similar to those in genital infection in monkeys and humans result. We have developed a model of ascending genital

Transcription

1 INFECTION AND IMMUNITY, Sept. 1994, p /94/$ Copyright C 1994, American Society for Microbiology Vol. 62, No. 9 Mice Immunized with a Chlamydial Extract Have No Increase in Early Protective Immunity despite Increased Inflammation following Genital Infection by the Mouse Pneumonitis Agent of Chlamydia trachomatis STEVEN J. BLANDERl* AND ANTONIO J. AMORTEGUI2 University of Pittsburgh Medical Center' and Magee Women's Hospital,2 Pittsburgh, Pennsylvania Received 8 November 1993/Returned for modification 15 April 1994/Accepted 8 June 1994 We have determined that immunization with a detergent extract of the mouse pneumonitis agent of Chlamydia trachomatis fails to induce a protective inflammatory immune response following genital infection by C. trachomatis. We demonstrated that mice immunized with the detergent extract have increased cutaneous delayed-type hypersensitivity and increased splenic T-cell proliferation in response to the chlamydial extract. After genital infection by C. trachomatis, extract-sensitized mice had significantly increased genital inflammation (P = 0.044) compared with controls. The inflammation was characterized by significantly increased eosinophils in the genitalia (P < ) and increased genital edema (P < ). However, the increased genital inflammation of extract-sensitized mice provided no increase in protection against infection (P = 0.92). Chlamydia trachomatis is an important cause of ocular and genital infections worldwide. In developing countries, C. trachomatis is the most common infectious cause of blindness. In 1973, an estimated 400 million people suffered from trachoma, with blindness resulting in 2 million of them (32). In the United States and other developed countries, C. trachomatis is the most common sexually transmitted pathogen (28). In the United States, more than 3 million new infections occur each year (27). Among females, C. trachomatis is a frequent cause of salpingitis, which can cause tubal occlusion and damage of ciliary transport, leading to infertility. The risk of infertility is 10% with the initial episode, 30% with two episodes, and 50% with three or more episodes (6). This increasing risk of infertility is thought to result from increased inflammation following repeated exposure to chlamydial antigens. For example, humans or monkeys immunized by parenteral vaccination with killed C. trachomatis have an increased inflammatory response following ocular infection by C. trachomatis (10, 35, 41). In some human vaccine trials, children treated with vaccine preparations of killed C. trachomatis had an increased rate of trachoma compared with children treated with a placebo (41). Studies have examined the role of chlamydial antigens in the inflammatory response but have not examined whether this inflammation is protective or deleterious. For example, ocular inoculation of detergent extracts of a guinea pig biovar of Chlamydia psittaci, the guinea pig inclusion conjunctivitis agent, stimulates ocular inflammation in previously infected animals (33, 36). The inflammatory response consists primarily of lymphocytes and mononuclear macrophages. Eosinophilic infiltrates are a part of this inflammatory infiltrate. The inflammatory response to detergent extracts resembles that found in human trachoma (1), but the role of this response in protective or deleterious immunity is unexplored. Recent studies show that infection of mice with human * Corresponding author. Mailing address: University of Pittsburgh Medical Center, 200 Lothrop St., W636, Pittsburgh, PA Phone: (412) Fax: (412) strains of C. trachomatis is highly dependent on the stage of murine estrus (12). Uniform infection at a given time is essential for comparisons among animals. Progesterone treatment of both experimental and control groups affects the immune state in a comparative manner. Without hormonal pretreatment, only the ectocervix is infected, while with it we observe an ascending infection (2). In addition, the 4- to 5-day estrus cycle of the mouse makes histopathological comparisons among individual mice problematic. Hormonal pretreatment controls for the variable state of estrus so that histopathological comparisons are more comparative in the weeks following infection. Examination of hormonally pretreated mice is akin to examination of women taking birth control pills. Infection of mice with the mouse pneumonitis (MoPn) agent of C. trachomatis provides an excellent model of infection with a strain of C. trachomatis indigenous to mice (2, 15, 19, 38). Inflammation (2), protective immunity, and infertility (15) similar to those in genital infection in monkeys and humans result. We have developed a model of ascending genital infection in the mouse, using the MoPn agent of C. trachomatis. We pretreat experimental and control mice with progesterone (34) and then intravaginally infect them with the MoPn agent. The infection rate is 100%, the infection tends to ascend the genital tract, inflammation of the genital tract results, inflammation and infection are maximal 1 week after infection, and the mouse resolves the infection over 3 to 4 weeks. The inflammation in the murine model of genital infection is similar to that observed in humans and monkeys (21, 23). Using our murine model, we demonstrated that mice sensitized to chlamydial detergent extract antigens have increased genital inflammation that fails to limit chlamydial infection. This demonstrates that during genital infection the increased inflammation induced by previous exposure to these antigens provides no protective immunity and confirms the importance of these antigens in genital infection. (This paper was presented in part at the 93rd General Meeting of the American Society for Microbiology, 16 to 20 May 1993, Atlanta, Ga.)

