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The Journal of Immunology, 2006, 176: 4959-4967.
Copyright © 2006 by The American Association of Immunologists

STAT1 in Peripheral Tissue Differentially Regulates Homing of Antigen-Specific Th1 and Th2 Cells1

Zamaneh Mikhak, Carolyn M. Fleming, Benjamin D. Medoff, Seddon Y. Thomas, Andrew M. Tager, Gabriele S. Campanella and Andrew D. Luster2

Division of Rheumatology, Allergy and Immunology, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Th1 and Th2 effector CD4+ T cells orchestrate distinct counterregulatory biological responses. To deliver effective tissue Th1- and Th2-type responses, Th1 and Th2 cell recruitment into tissue must be differentially regulated. We show that tissue-derived STAT1 controls the trafficking of adoptively transferred, Ag-specific, wild-type Th1 cells into the lung. Trafficking of Th1 and Th2 cells is differentially regulated as STAT6, which regulates Th2 cell trafficking, had no effect on the trafficking of Th1 cells and STAT1 deficiency did not alter Th2 cell trafficking. We demonstrate that STAT1 control of Th1 cell trafficking is not mediated through T-bet. STAT1 controls the recruitment of Th1cells through the induction of CXCL9, CXCL10, CXCL11, and CXCL16, whose expression levels in the lung were markedly decreased in STAT1–/– mice. CXCL10 replacement partially restored Th1 cell trafficking in STAT1-deficient mice in vivo, and deficiency in CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, impaired the trafficking of adoptively transferred Th1 cells in wild-type mice. Our work identifies that STAT1 in peripheral tissue regulates the homing of Ag-specific Th1 cells through the induction of a distinct subset of chemokines and establishes that Th1 and Th2 cell trafficking is differentially controlled in vivo by STAT1 and STAT6, respectively.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The homing of lymphocytes to distinct anatomical sites plays a critical role in their function. Naive and central memory T cells recirculate through secondary lymphoid organs in search of their cognate Ags, while effector T cells home to tissue sites to mount host defense and regulate inflammation (1). Effector CD4+ T cells mediate Th1- and Th2-type immune responses. Th1 cells secrete IFN-{gamma} and TNF, regulate IgG2a class switching, and coordinate cell-mediated immunity against intracellular pathogens. Th2 cells secrete IL-4, IL-5, IL-9, and IL-13, regulate IgE class switching, and mediate the allergic response and the host defense against parasites (2, 3). To deliver effective tissue Th1- and Th2-type immune responses, the recruitment of Th1 and Th2 cells into tissue must be differentially regulated since the Th1 response suppresses Th2 inflammation and the Th2 response suppresses Th1 inflammation (4, 5, 6, 7).

Chemokines and their receptors expressed on T cells play an important role in controlling T cell trafficking patterns. For example, CCL21 and its receptor CCR7 regulate homing of T cells from peripheral tissues into afferent lymphatics (8, 9). Distinct subsets of chemokine receptors have been described on Th1 and Th2 cells and have been postulated to control the differential trafficking of these cells in vivo (1, 10). Th1 cells have been shown to differentially express CCR5, CXCR3, CXCR6, and CX3CR1, and traffic to sites of Th1 inflammation where the ligands for these chemokine receptors are up-regulated (11, 12, 13). In contrast, Th2 cells have been shown to differentially express CCR3, CCR4, and CCR8 and traffic to sites of allergic inflammation where the ligands for these receptors are expressed (11, 12, 14, 15, 16).

Although these correlations are suggestive, chemokine receptors are not unique markers of Th1 and Th2 cells (17, 18) and, furthermore, mice deficient in these chemokines and/or chemokine receptors have not revealed that lack of any one receptor/ligand pair completely abrogates the trafficking of Th1 and Th2 cells in vivo (19, 20). This has led us to the hypothesis that recruitment of effector CD4+ T cells is coordinately regulated by subsets of chemokines expressed at sites of Th1- and Th2-type inflammation and their corresponding subsets of chemokine receptors expressed on Th1 and Th2 cells. In this regard, we have previously shown that STAT6 is a master regulator of Th2 cell trafficking in vivo. STAT6–/– mice did not express Th2-type chemokines in the lung and as a result failed to recruit Ag-specific Th2 cells into the lung following Ag challenge (21). In the present study, we asked whether a master transcriptional regulator similarly controls Th1 cell trafficking and if, in fact, Th1 and Th2 cell trafficking is differentially controlled by these regulators in vivo. We have found that STAT1, a transcription factor important for IFN-{gamma} signal transduction, controls Ag-specific Th1 but not Th2 cell trafficking into the lung following Ag challenge, while STAT6, which controls Th2 cell trafficking, has no effect on the trafficking Th1 cells. Our study established for the first time that Th1 and Th2 cell trafficking is differentially regulated in vivo by STAT1 and STAT6, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

