INFECTION AND IMMUNITY, Nov. 1982, p. 699-705 Vol. 38, No. 20019-9567/82/110699-07$02.00/0

Effect of Estradiol on Chlamydial Genital Infection of FemaleGuinea Pigs

ROGER G. RANK,* HAROLD J. WHITE,t AUBREY J. HOUGH, JR., JAMES N. PASLEY, ANDALMEN L. BARRON

Departments of Microbiology and Immunology, Pathology, and Physiology and Biophysics, University ofArkansas for Medical Sciences; and the Medical Research Service, Veterans Administration Medical Center,

Little Rock, Arkansas 72205

Received 15 January 1982/Accepted 26 April 1982

Female guinea pigs were treated daily with 1 mg of ,-estradiol-3-benzoateintramuscularly beginning 14 days before intravaginal inoculation with the chla-mydial agent of guinea pig inclusion conjunctivitis and continuing during thecourse of the infection. Treatment with estradiol was found to markedly influencethe course of genital infection with the chlamydial agent of guinea pig inclusionconjunctivitis, producing infections of greater intensity and longer duration thanthose in control animals. Moreover, pathogenesis was altered in that ascendinginfection was observed, resulting in endometritis, cystic salpingitis, and cystitis.Infection in the controls was limited to the cervix and vagina. Estradiol treatmentincreased the apparent number of infected cells in the cervix and vagina asdetected by histopathology and immunofluorescent staining. Humoral and cell-mediated immune responses to the chlamydial agent of guinea pig inclusionconjunctivitis were comparable in estradiol-treated and untreated animals. Thesedata indicate that hormonal manipulation may have profound effects on the courseof chlamydial genital infections.

Chlamydia trachomatis has been recognizednow for several years as a major cause ofvenereal disease in both men and women (19,20). It has been reported that as many as 11% ofnormal sexually active women may have chla-mydial infections of the cervix, although themajority of these infections are asymptomatic(21). In some cases, however, these infectionsmay progress into salpingitis (10) and even peri-tonitis and perihepatitis (13). The reason thatsome women develop these disseminated infec-tions, whereas others do not, is unknown.An important factor which may influence sus-

ceptibility to enhanced infection in women is theeffect of reproductive hormones on the genitaltract (6). Hormonal manipulation has been impli-cated in the alteration of other genital infections.The development of vaginal candidiasis wasassociated in two studies with the concomitanttaking of oral contraceptives (4, 14), whereasmice treated with progesterone were found tohave higher mortality rates when infected in thevagina with herpes simplex virus type II (2).Kita et al. (8) noted that female mice were moresusceptible to genital infection with Neisseriagonorrhoeae when challenged during late proes-

t Present address: Biomedical Department, General MotorsTechnical Center, Warren, MI 48090.

trus, or early estrus, the times when estrogenlevels are the highest.

In the present study, we investigated theeffect of estrogen treatment on genital infectionof female guinea pigs with the chlamydial agentof guinea pig inclusion conjunctivitis (GPIC).GPIC, a Chlamydia psittaci agent, has beenfound to produce an infection in guinea pigsrather similar to C. trachomatis genital infectionin women (3). Moreover, guinea pigs are suitableas a model for the study of hormonal influenceson genital infections since their reproductivefunction resembles that of the human femalewith regard to their long estrous cycle, sponta-neous ovulation, and actively secreting corpusluteum (1, 22). To determine the possible effectof female sex steroids on chlamydial genitalinfections, we treated female guinea pigs withestradiol before and during the course of GPICinfection. Estradiol was selected because it isone of the major reproductive hormones and amajor component of certain oral contraceptives.

MATERIALS AND METHODS

Experimental animals. Hartley strain female guineapigs weighing 400 to 500 g and about 3 months of agewere obtained from Simonsen Laboratories, Inc., Gil-roy, Calif. Although we have found this stock to befree of natural GPIC infection, we nevertheless as-sessed all animals for antibody to GPIC before inclu-

699

700 RANK ET AL.

TABLE 1. Experimental design

Expt Treatment GPIC No. ofinfected animals

1 Estradiol + 5Estradiol - 3Sesame oil + 3

2 Estradiol + 5Sesame oil + 6

3 Estradiol + 14Estradiol - 2Sesame oil + 3

sion in experiments. Animals from this source routine-ly yielded negative results. All animals were housedindividually in cages covered with a fiber glass filterand were provided food and water ad libitum in an

environmentally controlled room at 24°C with a 12:12light-dark cycle.

