB regulation (40
), and this cytokine is present in the GT during MoPn infection (45
). Thus, many factors that could suppress as well as enhance ECAM expression are present in the lower GT. Since the response of endothelial cells to inflammatory stimuli differs depending on the tissue (16
), further studies of the GT endothelium are needed in order to determine which factors are involved in maintaining this delicate balance.
Another possibility for the diminished expression of ECAM in the lower GT region may not be active down-regulation or inhibition of ECAM at that site but rather the lack of continued stimulation of the endothelium to maintain adhesion molecule expression. For instance, leukocytes themselves appear to stimulate endothelial cells to up-regulate expression of ECAMs. This was shown to be mediated through the release of inflammatory mediators such as TNF-α. Horie et al. showed that the TNF-α induced up-regulation of ICAM-1 and that VCAM-1 was blunted in SCID mice (17
). Adoptive transfer experiments revealed that only a T-cell-enriched population could reconstitute ECAM expression. This study also supported the finding that TNFRp55−/− mice were unable to express VCAM-1 in response to TNF-α administration (29
). Likewise, the increased production of TNF-α seen in the GT soon after MoPn inoculation (11
) most likely participates in the expression of ECAMs. TNF-α primarily utilizes the NF-κB pathway to induce expression of ECAMs, but other pathways for up-regulating the expression of VCAM-1 exist (25
). Recently, the cross-linking of ICAM-1 on the surfaces of human umbilical vein endothelial cells (HUVECs) was shown to induce VCAM-1 but not E-selectin through a pathway that is independent of NF-κB (25
). Finally, CD40L has also been shown to mediate the induction of ECAMs on HUVECs (21
). Thus, activated T lymphocytes recruited to the upper GT may be responsible for maintaining the expression of ECAM at that site both through cell contact and through the release of soluble mediators. Concurrently, the lack of CD4 lymphocyte recruitment to the lower GT may be due to the inability to maintain ECAM expression.
Other subsets of leukocytes are also recruited to GT during infection (Table ) and may participate in the extended expression of ECAMs in the upper GT. Stagg et al. recently associated the increased recruitment of an antigen-presenting cell (APC) population to the GT of BALB/c mice with more-efficient clearing of chlamydiae compared to that for C3H mice (41
). The authors suggested that the presence of these APCs in the local genital mucosa boosted the antichlamydial immune response. One could postulate that local T-cell activation may be necessary to provide a stimulus to endothelial cells for the continued recruitment of lymphocytes, as was suggested by Nakabayashi and colleagues (28
). In that report, the absence of major histocompatibility complex class II expression abolished the influx of lymphocytes to the salivary glands of transforming growth factor β knockout mice as well as VCAM-1 expression on the local endothelium. We also observed increased recruitment of a CD11b-positive population, which most likely contains neutrophils and monocytes that could potentially act as APCs and maintain antichlamydial T-cell activation locally.
Although the lack of ECAM expression by T lymphocytes may, in part, explain the selective recruitment of CD4 cells to the upper GT, it does not exclude a mechanism for the damping of immune responses in the lower GT. The lower GT, like the intestinal tract, is a mucosal surface that is in continual contact with potential pathogens and other commensal organisms. Continued exposure to endotoxin or inflammatory cytokines may induce excessive endothelial cell activation. To compensate for this, others have shown that human intestinal microvascular cells display an abbreviated response to LPS, as measured by the length of time ECAMs were expressed following exposure, compared to HUVECs (15
). Our findings suggest that the endothelium in the lower GT is hyporesponsive to LPS with respect to adhesion molecule expression. On the other hand, it is also possible that the endothelia in the lower and upper GTs respond equally to an inflammatory stimulus but that other tissue cells secrete modulatory molecules that could act on the endothelium or other cells such as lymphocytes. For instance, intestinal epithelial cells have been shown to inhibit intraepithelial T-cell responses (49
). As for the genital mucosa, epithelial cells from various regions of the GT differ in their responses to chlamydial infection. Recently, Wyrick et al. reported that polarized endometrial epithelial cells did not produce TNF-α, IL-1, or IL-8 following infection with Chlamydia
). In contrast, these authors, as well as Rasmussen and colleagues (38
), found that endocervical epithelial cells did produce these cytokines following infection.
Transforming growth factor β is another cytokine that displays immunosuppressive properties and may also neutralize an inflammatory response. Darville et al. reported peak levels in GT secretions 7 days after infection, and these levels diminished to baseline throughout the remainder of the infection course (10
). This finding correlates with our evidence for a temporary suppression of the inflammatory response in the lower GT. In Fig. , lymphocyte adhesion was increased on day 7 after infection, diminished temporarily on day 14, and then reacquired the ability to again mediate adhesion on day 21. In addition, the expression of MAdCAM-1 followed a similar pattern, with peaks observed on days 7 and 21 (Table ). Likewise, this pattern of expression was also noted at the mRNA level using reverse transcription-PCR (data not shown). Furthermore, the increased adhesion seen late in the course of infection in the lower GT was not due to fibrin deposition since immunohistochemical staining did not reveal increased fibronectin and since blocking the interaction of lymphocytes with the fibronectin-binding epitope did not diminish adhesion (data not shown). Taken together, these data suggest the presence of some mechanism that temporarily suppresses ECAM expression and possibly the inflammatory response in the lower GT.