2 3618 BLANDER AND AMORTEGUI MATERUILS AND METHODS Animals. Specific-pathogen-free female 6- to 7-week-old BALB/cByJ mice were obtained from Jackson Laboratory (Bar Harbor, Maine). On arrival, all mice were screened for chlamydial infection by vaginal culture for C. trachomatis, and no infection was found. Mice were housed in filter top cages and supplied food and water ad libitum in an environmentally controlled room at 22 C with a 12-h light-12-h dark cycle. One week prior to use, the animals were observed in the vivarium for signs of illness. Bacteria. The MoPn biovar of C. trachomatis (ATCC VR- 123; American Type Culture Collection, Rockville, Md.) was grown on McCoy cell (ATCC CRL 1696) monolayers and purified by using a modification of previously described procedures (7, 14, 26). Aliquots for later infection were frozen in 0.22 M sucrose-10 mm NaH2PO4-3.8 mm KH2PO4-5 mm glutamic acid, ph 7.4 (SPG), and stored at -70 C. Aliquots were thawed at 37 C immediately prior to use. The inclusionforming units (IFU) of the aliquots were enumerated as described below. To prepare a solution for sham infection, we followed the same protocol as given above but did not infect McCoy cell monolayers with chlamydiae. For detergent extraction, elementary bodies were further purified by a Renografin-76 (Squibb Diagnostics, Cranbury, N.J.) density gradient. The elementary bodies were applied first to 30% Renografin and pelleted at 90,000 x g for 30 min at 15 C. The pellet was resuspended in SPG, applied to a 30 to 44% discontinuous Renografin gradient, and pelleted at 90,000 x g for 90 min at 150C. Preparation of the extract of C. trachomatis. Renografinpurified elementary bodies were incubated in 0.5% (18.5 mm) Sarkosyl-10 mm dithiothreitol for 30 min on ice (3). The solution was diluted 1:13, below its critical micellar concentration (13.7 mm [20]), so that dialysis later would effectively remove the detergent. The solution was ultracentrifuged at 98,000 x g for 120 min at 15 C to pellet the extracted elementary bodies. The supernatant was extensively dialyzed against M Na2HPO M NaH2PO M NaCl, ph 7.4 (phosphate-buffered saline [PBS]), at 40C and concentrated with a 30,000-molecular-weight cutoff Centriprep-30 concentrator (Amicon, Beverly, Mass.). A bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, Ill.) was used to assess mass. Evaluation of the extract of C. trachomatis. The proteins contained in the chlamydial extract were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and by Western blotting (immunoblotting), using previously described techniques (5). We applied 76,ig of extract proteins to a 12.5% gel. We used the major outer membrane protein of the MoPn agent of C. trachomatis to help identify the location of the major outer membrane protein among extract proteins in the Western blot. The major outer membrane protein was purified by previously described techniques (7). We applied 50,ug of extract proteins to two lanes and 3,ug of the major outer membrane protein to one lane of a 12.5% gel and separated the proteins by SDS-PAGE. The proteins were electrophoretically transferred to an Immobilon transfer membrane (Millipore Corp., Bedford, Mass.), and each lane was cut into a membrane strip. Serum containing a 1:20 dilution of polyclonal antibody directed against chlamydial antigens or a 1:20 dilution of the GroEL-homologous heat shock protein of the MoPn agent was reacted with the strips. The former was obtained 1 week after reinfection from mice intravaginally infected with C. trachomatis twice, 5 weeks apart. The latter was obtained from heat shock protein-immunized INFEC7. IMMUN. mice. The mice were twice immunized, 3 weeks apart, with purified chlamydial heat shock protein mixed in monophosphoryl lipid A plus synthetic trehalose dicorynomycolate adjuvant (Ribi Immunochemical, Hamilton, Mont.). Serum was obtained 3 weeks after the booster dose. The heat shock protein was purified as previously described (4). The strips were visualized by an alkaline phosphatase enzymatic reaction as previously described (5). Immunization of mice with the extract of C. trachomatis. We immunized mice by subcutaneous inoculation of 150,ug of chlamydial extract mixed in incomplete Freund's adjuvant (IFA) and administration of a booster dose 3 weeks later with 50,ug mixed in IFA. Control mice were sham immunized with PBS diluting buffer mixed