Infection of guinea pigs. A suspension consistingprimarily of elementary bodies was prepared frominfected yolk sacs in sucrose-potassium-glutamatebuffer (pH 7.2) containing 0.5 mg of gentamicin per mland 0.5 mg of vancomycin per ml as described previ-ously (16). All animals were infected by intravaginalinoculation of 0.05 ml of the particle suspension,containing approximately 5.2 x 105 50% egg lethaldoses. A 23-gauge needle, 1 in. (ca. 2.54 cm) in lengthwith the tip blunted with solder was used for inocula-tion (3). The course of the infection was assessed byobtaining vaginal wall scrapings with a dental spatulaand making a smear on a glass slide. Smears werefixed in methanol and stained with Giemsa. Onehundred cells were counted, and the percent contain-ing inclusions was recorded as the inclusion score.

Detection of serum antibodies to GPIC. Antibodytiters to GPIC were determined by indirect immunoflu-orescence by using fluoroscein-conjugated rabbit anti-guinea pig immunoglobulin G (Miles Laboratories,Inc., Elkhart, Ind.) and a procedure described else-where (16).

Allergic contact dermatitis response to oxazalone.Cell-mediated immunity was routinely assessed by theresponse ofguinea pigs to sensitization with oxazolone(4-ethoxymethylene-2-phenyl oxazolone) (16). Guineapigs were sensitized on the ear with 0.2 ml of 10%oxazolone in acetone and challenged on a shaved flank7 days later with 0.2 ml of 0.125% oxazolone inacetone-corn oil (4:1). The reactions were assessed bythe intensity of erythema and edema at 24 h afterchallenge.

Delayed-type hypersensitivity to GPIC antigen. GPICantigen was prepared by heat inactivation (100°C, 30min) of a suspension of GPIC grown in McCoy cells(12). Control antigen consisted of heat-inactivatedMcCoy cells treated like the chlamydial suspension.The antigen (0.1 ml) was inoculated intradermally intothe pinna of the ear with a 30-gauge needle (9). Earthickness was measured with a Schnelltaster dial mi-crometer (H. C. Kroplin, Hessen, West Germany)before antigen injection and at 24, 48, and 72 h afterchallenge. The amount of induration was determinedby subtracting the ear thickness just before challenge

from the ear thickness at the various times afterchallenge. The maximum ear thickness routinely oc-curred 24 h after challenge. We have found that thismethod produces more consistent results than injec-tion of antigen into the flank and is a more accuratemeasure of induration.

Histopathology. Tissues were collected at necropsyfrom animals that had died or were killed underanesthesia at selected times. All tissues were fixed in10% buffered Formalin (pH 7.2) before sectioning.Tissues were embedded in plastic as described previ-ously (25). Sections were cut at 2.0 p.m thickness withglass knives mounted on a JB-4 microtome (DupontSorvall, Newtown, Conn.) and were stained witheither hematoxylin and eosin or toluidine blue (pH4.0).Immunofluorescence microscopy. Frozen sections of

vagina and cervix were cut on a Cryo-cut microtomeunit (American Optical, Buffalo, N.Y.) set at 6.0 p.m.Sections were fixed with acetone and stored at -70°Cbefore staining by the indirect procedure with a 1:10dilution of guinea pig serum containing antibodies toGPIC (titer, 160) followed by fluorescein-labeled rab-bit anti-guinea pig immunoglobulin G. The sectionswere counterstained with 0.1% Evans blue. Additionalsections from each specimen were incubated witheither normal guinea pig serum or phosphate-bufferedsaline (pH 7.2) in place of the guinea pig anti-GPICserum to serve as negative controls.Radioimmunoassay of estradiol. Estradiol levels

from pooled guinea pig plasma samples were measuredafter ether extraction by radioimmunoassay by thedextran-coated charcoal method (11). Estradiol wasmeasured with the use of a 17B-estradiol kit kindlyprovided by Wien Laboratories Inc., Succasunna,N.J., with a sensitivity of 10 pg/ml.

