INFECTION AND IMMUNITY, Mar. 2002, p. 1538–1546
0019-9567/02/$04.00?0 DOI: 10.1128/IAI.70.3.1538–1546.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 3
Chemokine Expression Patterns Differ within Anatomically Distinct
Regions of the Genital Tract during Chlamydia trachomatis Infection
Heather K. Maxion and Kathleen A. Kelly*
Department of Pathology and Laboratory Medicine, UCLA Medical Center, Los Angeles, California 90095-1732
Received 15 August 2001/Returned for modification 2 October 2001/Accepted 27 November 2001
Untreated infections with Chlamydia trachomatis commonly result in ascending infection to fallopian tubes
and subsequent immune-mediated tubal pathology in females. The proposed immune-mediated injury may be
associated with the increased recruitment of CD4 cells to the upper genital tract (GT) (oviducts) in comparison
to the lower GT (cervix) during infection, as shown in animal models. To understand the mechanisms
responsible for this biased recruitment of CD4 cells within the GT, we characterized chemokine expression
patterns in the upper and lower GTs in mice during infection with the murine pneumonitis biovar of Chlamydia
trachomatis. Enzyme-linked immunosorbent assays of supernatants from GT homogenates revealed that the
levels of the Th1-associated chemokines CXCL9 (monokine induced by gamma interferon), CXCL10 (inter-
feron-inducible protein 10), and CCL5 (RANTES) were significantly higher in the upper GT than in the lower
GT after infection, while the CCL3 (macrophage inflammatory protein 1?) level was not increased. In contrast,
the level of chemokine CCL11 (eotaxin) was significantly elevated in the lower GT later in the course of
infection. Increased levels of mRNA confirmed the selective differences in chemokine expression within the
upper and lower GTs. The increased levels of Th1-inducible chemokines in the upper GT were not due to
differences in the magnitude of infection or progesterone pretreatment. These data demonstrate that the upper
and lower regions of the GT respond differently to Chlamydia infection.
The gram-negative bacterium Chlamydia trachomatis is sex-
ually transmitted and infects squamous epithelial cells in the
female cervix. From here, the bacteria ascend and establish
infection in columnar cells of human fallopian tubes or ovi-
ducts in mice. Left untreated, human infection can lead to
pelvic inflammatory disease, fallopian tube injury, and infertil-
ity (22). Oviduct injury, characterized by hydrosalpinx, and
infertility are also seen in murine infections (33). The devel-
opment of T-cell-mediated immunity is one of the most crucial
elements required for the effective clearance of this pathogen.
A CD4 Th1 response is necessary for Chlamydia eradication,
and this finding has been demonstrated by prolonged infection
in gamma interferon knockout mice (23) as well as in mice
injected with blocking antibodies against the Th1-inducing cy-
tokine interleukin 12 (IL-12) (23). Additionally, the ability to
clear infection in nude mice is restored following the adoptive
transfer of an anti-C. trachomatis murine pneumonitis biovar
(MoPn) CD4 Th1 clone (15). Similarly, IL-10 knockout mice
exhibit a shorter course of infection (37). Thus, the regulation
of Th1 and Th2 responses in the genital tract (GT) during
Chlamydia infection is a crucial factor controlling the duration
of infection and subsequent tubal pathology.
Chemokines are a rapidly growing family of small chemo-
tactic molecules that are specific for various subsets of lym-
phocytes as well as other types of leukocytes. Increasing evi-
dence suggests that chemokines play an important role in the
regulation of Th1 and Th2 responses in vivo. These responses
appear to be directed by the differential expression of chemo-
kine receptors on Th1- and Th2-cell subsets (21). Many studies
have demonstrated patterns of either Th1- or Th2-associated
chemokines in diseased tissues previously shown to contain
large infiltrates of either Th1 or Th2 cells (9). For instance, in
the Th1-mediated disease multiple sclerosis, high levels of che-
mokines CXCL10 (interferon-inducible protein 10 [IP-10]),
CXCL9 (monokine induced by gamma interferon [MIG]), and
CCL5 (RANTES) are found in the cerebrospinal fluid (30).