in IFA. Intravaginal infection. Mice were subcutaneously inoculated with 2.5 mg of medroxyprogesterone acetate (Depo-Provera) 1 week before and on the day of infection (34). They were anesthetized with 1.5 mg of pentobarbital sodium (Nembutal) on the day of infection. The pentobarbital sodium induced anesthesia for 45 to 60 min. The mice were then intravaginally inoculated with 2.5 x 106 IFU of the MoPn agent of C. trachomatis in 40,lI of SPG or sham infected. A Hamilton gas-tight syringe with a repeating dispenser and point style 5 needle was used for intravaginal inoculation. The mice were kept in a Trendelenburg position until they awakened. Cutaneous delayed-type hypersensitivity. Cutaneous delayed-type hypersensitivity was assessed by previously described techniques (25). Five micrograms of extract in a 50-,u volume was inoculated into one hind footpad of a mouse. As a control, 50,ul of PBS diluting buffer was inoculated into the other footpad. The thickness of each footpad was measured 24 h later with a Manostat dial caliper. The mean footpad edema was calculated from the difference between the mean of edema in antigen-inoculated footpads and the mean of edema in diluting buffer-inoculated footpads. Incubation of splenic T cells. The spleen was aseptically removed en bloc. A single cell suspension was prepared by passage through 200-jim sterile wire mesh and a 23-gauge needle. Erythrocytes were hypotonically lysed. Nylon wool (Polysciences, Warrington, Pa.)-enriched splenic T cells (9) were aliquoted, 105 cells per well, in a 100-,u volume in 96-well tissue culture plates (Corning Glass Works, Corning, N.Y.). Sufficient antigen-presenting cells remain after a single passage on nylon wool so that additional antigen-presenting cells are unnecessary. Triplicate wells were incubated in a humidified 37 C, 5% CO2 incubator for 3 days with either 106 UVirradiated elementary bodies, 10 jig of extract of C. trachomatis per ml, or medium alone (control wells). The cells were pulsed 16 h before harvest with 1 jici of [3H]thymidine per well and harvested on glass-fibered scintillation paper, and a emissions were counted in a scintillation counter. The mean counts per minute of the triplicate wells were calculated for each mouse. Lymphocyte culture medium consisted of RPMI 1640 (Gibco, Grand Island, N.Y.), 2 mm L-glutamine, 0.05 mm 2-mercaptoethanol, 10 mm HEPES (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid), 0.1 mm nonessential amino acids, 1.0 mm sodium pyruvate, 100 U of penicillin per ml, 100 jig of streptomycin per ml, and 10% heat-inactivated fetal calf serum (38). T cells were enumerated in a hemacytometer, and viability was assessed by trypan blue exclusion. Assessment of chlamydial infection. C. trachomatis organisms in the genitalia were enumerated by using a modification of a technique previously described for quantitation of C. trachomatis in the lung (38). After infection, mice were anesthetized with pentobarbital sodium and then killed by cervical dislocation. The genitalia were dissected en bloc by a sterile

3 VOL. 62, 1994 C. TRACHOMATIS EXTRACT-INDUCED GENITAL INFLAMMATION 3619 technique. The genital tissues were thoroughly ground with a mortar and pestle, and the ground tissue was serially diluted in Hanks' balanced salt solution. We incubated the dilutions on McCoy cell monolayers in duplicate wells of 16-well Lab-Tek tissue culture slides (Nunc, Inc., Naperville, Ill.). The growth medium was supplemented with 50,ug of vancomycin and 2.5,ug of amphotericin B (Fungizone) per ml. Thirty hours later, the wells were fixed in cold acetone and then incubated for 30 min with a fluorescein-conjugated antichlamydial monoclonal antibody (Kallestad Diagnostics, Chaska, Minn.). The IFU were enumerated with a microscope under UV light. Assessment of ascending genital infection. We pretreated six mice with medroxyprogesterone acetate, and six mice were untreated. We killed the mice and extracted the genitalia en bloc 1 week after infection. The genitalia were sectioned 1 mm above the bifurcation of the uterus into a vaginal-cervical and uterine-ovarian piece of tissue. Each piece was cultured separately on McCoy cell monolayers as described above. Histopathology. The genitalia were removed en bloc, fixed in PBS-buffered formaldehyde, embedded in paraffin, sectioned longitudinally, stained with hematoxylin and