Experimental design. Three different experimentsare described in this investigation and are outlined inTable 1. Appropriate animals were injected subcutane-ously daily with 1 mg of ,B-estradiol-3-benzoate (SigmaChemical Co., St. Louis, Mo.) dissolved in 0.1 ml ofsesame oil (Dragon Brand, D. Y. Import Co., Inc.,Los Angeles, Calif.) or sesame oil alone, beginning 14days before infection. Injections were given at approx-imately the same time each day. All animals wereinfected with GPIC and sensitized with oxazolone onday 0. Vaginal scrapings were routinely collected at 3-day intervals beginning on day 3 for the determinationof inclusion scores. In experiment 3, animals werekilled for histopathology at the times indicated in Fig.2.Plasma samples taken from the five estradiol-treated

animals in the experiment 1 on days 7, 14, 21, and 28were pooled by day for the determination of estradiollevels. Since comparable samples from sesame oil-treated guinea pigs in the first experiment were notavailable, samples from a sesame oil-treated group inanother experiment not reported here (days 7, 14, 21,and 28) were used to obtain control levels.

RESULTSEffect on course of infection. In experiment 1,

the course of GPIC infection in estradiol-treatedanimals was found to last about 9 days longerthan in sesame oil-treated animals (Fig. 1A).

INFECT. IMMUN.

ESTRADIOL EFFECT ON CHLAMYDIAL INFECTION 701

c 20-I

15 _ / o N _ 1000

10 100

5 10

/~~I105 10 15 20 25 30 35

Days after infection

FIG. 1. Comparison of course of GPIC infection inguinea pigs treated with estradiol (0) and in guineapigs treated with sesame oil alone (0). The develop-ment of serum antibody in estradiol-treated animals(A) or sesame oil-treated animals (A) is also represent-ed. Each point represents the mean of all the animalsin the group. The inclusion scores are the arithmeticmeans, and the antibody titers are the geometricmeans. (A) Experiment 1: estradiol, five animals;sesame oil, 3 animals. (B) Experiment 2: estradiol, 5animals; sesame oil, 6 animals.

Moreover, a higher number of inclusions was

detected in smears obtained from estradiol-treat-ed guinea pigs at each time point after day 5. Thecourse of the infection in estradiol-treated guin-ea pigs was significantly different [F(1,6) =

32.11, P < 0.002] than that of the control groupwhen the data were compared by a two-factor(treatment group, days) analysis of variancewith repeated measures on one factor (days).When the experiment was repeated (experiment2), similar results were obtained (Fig. 1B). Inthis experiment, the estradiol-treated guineapigs remained infected about 15 days longer thandid the control animals. These data were alsostatistically significant [F(1,9) = 46.80, P <0.001]. The difference in prolongation of theinfection in the two experiments may be ex-

plained by the result of one sesame oil-treatedanimal in experiment 1 having an infectioncourse somewhat longer than usual.

Effect on systemic immune response. Whensera from each of the infected groups wereassessed for antibodies to GPIC in each of thetwo experiments, no differences were seen in thedevelopment of antibodies or the titers attained(Fig. 1A and 1B). Antibodies were first detected

in both groups on day 14 and reached peak levelson about day 28.

Cell-mediated immunity to a contact allergenas well as GPIC antigen was also evaluated inexperiment 1. All animals responded to sensiti-zation with oxazolone with positive reactionsafter challenge on day 7 of infection (Table 2).When the guinea pigs were skin tested withGPIC antigen on day 15, four of five infected,estradiol-treated and three of three infected,sesame oil-treated animals responded with posi-tive reactions. There was no significant differ-ence between the response of infected, estradi-ol-treated and infected, sesame oil-treatedanimals when the data were compared by a one-tailed t test (P > 0.05). However, both groupshad significantly greater reactions when com-pared with uninfected estradiol-treated animals(P < 0.001).

Alteration of pathogenesis of chlamydial infec-tion. All guinea pigs from experiment 1 weresacrificed on day 42 after the infection hadsubsided and were examined for any grosspathological changes. Interestingly, in four offive infected, estradiol-treated animals, fallopiantube cysts filled with clear fluid were observedand were generally found to be attached to otherorgans by adhesions. This was an unusual find-ing since we have never seen fallopian tube cystsin uninfected animals and have seen fallopiantube cysts in only two immunologically intactanimals infected with GPIC out of approximate-ly 60 which have been examined. No pathologi-cal changes were associated with the fallopiantubes of infected, sesame oil-treated animals orthree animals treated with estradiol but notinfected.