DO11.10 mice, in the BALB/c background, transgenic for the TCR recognizing OVA peptide 323–339 (pOVA 323–39), and STAT6–/– mice, in the BALB/c background, were purchased from The Jackson Laboratory and bred in our animal facility. STAT1–/– mice were a gift from D. E. Levy (New York University School of Medicine, New York, NY) (22); T-bet–/– mice were a gift from L. Glimcher (Harvard Medical School, Boston, MA) (23). STAT1–/– and T-bet–/– mice, also in the BALB/c background, were subsequently bred at Charles River Laboratories. Wild-type (WT)3 BALB/c mice, between 6 and 8 wk of age, were obtained from The Jackson Laboratory. C57BL/6 mice, congenic for Thy1.1 and Thy1.2, were purchased from The Jackson Laboratory. OTII (Thy1.1) mice were a gift from P. Shrikant (Roswell Park Cancer Institute, Buffalo, NY), OTII (Thy1.2) mice were a gift from L. Lefrancois (University of Connecticut School of Medicine, Farmington, CT), CXCR3–/– mice (in the C57BL/6 background) were a gift from C. Gerard (Harvard Medical School) (24), and CXCL9–/– mice (in the BALB/c background) were a gift from J. Farber (National Institutes of Health, Bethesda, MD) (25). CXCR3–/–OTII and Thy1.1/Thy1.2 mice were bred in our laboratory. All experiments were done according to protocols approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Generation of Th1 and Th2 cells

Th1 and Th2 cells were generated as described previously (26). Briefly, CD4+ T cells were isolated from spleen and pooled lymph nodes of DO11.10 mice using CD4 Dynabeads (Dynal Biotech). For experiments involving OTII and CXCR3–/–OTII mice, naive Th cells were isolated using the CD4+CD62L+ T Cell Isolation kit (Miltenyi Biotec). Purified CD4+ T cells (2 x 105 cells/ml) were activated in the presence of irradiated (3000 rad) splenocytes (2 x 106 cells/ml), 1 µg/ml pOVA323–339, and 1 µg/ml anti-CD28 (BD Pharmingen) in a 24-well plate. Th2 cells were generated by activating the cells in the presence of 1000 U/ml IL-4 (PeproTech) and anti-IFN-{gamma} (R46A2) at inhibitory concentrations. Th1 cells were generated by activating the cells in the presence of 100 U/ml IL-12 (PeproTech) and anti-IL-4 (11B11) at inhibitory concentrations. Cells were fed with IL-2 (5–10 U/ml; PeproTech) initially on day 2 and then daily until used between days 5 and 7.

Intracellular staining

Aliquots of cells (0.5 x 106 cells) were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml; Sigma-Aldrich) at 37°C for 1 h. The reaction was stopped using GolgiStop (BD Biosciences) at 1.3 µl/ml at 37°C for 3 h. Cells were washed and nonspecific staining was minimized by blocking with anti-mouse CD16/CD32 (BD Pharmingen) for 10 min on ice. The cells were washed and permeabilized with Fix and Perm fixation medium A and B (Caltag Laboratories) and stained intracellularly with 2.5 µl of PE-conjugated rat anti-mouse IL-4 (0.2 mg/ml; BD Pharmingen) and 4 µl of FITC-conjugated IFN-{gamma} (0.5 mg/ml; BD Pharmingen) and isotype controls. The level of intracellular cytokine staining was determined by flow cytometry to assess the degree of Th1 and Th2 polarization.

CFSE staining

To evaluate proliferation of adoptively transferred cells, cells were stained with CFSE, washed twice in PBS, and suspended in RPMI 1640 at 20 million cells/ml. Three microliters of CFSE was added to each milliliter of cells. After incubation at 37°C for 20 min, cells were overlaid on an equal volume of FBS and centrifuged at 1300 rpm for 10 min. The cell pellet was resuspended in RPMI 1640 medium to a concentration of 20 million cells/ml and incubated at 37°C for 15 min. Cells were washed in PBS twice and resuspended to a concentration of 10 million/ml for adoptive transfer. Level of CFSE staining was determined by flow cytometry. Cells were transferred into WT, STAT1–/–, and STAT6–/– mice via tail vein injection followed by four daily OVA challenges. Single-cell suspensions of the bronchoalveolar lavage (BAL) and lung were PE stained for the mouse D011.10 TCR (KJ1-26; Caltag Biochemicals) and allophycocyanin stained for CD4 (BD Pharmingen). Flow cytometry was performed and Ag-specific T cells positive for CD4 and DO11.10 TCR were identified. The extent of cellular proliferation was determined by analyzing CFSE staining of these Ag-specific cells by flow cytometry.

Transfer of Th1 or Th2 cells and OVA challenges

Th1 or Th2 cells were harvested on days 5–7, washed twice with PBS, and 5 x 106 cells were injected i.v. via the tail vein into naive BALB/c (WT), STAT1–/–, STAT6–/–, T-bet–/–, or CXCL9–/– mice. After transfer, mice were challenged once a day (25 min/day) with 5% OVA solution or PBS using a nebulizer (Pulmo Aide; DeVil Biss). For CXCL10 replacement, CXCL10 was used at 5 µg/50 µl of PBS and administered intranasal after anesthesia using isoflurane. CXCL10 was prepared as previously described (27). In separate experiments, Th1 cells were collected on day 5, resuspended at 4 x 105 cells/ml and treated with pertussis toxin (PTX) (100 ng/ml; Sigma-Aldrich) or vehicle (distilled H2O) for 24 h. Cells were then collected, washed with PBS, and 5 x 106 cells were adoptively transferred (i.v. via the tail vein) into WT mice and challenged with aerosolized OVA as above for 4 days.

