CCL5, CCR5, and IFN-γ mRNAs were measured by quantitative RT-PCR analysis after genital chlamydial infection. A significant increase in CCR5, CCL5, and IFNγ gene expression in the spleen and ILN was observed 7 days after genital infection when compared with levels before infection (Figure 1). These mRNA levels modestly declined at inductive sites 14 days after infection. CCR5, but not CCL5 or IFN-γ mRNA expression by fallopian tube-, uterus-, and cervix- derived lymphocytes were considerably higher than levels before infection. Indeed, CCR5 mRNA expression by fallopian tube lymphocytes was significantly higher 7 and 14 days post infection. These data suggest that increases in CCL5, CCR5, and IFN-γ mRNA expression during early stages of infection at inductive sites (i.e., spleen and ILN) preceded CCR5 expression at effectors sites (i.e., fallopian tube(s), uterus, and cervix). The pattern of CCL5 and CCR5 as well as IFN-γ illustrates how this chemokine axis coincides with both the innate (recognition phase, 0 to 7 days) and adaptive (activation, effector, and decline/homeostasis phases) immune responses to this pathogen and associated inflammation.

We next characterized C. muridarum-specific proliferative responses of T helper cells isolated from the spleen, ILNs, fallopian tube(s), uterus, and cervix, 42 days after challenge. CD4⁺ T cells isolated from these mucosal and systemic immune compartments of C. muridarum-infected and control Ab-treated mice exhibited marked increases in Ag-specific proliferative responses compared to disease-free (uninfected) or C. muridarum-infected and anti-CCL5 Ab-treated mice (Figure 2). Notably, systemic and mucosal inductive sites of the spleen and ILN, respectively, contained Ag-specific CD4⁺ T cells that significantly proliferated after C. muridarum re-stimulation. These results suggest that CCL5 is required for optimal generation of Chlamydia-specific CD4⁺ T cells that proliferate after Ag recognition.

To test the role of CCL5 in Th1-biased humoral responses to genital infection, we measured Chlamydia-specific IgG1, IgG2a, IgG2b, IgG3 and IgM Abs in sera as well as Ag-specific IgA and IgG Abs in vaginal washes. Infected mice that received control Ab displayed significantly higher levels of Chlamydia-specific serum IgG2a, followed by IgG2b responses than compared to similar mice that received anti-CCL5 Ab or mock-infected (i.e., naïve) mice (Figure 3). Analysis of vaginal secretions revealed a significant increase in Ag-specific IgA Ab responses in C. muridarum-challenged mice that received control Ab when compared with similar mice that received anti-CCL5 Ab or uninfected animals. These results indicate C. muridarum infection enhanced Th1-biased humoral responses and CCL5 blockade attenuated Chlamydia-specific IgG2a serum responses as well as vaginal IgA responses.

Genital Chlamydia infection up-regulated Ab responses as well as splenic and ILN CD4⁺ T cell proliferative responses. We next examined whether these effects were mediated in part through T helper cytokine responses. IL-2 secreted by Ag-stimulated splenic CD4⁺ T cells from infected mice that received control Ab was significantly higher than levels from cells isolated from similar mice that received anti-CCL5 Ab or uninfected mice (Figure 4). The secretion of IFN-γ from splenic, but not ILN, CD4⁺ T cells from C. muridarum-infected and control Ab-treated were considerably higher than T helper cells isolated from similar mice treated with anti-CCL5 Ab or uninfected animals. Chlamydial infection of mice lead to the development of CD4⁺ T cells that significantly secreted IL-6, IL-10, and GM-CSF, but not IL-4, in response to C. muridarum re-stimulation. Anti-CCL5 Ab-treated mice infected with C. muridarum resulted in T helper cells with reduced IL-6, IL-10, and GM-CSF secretion following Ag re-stimulation when compared to CD4⁺ T cells from similar mice treated with control Ab; however, the secretion pattern of Th2 cytokine responses by CD4⁺ T cells isolated from ILNs did not significantly change. These results indicate that C. muridarum infection invoked splenic, but not ILNs, Chlamydia-specific CD4⁺ T cells that secreted Th1 cytokines as well as IL-6, IL-10, and GM-CSF, which were reduced by CCL5 blockade. The extent of chlamydial infection, determined by C. muridarum detected in cervico-vaginal swabs, was significantly higher in mice receiving anti-CCL5 Ab treatment than compared to control mice (Figure 5). However, all mice resolved chlamydial infection in < 90 days.

Stocks of the Chlamydia muridarum (trachomatis agent for mice) were prepared by propagating elementary bodies (EBs) in McCoy cells and used to infect mice as previously described 49. The stocks were tittered by infecting McCoy cells with varying dilutions of EBs. The infectious titer was expressed as inclusion-forming units per milliliter (IFU/ml).