eosin, and examined microscopically. The evaluation was single blinded. The genital sections examined include the cervix, endometrium, myometrium, fallopian tubes, and ovaries. We qualitatively assessed lymphocytes, eosinophils, plasma cells, and polymorphonuclear neutrophils on a scale of 0 to +4. The approximate range of cells this scale represents per low-powered field for each section examined was as follows: 0 = no cells; + 1 = 1 to 5 cells; +2 = 6 to 10 cells; +3 = 11 to 25 cells; +4 = >25 cells. Squamous metaplasia and tubal exudates were evaluated as 0, if absent, or +2, if present. Tubal edema was measured with a fine-scaled ruler to the nearest 0.5 mm immediately after removal of the genitalia from the pelvis. The uterus of an uninfected, untreated mouse has an average diameter of 1 mm. We evaluated tubal edema as + 1 for each 0.5 mm of diameter greater than 1 mm in diameter. We calculated the inflammation index by dividing the sum of inflammation scores (lymphocytes, eosinophils, plasma cells, polymorphonuclear neutrophils, squamous metaplasia, and tubal edema) for each section of the genitalia (cervix, endometrium, myometrium, fallopian tubes, and ovaries) by the number of genital sections examined. Statistics. The results were analyzed with SigmaStat statistical software (Jandel Scientific, San Rafael, Calif.). RESULTS Proteins contained in the extract of C. trachomatis. We evaluated the proteins contained in our detergent extract of the MoPn agent of C. trachomatis by SDS-PAGE and Western blotting. Figure 1, lane B, demonstrates that the extract contains multiple protein bands in the Coomassie blue-stained gel. The polyclonal antiserum from previously infected mice reacts with at least three bands (Fig. 2, lane B), one of which is located at the 42-kDa position of the purified major outer membrane protein located in Fig. 2, lane D. The polyclonal antiserum from mice previously immunized with the GroELhomologous heat shock protein of the MoPn agent reacts with a single band with an apparent molecular weight of 62,000 (Fig. 2, lane C). The chlamydial extract contains the major outer membrane protein and a GroEL-homologous heat shock protein. Medroxyprogesterone acetate-treated mice have an ascending genital infection. We assessed whether mice pretreated with medroxyprogesterone acetate and intravaginally infected with the MoPn agent have an ascending genital infection. We A B FIG. 1. SDS-PAGE of the detergent extract of the MoPn agent of C. trachomatis. L-ane A contains molecular size markers. The apparent molecular weights (10') are indicated to the left of lane A. In lane B, 72 jig of extract proteins was applied to SDS-PAGE, and the gel was stained with Coomassie brilliant blue R. evaluated IFUs in the lower genital tract (vaginal-cervical section) and the upper genital tract (uterine-fallopian tubeovarian section) in mice treated with medroxyprogesterone acetate and untreated mice. Mice pretreated with medroxyprogesterone acetate had a 100% rate of infection throughout the genitalia (Table 1). The quantity of chlamydiae in the mice untreated with hormone was less than that in hormonally treated animals in both the upper and lower genital tracts. Only two of six untreated mice had upper tract infection, although one animal had an aberrantly high result (6.1 x 106 IFU in the upper genital tract). Overall, there was a significant increase in the rate of infection of the upper genital tract in medroxyprogesterone acetate-treated mice. In addition, compared with uninfected mice (Fig. 3A to C), infected mice had inflammation throughout the genital tract (Fig. 3G to I). Thus, mice pretreated with medroxyprogesterone acetate and intravaginally infected with the MoPn agent have an ascending genital infection. Cutaneous delayed-type hypersensit'ivi'ty to chlamydial ex- A Y 27.2.:!!., i B CD FIG. 2. Western blot of the detergent extract of the MoPn agent of C. trachomatis. Lane A contains molecular size markers. The apparent molecular weights (103) are indicated to the left of lane A. Lanes B and C contain extract proteins of the MoPn agent. Lane D contains purified major outer membrane protein of the MoPn agent. Lanes B and D were reacted with antiserum from a mouse genitally reinfected by the MoPn agent. Lane C was reacted with antiserum from a mouse immunized with the GroEL-homologous heat shock protein of the MoPn agent.