These data suggest that the chemokine profile plays a central
role in determining the predominant T-cell subset associated
with a particular disease or infection.
Chemokines also provide fine specificity for the direction of
cellular recruitment to discrete anatomical regions within a
given tissue. For example, site specificity has been noted at
mucosal surfaces, where CCL25 (thymus-expressed chemo-
kine) has been shown to localize to the epithelium of the small
intestine but not the large intestine (19). Many chemokines
have been detected in the endometrial epithelium within the
female GT in humans, including CCL3 (macrophage inflam-
matory protein 1? [Mip-1?]) (1), CCL5 (RANTES) (2), CCL2
(monocyte chemotactic protein 1 [MCP-1]) (4), and CCL11
(eotaxin) (13). However, it is not known whether chemokine
expression differs within functionally discrete regions of the
GT. It was previously shown that a significantly larger number
of CD4 cells are recruited to the oviducts (upper GT) than to
the cervical-vaginal region (lower GT) of mice infected with
MoPn (18). To investigate the basis for the increased recruit-
ment of CD4 cells to the upper GT, we evaluated the expres-
sion of chemokines associated with Th1 and Th2 responses in
the upper and lower GTs during infection.
MATERIALS AND METHODS
Infection. Female BALB/c mice, 4 to 6 weeks old, were purchased from Harlan
Sprague-Dawley (Indianapolis, Ind.) and were housed according to American
* Corresponding author. Mailing address: UCLA Medical Center,
Department of Pathology and Laboratory Medicine, 10833 Le Conte
Ave., Mailroom A2-179 CHS, Los Angeles, CA 90095-1732. Phone:
(310) 206-5562. Fax: (310) 794-4863. E-mail: firstname.lastname@example.org.
Association of Accreditation of Laboratory Animal Care guidelines. Experimen-
tal procedures were approved by the UCLA Institutional Animal Care and Use
Committee. All mice were first injected subcutaneously with 2.5 mg of medroxy-
progesterone acetate (Depo-Provera; Upjohn, Kalamazoo, Mich.) in 100 ?l of
sterile phosphate-buffered saline. Medroxyprogesterone acetate drives mice into
a state of anestrus, thus eliminating the variability in the rate and severity of
infection due to the estrous cycle (26). Seven days later, while under sodium
pentobarbital anesthesia, all mice were inoculated with 107inclusion-forming
units (IFU) (50% infective dose, 1.5 ? 103IFU) of MoPn grown in McCoy cells.
Mice were killed on days 3, 7, 14, 21, and 35 after inoculation. Infection was
monitored by examining cervical-vaginal swab samples (Dacroswab type 1; Spec-
trum Labs, Houston, Tex.) obtained immediately before mice were killed and
homogenates of tissue samples obtained from the upper and lower GTs. Swab
samples were stored in sucrose-phosphate buffer at ?70°C until analyzed. Tissue
homogenate samples were also frozen at ?70°C until analyzed.
Tissue homogenates. GT tissues were divided into the cervical-vaginal region
(lower GT) and oviducts (upper GT) with the ovaries removed. Uterine horns
were not included in our analysis. Tissue sections from individual mice were
placed in 1 ml of a protease inhibitor buffer (1 ?g each of antipain, aprotinin,
leupeptin, and pepstatin A/ml and 2 mM phenylmethylsulfonyl fluoride in sterile
phosphate-buffered saline) (Sigma, St. Louis, Mo.) and homogenized as previ-
ously described (3) by using a hand-held homogenizer (Omni International,
Warrenton, Va.). Aliquots of each homogenate were removed for isolation of
chlamydiae. The remaining homogenate volumes were sonicated at 20 kHz for 1
min and then centrifuged at 900 ? g for 15 min at 10oC to remove cellular debris.
