Virtually everything we know about the host response to chlamydial genital infections has been derived from studies of the female, both in humans and in animal models. Painfully little is known about the immune response of the male, and even less is known about the local host response in the male urethra. Therefore, in this study, we used the model of the male guinea pig infected with C. caviae to further characterize the immune responses to chlamydial infection, especially the local antibody, cytokine/chemokine, and cellular responses in the penile urethra, the primary target infection site in males.
Initially, we determined the infection course in male guinea pigs inoculated with different doses of chlamydiae in order to determine the optimal dose for our studies. Not surprisingly, we found that guinea pigs infected with higher doses of the GPIC agent had increased levels of infection and earlier onset. A similar dose response in the course of cervical chlamydial infection was seen in the female (24
). We did not see any obvious clinical signs of disease, such as significant weight loss or urethral exudate.
Upon histopathologic examination of the penile urethra, there was clear evidence of a marked inflammatory response directed to the site of infection. As in the female (25
), the initial response was an acute inflammatory response peaking at 4 days after infection. A chronic inflammatory response developed concomitantly and remained elevated through day 14. Of note was the observation of a large number of plasma cells from days 4 to 14. This was quite interesting in that plasma cells are a hallmark of the histopathologic response of the female in the cervix and Fallopian tubes. Epithelial erosion occurred along with PMN infiltration into the male urinary tract, and fibrotic changes of the urethral mucosa appeared during the peak response of chronic inflammation. While some PMNs were noted in the lumen, there was no gross evidence of pyuria, although we did not examine the urine for leukocytes. Since fibrosis was only a rare event seen between days 7 and 14, there was no obvious scarring in the male urinary mucosa. This is in contrast to the infection in females, where the fibrosis in the Fallopian tubes is responsible for the severe consequences of chlamydial infection (25
). These data clearly demonstrate that the particular morbidity associated with chlamydial infection in either sex depends on the nature of the specific anatomic site that is targeted. Although only the penile urethrae of male guinea pigs with chlamydial infection were examined for urethritis in the present study by histopathology, Rank et al. (26
) previously observed that cystitis resulted from ascending infection to the bladder following intraurethral inoculation of chlamydiae into male guinea pigs; however, this occurrence was relatively rare in immunologically intact animals. In our earlier study, we also examined the vas deferens, epididymis, seminal vesicles, and prostate glands by histopathology and found no apparent inflammation or organisms in any of these tissues in normal animals. Only in one animal, which was immunosuppressed, did we find inflammation in the vas deferens and epididymis. In general, the nature and kinetics of the pathological response in the male guinea pig are virtually the same as those seen in the female cervix, endometrium, and oviducts (25
The only other animal model for infection of males via the urethral route in which histopathology was examined is the mouse infected with Chlamydia muridarum
). In a detailed study, Pal and colleagues were able to detect organisms in the urethrae, bladders, epididymides, and testes of infected mice, in contrast to GPIC infection of male guinea pigs. Moreover, they were able to detect organisms in the urethra and bladder for as long as 7 weeks after infection, considerably longer than we have seen in the guinea pig model. The histopathologic response was very similar to that of the guinea pig, with an acute inflammatory response developing later into a chronic inflammatory response.
The induction of a strong acute inflammatory response is supported by the expression of key proinflammatory cytokines, such as IL-8, IL-1β, and TNF-α, concomitant with the appearance of PMNs in the tissue. The levels of IL-8, IL-1β, and TNF-α were considerably lower following challenge infection than after primary infection, but this would be expected because of the lower number of chlamydiae present as a result of the adaptive immune response. Each of these cytokines is dependent on the interaction of chlamydiae with the host cell (16
). The increase in the expression of CCL2 (MCP-1) would suggest that other cells, such as NK cells and dendritic cells, are also recruited to the urethra during a primary infection.