4 3620 BLANDER AND AMORTEGUI INFECT. IMMUN. TABLE 1. Ascending genital infection by the MoPn agent of C. trachomatis in mice pretreated with medroxyprogesterone acetate Conditiona Genital section examinedb Median Rate of Pe IFUc infection' No medroxyprogesterone acetate Vagina-cervix 9.5 x 102 4/6 (67) Medroxyprogesterone acetate Vagina-cervix 3.4 x 104 6/6 (100) 0.23 No medroxyprogesterone acetate Uterus-fallopian tubes-ovaries 0.0 2/6 (33) Medroxyprogesterone acetate Uterus-fallopian tubes-ovaries 0.94 x 104 6/6 (100) a Some mice were subcutaneously inoculated with 2.5 mg of medroxyprogesterone acetate 1 week before and on the day of infection, while others received no medroxyprogesterone acetate (controls). Mice were intravaginally infected by the MoPn agent of C. trachomatis. b One week after infection, the genitalia were removed en bloc and sectioned 1 mm above the bifurcation of the uterus. Each section of the genitalia (vagina-cervix and uterus-fallopian tubes-ovaries) was enumerated for IFU. cthe median quantity of IFU in each section of the genitalia was calculated. d The ratio of mice with detectable IFU in each genital section per quantity of mice infected was calculated. The percentage is given in parentheses. Fisher's exact test, two tailed. tract in extract-immunized mice. We examined whether mice immunized with the antigens of the C. trachomatis extract would have increased cutaneous delayed-type hypersensitivity. We also examined whether any differences would persist after chlamydial genital infection. Mice were immunized with 150,ug of chlamydial extract mixed in IFA, and their footpads were tested 3 weeks later. Sham-immunized control mice received PBS diluting buffer mixed in IFA. Immunized mice demonstrated significantly greater footpad edema than sham-immunized controls (P < 0.01; Table 2). Three weeks later, immunized mice were given a booster of 50,ig of extract mixed in IFA. Control mice that were not previously skin tested received a second sham immunization. Three weeks after the booster dose, they were intravaginally infected with C. trachomatis. One week after genital infection, the difference in cutaneous delayed-type hypersensitivity between immunized and control mice persisted. Immunized mice had significantly greater footpad edema than controls (P < 0.01; Table 2). Immunization with the extract induced cutaneous delayed-type hypersensitivity to the extract. Chlamydial genital infection induced cutaneous delayed-type hypersensitivity to the extract, but the response was less than that in infected mice that had been previously immunized with the extract. Splenic T cells of extract-immunized mice have increased proliferation in response to the extract. We examined whether mice immunized with the extract and then infected by C. trachomatis would have greater splenic T-cell proliferation in response to extract than control mice. In two independent experiments, we immunized or sham immunized mice. Three weeks later, the mice were genitally infected with C. trachomatis. Splenic T-cell proliferation was assessed 1 week after infection. Immunized and control mice had similar splenic T-cell proliferation in response to elementary bodies (P = 0.68; Table 3). However, extract-immunized mice had significantly greater splenic T-cell proliferation in response to the extract (P = 0.045). Immunized mice had a mean stimulation index (in response to the extract) that was 3.1 times greater than that of controls. One week after chlamydial genital infection, mice with previous exposure to chlamydial extract had significantly greater splenic T-cell proliferation in response to extract than sham-immunized controls. Extract-sensitized mice have increased inflammation, particularly, increased eosinophilia and edema. We examined whether mice immunized with the antigens of the C. trachomatis extract would have increased inflammation after chlamydial genital infection. Also, we assessed the particular elements of the inflammatory response that were affected by sensitization to the antigens. Sensitized and control mice were infected 3 weeks after the booster dose. Genital histopathology was examined before and 1 week after infection. Before infection, there was a moderate increase in the total genital inflammation index of sensitized versus control animals, although the results did not achieve significance (P = 0.080; Table 4). Sensitized mice had modest