In experiment 3, we investigated further theextent of the pathological changes apparentlycaused by GPIC in animals treated with estradi-ol. Fourteen guinea pigs were treated with estra-diol as above and were infected with GPIC.Since there was no apparent effect of sesame oilon the course of GPIC genital infection, we

TABLE 2. Cell-mediated immune responses toGPIC antigen and oxazolone in estradiol-treated

guinea pigsDelayed hypersensitivity

Treatment GPIC response to:infected

Oxazolone GPIC

Estradiol + 5/5a 4/5 (4.5 + 1.3)bEstradiol - 3/3 0/3 (0.3 + 0.3)Sesame Oil + 3/3 3/3 (6.2 + 1.1)

a Number responding/number challenged at 24 hafter challenge.bChange in ear thickness (1 unit = 0.1 mm) ±

standard deviation.

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702 RANK ET AL.

GROUP DAY AFTER VAGINA CERVIX UTERUS FALLOPIAN BLADDERINFECTION TUBE

ESTRADIOL- 5 (1)INFECTED 10 (2)

* * S 0

so So 59 00

15 (2) es so so 60

21 (4)25 (3)

5550 550 9900 699go

0

00

00

*0oo

i@@ goo goo g0o 9o930 (2) of so IO IO

SESAME OIL- 10 (1)INFECTED 15 (1)

21 (1)

ESTRADIOL 10 (1)ALONE 21 (1)

0 0

0 0

0 0

0 0

0 0

so

0

0

0

0

0

FIG. 2. Summary of histopathology seen in guinea pigs sacrificed at various times after infection with GPIC.The determinations were made based on the observation of tissues stained with either hematoxylin and eosin ortoluidine blue (pH 4.0). The numbers within parenthesis represent the numbers of animals sacrificed at each timepoint. Each circle represents the observation of tissue from a single animal. Symbols: (0) GPIC inclusions weredetected in the presence of inflammation, (9) only inflammation was observed, and (0) absence of either GPICinclusions or inflammation.

included only three animals infected with GPICand treated with sesame oil to confirm the lackof vehicle effect. Two other animals were givenestradiol but were not infected. Animals fromeach group were sacrificed at various intervalsafter infection, and tissues were removed forhistopathological examination. Inflammationand chlamydiae were found in all areas of thegenital tract of estradiol-treated guinea pigs atvarious times after infection (Fig. 2). In contrast,the infection in control animals remained limitedto the cervix and vagina. The pathologicalchanges in these animals were identical to thechanges seen in other immunologically intactguinea pigs infected with GPIC as described indetail previously (3).

In general, the genital tracts from animalstreated only with estradiol (three animals inexperiment 1 and two animals in experiment 3)were larger and more hyperemic than those ofuntreated animals. Upon microscopical exami-nation, estradiol treatment did increase theamount of superficial stainable mucin, particu-larly in the exocervix. The mucin stained meta-chromatically with toluidine blue (pH 4.0).There was also an increase in the number ofglycogen-filled cells lining the vagina, exocer-vix, and endocervix. There was some decreasedcellularity of the uterine stroma. No inflamma-tion was ever detected in animals receiving onlyestradiol.The amount of inflammatory reaction was

greatly, increased in the cervix and vagina in

infected, estradiol-treated animals. Inflamma-tion with polymorphonuclear leukocytes andmononuclear cells was apparent in the epitheli-um of the exocervix and vagina beginning on day5. Mononuclear inflammation was observed inthe submucosa of the endocervix, whereas in-flammation with polymorphonuclear leukocyteswas prevalent in the superficial epithelium.There also appeared to be a greater number ofchlamydial inclusions present in the endocervi-cal epithelia of the estradiol-treated guinea pigscompared with sesame oil-treated guinea pigs.

In addition to cervicitis and vaginitis, an endo-metritis was observed in most infected, estradi-ol-treated animals. Chlamydiae were detected inthe superficial epithelium up to day 15, but notthereafter. Polymorphonuclear leukocytes in-flammation of the superficial epithelium of theendometrium was noted as well as focal collec-tions of polymorphonuclear leukocytes in theendometrial cavity.