For the first time in the male, the local cellular response was evaluated by flow cytometry. The data largely supported the observations from the histopathologic analysis. There was a strong influx of CD45+
cells beginning on day 4, increasing to a peak response on day 14. CD45+
cells would include PMNs and the various mononuclear cells, so this measure reflects the overall cellular response. Both CD4 and CD8 cells were observed in the urethra in parallel to the CD45 response. Interestingly, in contrast to the female guinea pig and mouse models (7
), there were significantly more CD8 cells than CD4 cells in the target tissue. The appearance of B cells was somewhat delayed in the primary infection, and they reached their peak level about 7 days after the T cells. Unfortunately, because of the lack of reagents for the guinea pig, no other cell surface markers or cell populations could be quantified. Upon challenge infection, CD45+
cells appeared by day 2, more quickly than in the primary infection but not to the same level. Surprisingly, the B-cell response appeared to constitute the majority of the response to the challenge infection, while there was only a trivial influx of CD4 and CD8 cells. This was somewhat different from the response in the female, in which there was only a minimal influx of T and B cells into the cervix and endometrium following challenge infection. Levels of both T and B cells were markedly increased in the oviduct following challenge in contrast to the primary infection, indicating a clear anamnestic response in the upper genital tract. Obviously, it is difficult to compare the male and female completely because of the anatomic differences, but the fact that there is a stronger anamnestic response of B cells than of T cells in the male urethra is of interest and suggests that memory B cells/antibodies may play an important role in the resolution of challenge infections in the male.
The presence of CD4 and CD8 cells certainly suggests that an active cell-mediated immune response develops in the male urethra, and this was supported by the strong proliferative response to chlamydial antigen by lymphocytes derived from the draining lymph nodes. The response paralleled the presence of T cells in the urethra. The likelihood that the T-cell response is a Th1 response was supported by the presence of IFN-γ, IL-2, and IL-12p40 in the urethra, paralleling the T-cell response in the genital tract. There was an increase in IL-10 levels; however, the absolute amount of IL-10 was far less than that of IFN-γ, and so the response was clearly a Th1 response. The increase in CCL5 (RANTES) levels also paralleled the T-cell influx in the urethra, a response that would be expected because CCL5 (RANTES) is a major T-cell chemokine. Upon challenge infection, only CCL5 (RANTES) appeared to be elicited at a relatively high level, in contrast to the other chemokines and cytokines. CCL5 (RANTES) has been found to be important for immunity to chlamydial infections (28
). The lower levels of IFN-γ, IL-2, and IL-12p40 following challenge are substantiated by the low T-cell response following challenge. While cell-mediated immunity has been found to be essential for the resolution of chlamydial genital infections in female guinea pigs and mice (18
), one cannot yet make the same statements regarding the male.
The other major observation in this study was the quantification of the local GPIC-specific antibody response in urethral secretions. GPIC-specific IgG and IgA levels increased by days 14 to 21 and remained relatively constant in the local secretions after the primary infection. Upon challenge, there was a marked increase in local IgG and IgA levels in secretions but only a modest increase in serum. The source of the antibodies detected in the urethral secretions of infected males is not yet clear. It is likely that IgG was derived from serum and IgA was produced locally, but that remains to be determined. It is noteworthy that there were numerous plasma cells in the submucosal area of the penile urethra, which could be the origin of local antibody production. The increase in the level of B cells in the urethra paralleled the increase in local antibody levels. Since the B-cell and antibody responses upon challenge infection were more intense than the T-cell response, it is tempting to speculate that this branch of the immune response is critical for immunity to reinfection. In fact, we reported that male guinea pigs which were depleted of B cells by cyclophosphamide treatment, leaving cell-mediated immunity intact, had a longer course of infection with increased pathology (26
). Moreover, Cunningham and colleagues recently demonstrated that polyimmunoglobulin receptor-mediated transport of IgA played an important role in the clearance of infection from the genital tracts of male mice (2
). In female guinea pigs, humoral immunity is essential for the recovery of chlamydial genital infection (25
), and immune serum-derived antibody contributed to protection against a genital tract infection (21
). Immunity to reinfection in the genital tracts of female guinea pigs was also dependent on an intact humoral immune response (19
In general, we have observed that the host response in the male urethra is similar both to the inflammatory response and to the humoral and cell-mediated immune responses to infection of the female genital tract. Nevertheless, there are two key differences between the male and the female. First, the CD8 T-cell response appears to be greater than the CD4 T-cell response in the male guinea pig. This is in contrast to the responses of the female guinea pig, in which the ratio of CD4 to CD8 cells is about 1:1 (23
), and the female mouse, in which the ratio is about 2:1 (7
). Second, upon challenge infection, the B-cell response is stronger than the T-cell response in the male guinea pig, in contrast to the responses in the female guinea pig and mouse (7
). The anamnestic B-cell response is accompanied by a sharp increase in both IgG and IgA levels in urethral secretions. We have observed previously that male guinea pigs have a high level of complete immunity for a longer time than female guinea pigs (13
). Whether these differences in the host response account for the “stronger” immunity in the male guinea pig remains to be determined.