increases in the lymphocytic, eosinophilic, and plasma cell indices, although the results did not achieve significance (P = 0.84, 0.13, and 0.19, respectively). However, in all of these indices the level of inflammation was low compared with that in mice after chlamydial genital infection (Table 4). One week after genital infection by C. trachomatis, there was markedly greater inflammation throughout the genital tract in sensitized mice than in sham-sensitized controls (Fig. 3). The total inflammation index of sensitized mice was significantly greater than that of controls (P = 0.044; Table 4). Eosinophilia and edema were significantly increased in sensitized mice (P < for each). Although the amount of lymphocytes was greater and the amount of plasma cells was less in sensitized mice, there was no significant difference from control mice (P = 0.31 and 0.071, respectively). The amounts of neutrophils, squamous metaplasia, and exudates did not differ between sensitized and control mice. Extract-sensitized mice had increased genital inflammation in response to genital infection by C. trachomatis, with particularly marked increases in genital eosinophils and edema. Immunization with the extract does not induce protective immunity against chlamydial genital infection. We examined whether immunization with the extract would induce protective immunity against genital infection with C. trachomatis. One week after chlamydial genital infection, we enumerated the quantity of C. trachomatis organisms in the genitalia of immunized and control mice. In two independent experiments, detergent extract-immunized and control mice demonstrated no significant difference in the quantity of C. trachomatis in the FIG. 3. Histopathology in the genitalia following chlamydial genital infection. Mice were untreated (A to C), extract sensitized (D to F), or sham sensitized (G to I). The last two groups were genitally infected by C. trachomatis. One week later, the genitalia were extracted en bloc and stained with hematoxylin and eosin. The cervix (A, D, and G), uterus (B, E, and H), and fallopian tubes and ovaries (C, F, and I) were examined by light microscopy.

5 L., F I-...,I 'C..-,., "-..i. 1,.':

6 3622 BLANDER AND AMORTEGUI TABLE 2. Cutaneous delayed-type hypersensitivity in mice sensitized with the extract of C. trachomatisa Mean footpad edema ± SD (mm)' Time of test Sensitized Control PC mice mice Before infection 0.70 ± ± <0.01 (n = 8) After infection 0.83 ± ± 0.15 <0.01 (n = 6) a Cutaneous delayed-type hypersensitivity before and after chlamydial genital infection of sensitized and sham-sensitized (control) mice was assessed. 'The footpad edema is the difference between the footpad edema in antigen-inoculated and control footpads. c t test, two tailed. genitalia (P = 0.68 and 0.99, respectively; Table 5). Overall, immunized mice had a log mean of 5.35 IFU in the genitalia, while controls had a log mean of 5.38 IFU (P = 0.92). Mice sensitized with the extract demonstrated no protective immunity against genital infection with C. trachomatis. DISCUSSION The association between inflammation and chlamydial detergent extract has been previously described for ocular infection, but the ability of this inflammation to limit chlamydial infection had not been examined (33, 36). In the present study, the composition of the detergent extract of the MoPn agent of C. trachomatis was similar to that of the detergent extract studied in the guinea pig ocular model of chlamydial disease (3). The MoPn extract contains a member of the 60-kDa class of heat shock proteins (GroEL homolog) which has been associated with the inflammatory response during chlamydial infection (18). Extract-sensitized mice had increased cellmediated immune responses (lymphocyte proliferation and cutaneous delayed-type hypersensitivity) to extract compared with those in control mice. Immunized mice, therefore, demonstrate heightened immune recognition of the immunizing agent. The increased inflammation in immunized mice after chlamydial genital infection is likely associated with this heightened immune recognition. There was, however, no protective immunity at the height of TABLE 3. T-cell proliferation in mice sensitized with C. trachomatis extracte Proliferation after infection Test mice Antigen6 P (n = 6) Mean cpm SI (103) (mean ± SEM)d Sham sensitized Medium 2.0 EB ± 2.8 Extract ± 0.25 Sensitized Medium 3.1 EB ± Extract ± a Splenic T-cell proliferation was assessed after chlamydial genital infection in sensitized and sham-sensitized mice. b EB, 106 elementary bodies per ml; extract, 10,ug of extract per ml. c t test of the mean stimulation index, two tailed. d The stimulation index (SI) is the mean of antigen-stimulated T-cell uptake of [3H]thymidine for triplicate wells divided by the mean of control stimulated T-cell uptake of [3H]thymidine for triplicate wells. The mean stimulation index is the mean of the individual stimulation indices. INFEc-r. IMMUN. genital infection, which occurs 5 to 10 days postinfection (24). Certainly, extract-sensitized mice had markedly increased inflammation then, yet the inflammation failed to inhibit the growth of C. trachomatis in the genitalia. Although there was an increased early inflammatory response without increased protective immunity, the effect of immunization on late inflammation and protective immunity requires further investigation. For example, chlamydial genital infection typically resolves over 3 to 4 weeks. If extract sensitization is deleterious to host defenses, there may be persistence of infection detectable later. The significant increases in genital inflammation in extractsensitized mice following chlamydial genital infection agrees with the previously described association of the detergent extract with ocular inflammation (33, 36). In particular, we found significant increases in eosinophilia and edema in the genitalia. We believe this to be of interest because increased eosinophils in C. trachomatis-infected tissues in animals (21, 22, 36) and humans (1, 16) have been described. For example, Watkins et al. (36) demonstrated the appearance of eosinophils in ocular tissue in guinea pigs previously infected in the eye by the guinea pig inclusion conjunctivitis agent and then challenged with a chlamydial detergent extract. Patton et al. (21, 22) found tissue eosinophilia in monkeys after repeated C. trachomatis infection in both a genital and a subcutaneous pocket model of infection. Eosinophilia was not observed after primary infection. Eosinophils are found in conjunctival biopsy specimens from patients with active trachoma (1). Eosinophils play a role in other infectious diseases. They are capable of killing helminths and protozoa (37) and may contribute to the inflammation following viral infection (13). Mycobacterium, another intracellular pathogen, frequently elicits eosinophilic responses in humans and is ingested by eosinophils in a murine model (8). Eosinophils are cytotoxic cells most abundant in tissues with an epithelial surface such as the lower genitourinary tract (37). They survive longer than neutrophils and may persist for weeks in tissue. They are abundant in toxic substances, including cationic proteins that are cytotoxic to host cells. Eosinophils secrete cytokines and may also serve as antigen-presenting cells. These characteristics and the presence of eosinophils during chlamydial infection in chlamydial antigen-sensitized hosts suggest that they have a potential role in the inflammatory response observed after repeated exposure to chlamydial antigens. Study of the role of eosinophils in chlamydial infection is warranted, including study of the persistence of eosinophils during chlamydial genital infection, the effect of eosinophilic cations on C. trachomatis, and the presentation of chlamydial antigens by eosinophils. For the murine model of leishmaniasis, a dichotomous immune response has been reported (11). Th2 T-helper cells are associated with deleterious inflammation, and Thl T- helper cells are associated with resolution of infection. Such a dichotomous response may also exist in chlamydial disease. The presence of eosinophils during infection in extract-sensitized mice may result from increased interleukin-5 production. Interleukin-5 is the principal cytokine associated with the recruitment of eosinophils (37). Interleukin-5 production is characteristic of Th2 cells. Th2 cells may play a role in the nonprotective inflammation we have observed during early chlamydial genital infection. Previous studies demonstrate that the mucosal immune response in the genital tract is affected by hormones (17, 29-31, 39, 40). For example, in the uterus, estrogen causes increased levels of immunoglobulin A-producing B cells and of antibody and production of secretory component (17, 30, 31, 39).