Salpingitis was not detected until day 15.Upon gross examination, unilateral enlargementof the fallopian tube was seen, although occa-sionally bilateral enlargement was detected. Ad-hesions between the ovary-fallopian tube com-plex and other organs (kidney, spleen, smallintestine, and peritoneum) were commonly ob-served. Microscopically, an acute inflammatoryresponse was seen in the lumen of the fallopiantube with chronic inflammation within the wall.When seen, organisms were present in eitherattached or shed epithelial cells of the fallopian

INFECT. IMMUN.

ESTRADIOL EFFECT ON CHLAMYDIAL INFECTION

FIG. 3. Immunofluorescent staining of chlamydial inclusions along the cervical epithelium. The tissue wasobtained from an estradiol-treated animal sacrificed on day 10 of infection. Note the almost confluent presence ofinclusions restricted to the superficial epithelial layer. x400.

tube wall. The pathological changes of the fallo-pian tube described above for experiment 3 areindicative of a pyosalpinx and probably lead tothe formation of a hydrosalpinx after the resolu-tion of the infection as seen in experiment 1.Some early fibroblastic proliferation was ob-served on the peritoneal surface of the fallopiantubes, resulting in the aforementioned adhe-sions. Focal peritonitis was also observed, al-though no organisms were detected in the perito-neum.As the infection continued, cystitis was noted

by day 21. Upon examination, a focal infiltrationof polymorphonuclear leukocytes along withmononuclear cells was observed in the bladderepithelium. Collections of mononuclear cellswere also seen in the submucosa.Frozen sections of vagina and cervix from the

above animals were also prepared by indirectimmunofluorescence techniques using guineapig anti-GPIC serum. Sections of both vaginaand cervix from estradiol-treated animalsstained very heavily along the epithelium ondays 10 and 15, producing almost a confluentlayer of cells with chlamydial inclusions (Fig. 3).Many inclusions were still detected on day 21. Incontrast, in infected, sesame oil-treated animals,only a rare inclusion could be detected on day10, and none were observed on day 15 or 21.Control sections in which either normal guineapig serum or phosphate-buffered saline was sub-

stituted for guinea pig anti-GPIC serum wereroutinely devoid of fluorescence. These datasupport the observations made by histopatholo-gy which suggested a greater number of cellsinfected in estradiol-treated animals.Serum estradiol levels. When the values ob-

tained for the pooled samples from individualdays from estradiol-treated animals were aver-aged, a mean of 531 pg/ml with a standarddeviation of 61 was determined. Pooled samplesfrom control animals averaged 128 ± 49 pg ofestradiol per ml. Estradiol levels were signifi-cantly higher (P < 0.05) in estradiol-treatedguinea pigs when compared to sesame oil-treat-ed animals by a one-tailed t test. No effort wasmade to coordinate temporal estrous cycle stagevariability among the control animals; thus sam-ples from control animals probably containedplasma from both estrous and diestrous animals.

DISCUSSIONIn this investigation, treatment offemale guin-

ea pigs with estradiol was found to modify thecourse of GPIC genital infection, producing in-fections of greater intensity and longer durationthan in control animals. A significant aspect ofthis study is the altered pathogenesis caused bytreatment with estradiol. In untreated guineapigs, the infection is limited mainly to the cervixand, to some extent, the vagina, with the exocer-vix as the major target organ (3). Furthermore,

703VOL. 38, 1982

704 RANK ET AL.

ascending GPIC infection has only been ob-served previously when animals were immuno-suppressed with cyclophosphamide (16, 25). Thepathology reported here is quite similar to thatseen in our cyclophosphamide immunosuppres-sion experiments. In the present investigation,we also observed a severe vaginitis and cervici-tis in animals treated with estradiol as well as acystic salpingitis in a high percentage of animals.However, we noted a more pronounced endo-metritis than in the immunosuppression studies.Here, too, adhesions between the fallopian tube-ovary complex and other organs were moreextensive than in our previous experiments,indicating more involvement of the peritoneum.Not only did the infection encompass the