Murine anti-CCL5 was produced as previously described 34. Briefly, rabbits were immunized with murine CCL5 (Pepro-Tech) and the hyper immune CCL5 antisera obtained was yielded titers of ~1:10⁶ such that 10 μl of rabbit CCL5 antiserum neutralized 20 ng of CCL5. This antiserum was titrated by direct ELISA, and no cross-reactivity was detected when tested against other CCR5 ligands, chemokines and cytokines. Subsequently, antisera were heat-inactivated and purified using an IgG isotype-specific protein A column (Pierce). Anti-CCL5 Ab titers were adjusted to 1 : 4 × 10⁵ (i.e., 50× dilution) in PBS (Ab solution) for CCL5 blocking experiments. Similarly prepared normal or preimmune rabbit serum was used to generate the control Ab solution.

Female BALB/c mice, ages 6 to 8 weeks, were purchased from Jackson Laboratory and used to establish a colony at the Morehouse School of Medicine animal facility. The animals were housed and maintained in isolator cages under specific-pathogen-free housing conditions. The guidelines proposed by the Committee for the Care of Laboratory Animal Resources Commission of Life Sciences-National Research Council were followed to minimize animal pain and distress. These studies were approved by the Morehouse School of Medicine Institutional Animal Care and Use Committee (IACUC). Seven days prior to infection, mice received 2.5 mg of medroxy-progesterone acetate (Depo-Provera; The Upjohn Co.) by subcutaneous route in 100 μl of PBS 50. Groups were intravaginally infected with 5 × 10⁴ IFU of C. muridarum under phenobarbital anesthesia, whereas uninfected control mice received only phosphate buffered saline (PBS). Mice (15 mice per group) received 100 μl of either control or anti-CCL5 Abs by intraperitoneal injection, 24 hrs before chlamydial infection and every 72 hrs thereafter. The extent of unresolved chlamydial infection was monitored by taking cervico-vaginal swabs from individual mice every week until 42 days after the challenge 22. Briefly,Chlamydia present in swabs were detected by infecting McCoy cells vaginal/swab rinses and staining infected monolayer with fluorescein isothiocyanate (FITC)-labeled, genus-specific, anti-Chlamydia Abs (Kallestad Diagnostics) to verify inclusion bodies (> 10 IFUs/ml) by direct immuno-fluorescence 49.

Vaginal cavities were rinsed three times with 50 μl of PBS to obtain mucosal secretions. Blood collected by retro-orbital bleeding and serum was separated following centrifugation. Serum and mucosal secretions were collected on 0, 7, 14, and 42 days post challenge and analyzed by ELISA. Following sacrifice by CO₂ inhalation, spleen, ILN, fallopian tube(s), uterus, and cervix tissues were aseptically removed and single-cell suspensions were prepared by passing tissues through a sterile wire screen to quantify mRNA expression and T helper responses. Reproductive tract tissues were further disrupted to generate single cell suspensions by stirring in collagenase type IV (Sigma) in RPMI 1640 (collagenase solution) at 37°C for 30 min. Lymphocytes were further purified using a discontinuous Percoll (Pharmacia) gradient, collected at the 40/75% interface. CD4⁺ T cells were enriched using Mouse CD4 Cellect^® plus columns according to manufacturer's protocol (Biotex Laboratories). Cell suspensions were washed twice in RPMI 1640 and lymphocytes were maintained in medium supplemented with 10 ml/L of nonessential amino acids (Mediatech), 1 mM sodium pyruvate (Sigma), 10 mM HEPES (Mediatech), 100 U/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin (Elkins-Sinn), 50 μM mercaptoethanol (Sigma), and 10% fetal calf serum (FCS) (Atlanta Biologicals).

Purified CD4⁺ T cells were cultured at a density of 5 × 10⁶ cells/ml with 10⁶ cells/ml γ-irradiated (3,000 rads) naïve splenic feeder cells in complete medium in the presence or absence of 10 μg/ml of UV-inactivated C. muridarum inclusion bodies (IBs) as Ag at 37°C in 5% CO₂. After 3 days of culture, cells were pulsed with BrdU labeling solution and incorporation was detected by ELISA (Roche Molecular Biochemical). Similarly, cell culture supernatants were collected 3 days after culture and assayed for cytokine secretion by Luminex assay 34.