Supernatants were filtered through 0.2-?m-pore-size Acrodisks (Gelman Sci-
ences, Ann Arbor, Mich.) to remove chlamydiae, and samples were stored at
?70oC until analyzed.
Isolation of chlamydiae from cervical-vaginal swab and tissue homogenate
samples. Swab samples were prepared as previously described. McCoy cell
monolayers in individual wells of 96-well plates were inoculated with 200 ?l of
the solution from vaginal swabs or homogenized GT tissue (18), followed by
centrifugation at 1,900 ? g for 1 h. The plates were incubated for 2 h at 37°C. At
this time, the isolation solutions were removed, fresh cycloheximide medium was
added, and the plates were incubated for an additional 32 h. The cultures were
then fixed with methanol. MoPn inclusions were identified by the addition of
anti-MoPn immune sera and anti-mouse immunoglobulin G conjugated to flu-
orescein isothiocyanate (ICN Immunobiologicals, Irvine, Calif.). The inclusion
bodies within 20 fields (?40) were counted under a fluorescence microscope, and
numbers of IFU per milliliter were calculated (17). Data were adjusted for IFU
per milligram of crude homogenized GT tissue (upper or lower).
Chemokine ELISAs. Recombinant protein and antibodies against CCL3 (Mip-
1?), CCL11 (eotaxin), CXCL9 (MIG), CXCL10 (IP-10), and CCL5 (RANTES)
were purchased from R&D Systems (Minneapolis, Minn.) and those against
CCL2 (MCP-1) were purchased from PharMingen (San Diego, Calif.) for use in
enzyme-linked immunosorbent assays (ELISAs). Upper and lower GT homog-
enates were added to duplicate wells of microtiter enzyme immunoassay plates
(Costar/Corning, Acton, Mass.) and assayed according to the manufacturer’s
protocol with the following exceptions. CXCL10 and CXCL9 primary antibody
concentrations were 1 and 2 ?g/ml, respectively, and secondary antibody con-
centrations were 0.5 ?g/ml. The recommended substrate was replaced with
1-StepTM Turbo TMB-ELISA substrate (Pierce Chemical Co., Rockford, Ill.).
The optical densities were read at 450 nm with a microplate reader (model 550;
Bio-Rad, Hercules, Calif.). Chemokine values were determined from a standard
curve generated with recombinant chemokines by using microplate reader soft-
ware. Chemokine values were corrected for total protein by using a micro-
bicinchoninic acid protein assay kit (Pierce).
Serum progesterone levels. Serum was collected from mice administered or
not administered medroxyprogesterone acetate. Progesterone levels were deter-
mined by a competitive electrochemiluminescence immunoassay with an Elecsys
2010 automated analyzer (Roche, Berkeley, Calif.).
mRNA isolation and SuperArray analysis. Total RNA was isolated from
paired GT tissues of mice according to the manufacturer’s protocol following
homogenization of tissues in RNAzol B (Tel-Test, Inc., Friendswood, Tex.) and
stored at ?80°C until use. Nonrad-GEArray kits specific for chemokine analysis
were purchased from SuperArray Inc. (Bethesda, Md.). Each kit provides a
matched set of membranes containing 23 chemokines plus controls. Probe syn-
thesis was carried out by using 10 or 7 ?g of mRNA per sample. The manufac-
turer’s protocol was followed for all steps. Following substrate addition, mem-
branes were exposed to X-ray film (Fuji, Tustin, Calif.) for 5 to 10 min. Data were
quantified by using a laser densitometer and ImageQuaNT software (Molecular
Dynamics, Sunnyvale, Calif.) to calculate the average integrated volumes of dots.