The expression of ECAM and the subsequent recruitment of leukocytes to an infection site are complex and involve many factors such as the types and levels of inflammatory cytokines and chemokines. A possible variable in the recruitment of cells at local sites during infection may be the dose of the organism that first comes in contact with the host mucosal surface. It is conceivable that the local mucosal surface may respond in proportion to the dose of the organism. Although this has not been addressed in vivo, some information has been reported on the basis of in vitro studies. Using chlamydial LPS, Ingalls et al. have shown that monocytes produce an increasing amount of TNF-α through ligation of CD14 (19
). It is possible that resident tissue macrophages within the GT may produce TNF-α when exposed to chlamydiae. In addition, Beekhuizen et al. (3
) showed that a proportional increase in VCAM-1 expression and leukocyte adhesion occurred in response to increasing numbers of staphylococcal organisms. However, the effect correlated to the number of organisms internalized within endothelial cells. Likewise, for chlamydiae, others have shown that adhesion or internalization alone is not enough to induce the release of inflammatory cytokines and chemokines (38
) and that replication of the organism within host cells is a necessary prerequisite. For this study, the numbers of organisms replicating within the various regions of the GT were used as a measure of the infectious burden and were found to be equivalent among the three GT regions over the course of infection (Fig. A). This data could also be interpreted as indicating that the dose used in this study may have “overloaded” the capacity of the CV region to respond and may explain the abbreviated expression of ECAM noted in Fig. B. In this case, using a lower dose may induce a more sustained inflammatory response in the lower GT. However, Darville has found that TNF-α levels do not significantly change with different infecting doses in the murine model (T. Darville, personal communication). Also, preliminary data from the guinea pig model have shown that the number of CD4 cells recruited to upper or lower regions for the GT did not differ between groups infected with 106
IFU (R. G. Rank, personal communication). Furthermore, a study using the cat model of Chlamydia psittaci
ocular infection also reported no change in the magnitude of the immune response in relation to dose but did report differences in the incidence of infection (44
). Since the local control of lymphocyte recruitment depends on a series of factors and since the infecting dose for humans is not known, further studies examining the relationship of the levels of cytokines, chemokines, and leukocyte influx to the infecting dose should aid in our understanding of the local human immune response to C. trachomatis
Based on our results and other published reports, we propose the following series of events as a possible scenario to explain the preferential recruitment of CD4 cells to the upper but not the lower GT. The infection of local epithelial cells causes the release of inflammatory mediators, including chlamydial LPS, that induces the local release of TNF-α early after infection (11
) and up-regulation of ECAMs. As suggested by others, an early influx of monocytes may also participate in the production of TNF-α and further stimulate the up-regulation of adhesion molecules (13
). As Chlamydia
-specific, activated T cells begin to immigrate into the infected areas, they in turn stimulate endothelial cells through cell contact and/or soluble mediators to maintain ECAMs. In the presence of the antigen, continual T-cell activation may occur through recently immigrated or resident APCs. In addition, locally recruited lymphocytes may induce the secretion of a specific array of chemokines through the activation of endothelial cells, as shown for HUVECs in vitro (26
), that could attract Th1 CD4 cells over other lymphocyte subsets. This could then create an amplification loop in the upper GT that would continue to preferentially recruit Th1 CD4 cells in the presence of a local antigen. In contrast, in the lower GT, endothelial cells may differentially respond to inflammatory stimuli by shortened expression of ECAMs. In addition, factors which may inhibit lymphocyte responses and interfere with amplified recruitment may be released, possibly by local epithelial cells. Other leukocytes, such as monocytes, NK cells (45
), and neutrophils, do not appear to possess the ability to sustain the amplification recruitment loop. The overall effect would result in the preferential recruitment of Th1 CD4 cells to the upper GT and the eradication of infection. Moreover, the accessibility of the upper GT to leukocyte recruitment may also predispose this site to the consequences of immune system-mediated pathology.
The ability to dampen immune responses in the lower GT, while beneficial to the host may also be of benefit to chlamydiae. One could hypothesize that following the induction of a local inflammatory response to Chlamydia
in the endocervix, an abbreviated inflammatory response could potentially result in the delayed clearance of chlamydiae from the lower GT. We noted a consistent delay in the clearance of organisms from the CV region compared to the UH and OD (Fig. ). Although it was not significant, this trend was observed in each experiment. Interestingly, there is also evidence for this in humans. Kiviat and colleagues performed both culture and direct immunofluorescent staining for Chlamydia
in the cervix, endometrium, and both fallopian tubes of each of a group of individuals suspected for PID. They reported that in over 50% of the patients, chlamydiae were detected in the cervix but not upper tract by both direct fluorescent-antibody assay DFA (52%) and culture (66%) (24
). A phenomenon such as this, if verified, may enhance chronic infection or persistence in the lower GT. Therefore, the difference in regulatory mechanisms of lymphocyte recruitment between the upper and lower regions of the GT may influence the clearance of chlamydiae and may also play a role in the development of tubal pathology.