Chlamydia-specific serum IgG subclass antibodies and vaginal wash IgA levels were quantified 6 weeks after challenge by ELISA 10. Briefly, 96-well Falcon ELISA plates (Fisher Scientific) were coated with 100 μl of anti-IgG or IgA Ab (BD-PharMingen) or 10 μg/ml of UV-inactivated C. muridarum IBs in PBS O/N at 4°C and blocked with 10% FCS in PBS for 2 hrs at RT. IgM, IgG subclasses or IgA standards, and experimental samples were serially added after diluted with PBS. After O/N incubation at 4°C and three washes using PBS containing 0.05% Tween 20 (PBS-T), Ag-specific titers of IgM, IgG, IgA, or IgG subclass Abs were determined following the addition of biotinylated detection Abs (BD-PharMingen). After incubation and wash steps, anti-biotin HRP Ab (Vector Laboratories Inc., Burlingame, CA) in PBS-T was added to detection wells and incubated for 1 hr at RT. Following incubation, all plates were washed 6 times and the color reaction was developed by adding 100 μl of 1.1 mM 2,2'-azino-bis(3)-ethylbenzthiazoline-6-sulfonic acid (Sigma) in 0.1 M citrate-phosphate buffer (pH 4.2) containing 0.01% H₂O₂ (ABTS solution).

The presence of the following T helper cell-derived cytokines in culture supernatants was determined by Beadlyte™ mouse multi-cytokine detection system kit (BioRad): interleukin (IL)-2, IL-4, IL-6, IL-10, granulocyte monocyte cell stimulating factor (GM-CSF), and interferon (IFN)-γ. Filter-bottom ELISA plates (BioRad) were rinsed with 100 μl of Bioplex assay buffer and the buffer was removed using a Millipore™ multiscreen separation vacuum manifold system set at 5 mm Hg. Analyte beads in assay buffer were added into wells, followed by 50 μl of serum or standard solution and incubated for 30 mins at RT with continuous shaking (at setting #3) using a Lab-Line™ Instrument Titer Plate Shaker (Melrose, IL). The filter-bottom plates were washed as before and the buffer was removed using a Millipore™ multiscreen separation vacuum manifold system. Subsequently, 50 μl of anti-mouse IL-2, IL-4, IL-6, IL-10, GM-CSF, or IFN-γ Ab-biotin reporter solution was added in each well and the plates were incubated with continuous shaking for 30 mins followed by centrifugation and washing. Next, 50 μl of streptavidin-phycoerytherin (PE) solution was added and the plates were incubated with continuous shaking for 10 mins at RT. 125 μl of Bio-plex assay buffer was added and Beadlyte™ readings were measured using a Luminex™ System and calculated using Bio-plex™ software (Bio-Rad). The cytokine Beadlyte™ assays were capable of detecting > 5 pg/ml for each analyte.

Total RNA from the spleen, ILNs, fallopian tube, uterus, and cervix leukocytes was isolated from mouse treated with anti-CCL5 or control Ab using Tri-reagent™ (Molecular Research Center, Cincinnati, OH). Potential genomic DNA contamination was removed from these samples by treatment with RNase-free DNase (Invitrogen) for 15 mins at 37°C. RNA was then precipitated and re-suspended in RNA secure (Ambion). cDNA was generated by reverse transcribing approximately 1.5 μg of total RNA using Taqman™ reverse transcription reagent (Applied Biosystems).

Mouse mRNA sequences of CCL5, CCR5, IFN-γ, and 18S rRNA were obtained from the NIH-NCBI gene bank database accession numbers NM03653, D83648, K00083, and X00686.1, respectively. These sequences were then used to design primers for real-time polymerase chain reaction (RT-PCR) analysis, which generated amplicons of 97, 100, 98, and 149 base pairs size, respectively, for CCL5 (sense-TCG TGT TTG TCA CTC GAA GG and antisense- GCT GAT GGC CTG ATT GTC TT), CCR5 (sense- CGA AAA CAC ATG GTC AAA CG and antisense- GGG AAG CGT ATA CAG GGT CA, IFN-γ (sense- ACT GGC AAA AGG ATG GTG AC and antisense- GTT CTC CTG TGG ATC GGG TA), and 18S rRNA (sense-GTA ACC CGT TGA ACC CAA TT and antisense- CAA TCC AAT CGG TAG TAG CG). Primers were designed using the primer 3 software program from Whitehead Institute at the Massachusetts Institute of Technology (MIT). Thermodynamic analysis of primers was conducted using the following computer programs: Primer Premier™ (Integrated DNA Technologies) and MIT Primer III (Boston, MA). The resulting primer sets were compared against the entire murine genome using the National Center for Biotechnology Information (NCBI) to confirm specificity and ensure that the primers flanked mRNA splicing regions. cDNA was generated as before and amplified with specific cDNA primers using SYBR^® Green PCR master mix reagents (Applied Biosystems). The copy number (> 10) of mRNA relative to 18S rRNA copies was evaluated by RT-PCR analysis using the BioRad Icycler and software (Hercules, CA).

Data were expressed as the mean ± standard error of mean (SEM), compared using a two-tailed student's t-test or an unpaired Mann Whitney U-test, and considered statistically significant if p < 0.01. When cytokine levels were below detection (BD) limit, they were recorded as one-half the lower detection limit (e.g., 5 pg/ml for IL-10) for statistical analysis.