entire genital tract, but it also spread to theurinary bladder. We had not previously exam-ined the bladder in our immunosuppression ex-periments with female guinea pigs, but we re-cently reported an extensive involvement of thebladder in male guinea pigs immunosuppressedwith cyclophosphamide (17). In the previousstudy (17), cystitis occurred in only a smallpercentage of immunologically intact guineapigs.Not unexpectedly, estrogen levels in the es-

tradiol-treated guinea pigs were higher than es-tradiol levels in sesame oil-treated animals.Obviously, the 1-mg estradiol dosage was phar-macological and produced considerably greaterplasma levels than would be expected via endog-enous secretion. It is still difficult at this point torelate these data obtained in the guinea pig to thepotential risk for women undergoing hormonalmanipulation. Only minimal information is avail-able in the literature describing hormonal influ-ences on chlamydial genital infections. Tuffreyand Taylor-Robinson (23) were only able toroutinely infect mice in the genital tract with theSA2 (F) strain of C. trachomatis when theytreated mice with progesterone. Hilton et al. (6)reported that women using oral contraceptiveshad a significantly higher rate of chlamydialinfections than did the control population. Ripaet al. (18) also observed that in women under 25years of age, a chlamydial cervicitis was foundmore often in users of oral contraceptives thanin nonusers. In contrast, Oriel et al. (15) foundno significant effects of oral contraceptives onthe incidence of chlamydial genital infections.Thus, hormonal manipulation may apparentlyeither increase the susceptibility to chlamydialinfection or induce the shedding of the organismfrom a latent infection so that the individualsbecome culture positive.Our data in this animal model system suggest

that alterations in hormonal balance might pro-vide a greater potential for more serious infec-tions. Moreover, because of the cyclical varia-

tion in hormone levels in the female, thepotential for exacerbated infections may be in-creased at specific times during the menstrualcycle, i.e., when ovarian estrogen production iselevated. This could be one possible explanationfor the development of chlamydial-induced pel-vic inflammatory disease in some women but notothers. The mechanism by which estradiolcauses the enhancement of infection has notbeen defined, but might be related to eitherhormonally induced immunosuppression or achange in either host cell numbers or host cellsusceptibility to infection.One explanation for the enhanced GPIC infec-

tion is that estradiol treatment modified the cellpopulation of the genital tract so that either thecells became more susceptible to infection or anincrease in the number of susceptible cells oc-curred. Among the changes in the genital tractinduced by estrogens are an increase in celldivision, protein synthesis, and nucleic acidcontent as well as an increase in glycogen con-tent of the cells (5, 24). Such changes might bemore compatible with chlamydial growth anddevelopment. Indeed, an accelerated rate ofhost cell division might make more cells avail-able for infection as infected cells are shed anddestroyed. This hypothesis could account forthe apparent high number of infected cells de-tected by indirect immunofluorescence in tissuesections from estradiol-treated animals as wellas in Giemsa-stained smears of vaginal scrap-ings.

In the present study we did not demonstrateany effect of estradiol treatment on the immuneresponse to GPIC. Serum antibody titers wereapproximately the same in estradiol-treated andcontrol animals. There were also no significantdifferences in delayed hypersensitivity to GPICbetween the two groups. These results are notsurprising since the major target organ of estra-diol is the genital tract and not the peripherallymphoid system (7). Although we did not deter-mine the antibody titers in genital tract secre-tions in the present investigation, we have ob-tained evidence that estradiol does indeedsuppress the local humoral immune response,resulting in a prolonged GPIC infection (1Sa).

It is quite possible that two separate mecha-nisms are responsible for the results observed.The increased intensity of the infection may bethe result of alteration of host cell susceptibilityor host cell numbers, whereas the prolongationof the infection may be caused by a suppressionof the local immune response in the genital tract.

ACKNOWLEDGMENTS

This study was supported by Public Health Service grantA113069 from the National Institute of Allergy and InfectiousDiseases and in part by Public Health Service BiomedicalResearch Support grant 5S07RR5350 from the General Re-

INFECT. IMMUN.

ESTRADIOL EFFECT ON CHLAMYDIAL INFECTION 705

search Support Branch, Division of Research Resources.We thank Estelle Moses for the preparation of the GPIC

skin test antigen, Lisa Kelly and Danny Smith for theirexcellent technical assistance, and Phillip Holcomb for statis-tical analyses.

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