7 VOL. 62, 1994 C. TRACHOAL4TIS EXTRACT-INDUCED GENITAL INFLAMMATION 3623 TABLE 4. Increased inflammation following genital infection by C. trachomatis in mice sensitized with an extract of C. trachomatis Test group Parameter Inflammation index' Total LC EOS PC PMN Sm Exud ED Uninfected Immune (n = 6) Median Avg rankc Control (n = 5) Median Avg rank pd wk postinfection Immune (n = 14) Median Avg rank Control (n = 13) Median Avg rank P < < a Mice were sensitized or sham sensitized (controls). Genital inflammation was evaluated before or after infection. b Total, overall inflammation; LC, lymphocytes; EOS, eosinophils; PC, plasma cells; PMN, polymorphonuclear cells; Sm, squamous metaplasia; Exud, exudate; ED, tubal edema. c Mann-Whitney rank sum test. d Mann-Whitney rank sum test. Estrogen and progesterone lower antibody and secretory component levels in cervicovaginal secretions (40). Progesterone causes decreases in levels of uterine antibody and secretory component (29, 39). Thus, hormones influence immune response in the genitalia. Progesterone pretreatment induced a consistent ascending chlamydial infection. Hormone levels vary during estrus, and the mucosal immune response is affected by hormones. Pretreatment with progesterone induces a uniform stage of estrus (diestrus) among mice, thereby removing this variable from the experimental paradigm. However, since both experimental and control mice receive the same hormonal pretreatment, differences in results are unlikely to be secondary to hormonal effects on immune response. Chlamydial infection at other stages of estrus may also be examined. A previous report describes intravaginal infection by the MoPn agent of C. trachomatis, and histological examination suggested that the infection is limited to only the ectocervical area (2). However, histological localization is less sensitive than direct culture of tissue for localizing infection. Even TABLE 5. Lack of protection against C. trachomatis infection in mice sensitized with the extract of C. trachomatis Log IFU Expt- Condition No. of mice (mean ± SEM) per genital pb specimen A Sensitized ± Control ± 0.18 B Sensitized ± Control ± 0.40 Overall Sensitized ± Control ± 0.26 a In two independent experiments, mice were sensitized or sham sensitized (controls). Mice were genitally infected with C. trachomatis, and 1 week later, C. trachomatis organisms in the genitalia were enumerated. bt test, two tailed. without hormonal pretreatment, we found that 33% of mice were infected in the upper genital tract. We have described a model of ascending chlamydial genital infection that is an improvement of previous murine models of chlamydial genital infection. Surgical inoculation of the MoPn agent in the periovarian and peribursal areas is used to infect the upper genital tract (15). The surgical route of infection is dissimilar to the intravaginal route of infection in women. A recent study shows that susceptibility to chlamydial genital infection is highly dependent on the stage of estrus (12), supporting the relationship between chlamydial infectivity and hormonal change in the host. Pretreatment with progesterone allows human strains of C. trachomatis to establish a genital infection in the mouse and prevents loss of target epithelial cells during the 5-day estrus cycle of the mouse (34). Hormonal pretreatment induces uniformity in the murine stage of estrus and allows standardization of infection in the experimental study. This is akin to examining a subpopulation of women on hormonal birth control. In addition, different quantities of inflammatory cells are found in the genitalia of uninfected mice at different stages of estrus. Pretreatment with progesterone controls the stage of estrus, removing the estrus-dependent histological variability during chlamydial infection. Thus, we control the stage of estrus by hormonally pretreating mice with medroxyprogesterone acetate. An ascending genital infection with inflammation throughout the genital tract results. ACKNOWLEDGMENTS We thank Lisa Bailey for outstanding technical assistance and Stephen Gregory, Elizabeth Wagar, and Charles Wira for expert advice and support. REFERENCES 1. Abu El-Asrar, A. M., J. J. Van den Oord, K. Geboes, L. Missotten, M. H. Emarah, and V. Desmet Immunopathology of trachomatous conjunctivitis. Br. J. Ophthalmol. 73: Barron, A. L., H. J. White, R G. Rank, B. L. Solof, and E. B. Moses A new animal model for the study of Chlamydia trachomatis genital infection of mice with the agent of mouse pneumonitis. J. Infect. Dis. 143:63-66.

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