Data were expressed as the average integrated volume of a sample relative to the
average integrated volume of a positive control (glyceraldehyde-3-phosphate
Immunohistochemical analysis. Tissues were harvested and prepared as pre-
viously described (18). Staining was carried out as previously described with the
following exceptions. After a tissue blocking step with rabbit serum, the primary
antibodies (goat anti-mouse CXCL10 [IP-10] and goat anti-mouse CCL11
[eotaxin]) (R&D Systems) were incubated on tissue sections for 45 min at room
temperature in a humidified chamber, and then the sections were washed. A
rabbit anti-goat immunoglobulin G antibody conjugated to biotin at 30 ?g/ml
(Antibodies Inc., Davis, Calif.) and streptavidin conjugated to horseradish per-
oxidase (Zymed, San Francisco, Calif.) were added next, and the tissue sections
were incubated for 45 min. Slides were developed as previously described (18).
Photographs were generated by scanning the microscope slides with an Olympus
DP10 color digital video camera.
Statistics. Statistical differences in chemokine protein levels were tested by
using two-way analysis of variance (ANOVA) and Tukey’s post hoc test. Statis-
tical differences in chemokine message levels were determined by using a paired
Student t test. The above statistical tests were suggested by and performed with
SigmaStat software based on the distribution of the data and sample size (Jandel
Scientific, San Rafael, Calif.). Groups were considered statistically different at a
P value of ?0.05.
Differential expression of chemokines in the upper and
lower GTs in response to C. trachomatis infection. To deter-
mine whether chemokine production differed between the up-
per and lower GTs during MoPn infection, we measured pro-
tein levels in tissue homogenates at weekly intervals that
spanned the induction phase (0 to 14 days) and the resolution
phase (14 to 35 days) of infection. We evaluated the expression
of chemokines that are generally associated with a Th1 re-
sponse, CXCL10 (IP-10), CXCL9 (MIG), CCL3 (Mip-1?),
and CCL5 (RANTES), or a Th2 response, CCL11 (eotaxin).
We also evaluated CCL2 (MCP-1), which has not been shown
to associate with any particular T-cell subset. As shown in Fig.
1A, the Th1-associated chemokines CXCL10 (IP-10), CXCL9
(MIG), and CCL5 (RANTES) were all induced by infection in
the upper GT. CXCL10 (IP-10) was measured at a significantly
elevated level on day 3 compared to the level in uninfected
mice. The level of CXCL9 (MIG) was significantly elevated on
day 7 and again later in infection, on day 35, whereas an
elevated level of CCL5 (RANTES) was maintained in the
upper GT from day 7 throughout the course of infection. In the
lower GT (Fig. 1B), the levels of both CXCL9 (MIG) and
CCL5 (RANTES) were elevated early in the course of infec-
tion and the CCL5 (RANTES) level was significantly increased
late in the course of infection compared to the results for
controls. Finally, CCL3 (Mip-1?) was not induced by infection
in either the upper or the lower GT. However, CCL3 (Mip-1?)
was expressed at a constitutively higher level in the upper GT
than in the lower GT.
We next examined the protein levels of CCL11 (eotaxin) and
CCL2 (MCP-1) in the upper and lower GTs with ELISAs. As
shown in Fig. 2A, CCL11 (eotaxin) expression was induced by
infection in the upper GT but only later in the course of
infection (day 14). Furthermore, the level of CCL11 (eotaxin)
was comparatively lower than the peak levels of Th1-associated
chemokines in the upper GT (Fig. 1A). CCL11 (eotaxin) was
also induced in the lower GT (Fig. 2B) by day 14 and, inter-
estingly, was expressed to a significantly higher degree in the
lower GT than in the upper GT during the resolution phase of
infection (Fig. 2). There was no difference in CCL2 (MCP-1)
expression between uninfected and infected tissues and be-
VOL. 70, 2002 CHEMOKINE EXPRESSION AND CHLAMYDIA GENITAL INFECTION1539
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Editor: J. D. Clements
1546MAXION AND KELLYINFECT. IMMUN.