Limulus amebocyte lysate assay for LPS

Five-percent OVA in PBS was serially diluted in PBS. Samples were added to the tubes from the Limulus assay kit (Charles River Endosafe). The solution was gently mixed and incubated at 37°C for 1 h and assessed for coagulation (the LPS content was found to be 0.6 endotoxin units/ml at 1 µg/ml OVA.)

BAL and lymphocyte isolation

BAL was performed 20–24 h after the final aerosol challenge with six 0.5-ml aliquots of PBS with 0.6 mM EDTA. Lungs were cut into small pieces and digested in RPMI 1640 medium plus 0.28 Wunsch U/ml Liberase Blenzyme (Roche) and 30 U/ml DNase (Sigma-Aldrich) for 45 min at 37°C. Single-cell suspensions of lymphocytes were made from the spleen, thoracic lymph nodes (TLN), peripheral lymph nodes (PLN), and digested lungs after passage through a 70-µm cell strainer (Fisher). Recovered cells were lysed with RBC lysis buffer (Sigma-Aldrich) and washed. Live cells were counted with a hemocytometer. Differential cell counts were determined using DiffQuik-stained cytocentrifuged cell preparations from BAL by counting two high-powered fields per sample. The investigator counting the cells was blinded to the treatment groups.

Quantitative PCR

Total RNA was isolated from BAL and tissue samples using RNeasy (Qiagen). RNA was converted to cDNA and analyzed by quantitative PCR as previously described (28) using the Mx4000 Multiplex Quantitative PCR System (Stratagene).

Flow cytometry

Cell suspensions from BAL, lung, lymph nodes, and spleen were analyzed by flow cytometry on a FACScan (BD Biosciences) cytofluorometer as described previously (29). Commercially conjugated Abs to CD4 (allophycocyanin; BD Pharmingen) and to the mouse DO11.10-transgenic TCR, KJ1-26 (PE; Caltag Biochemicals) were used for cell staining in experiments involving the adoptive transfer of Th1 cells generated from DO11.10-transgenic mice. To determine the number of Ag-specific Th cells, the total number of cells was multiplied by the percentage of cells positive for both CD4 and mouse D011.10 TCR. In experiments that involved the adoptive transfer of Th1 cells generated from OTII mice with or without CXCR3 deficiency, commercially conjugated Abs to Thy1.1 (PerCP; BD Pharmingen) and Thy1.2 (FITC; BD Pharmingen) were used. To determine the number of Ag-specific Th cells, the total number of cells was multiplied by the percentage of cells positive for both CD4 and mouse Thy1.1 or Thy1.2.

Histology

For histologic assessment, the chest was opened, the trachea was cannulated, and the lungs were maximally inflated with 10% formalin. The lungs were removed from the thoracic cavity and placed in 10% formalin. Multiple paraffin-embedded 5-µm sections were prepared and stained with H&E or diastase periodic acid Schiff (PAS). Staining for CXCL9 was conducted using frozen sections and anti-CXCL9 Ab (R&D Systems) (30). The lungs were maximally inflated as described above with 50% OCT (Cryomatrix) compound in normal saline as a cryoprotectant and frozen over liquid nitrogen. Cryostat sections were made and fixed in acetone for 10 min and air-dried. Slides were incubated with horse serum/avidin for 20 min and CXCL9 Ab was added at 1/50 for 1 h. Slides were rinsed in PBS, incubated with biotin/H2O2 for 20 min, rinsed in PBS again, and overlaid with biotinylated horse anti-goat IgG (Vector Laboratories) for 30 min. After washing in PBS, elite and PBS again, slides were incubated with avidin/biotin complex/alkaline phosphatase (DAKO) followed by AEC solution (Biocare Medical) for visualization and counterstained with hematoxylin.

Statistical analysis

All experiments were performed twice or more using greater or equal to three mice per group per each separate experiment. Comparisons were analyzed by a two-tailed Student’s t test using Microsoft Excel software for statistical significance. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag-specific Th1 cell trafficking is profoundly impaired in STAT1–/– mice

To determine the role of STAT1 in Th1 cell trafficking, we generated OVA-specific Th1 cells from DO11.10 mice, which are transgenic for the TCR recognizing OVA peptide 323–339 (pOVA323–39). OVA-specific Th1 cells were generated in vitro using pOVA323–339, IL-12, and anti-IL-4. We confirmed that these cells were functional Th1 cells by their preferential secretion of high levels of IFN-{gamma} and no IL-4. OVA-specific T cells were identified in tissue using the clonotypic Ab, KJ1-26, to the mouse D011.10 TCR.

To determine whether STAT1 is involved in trafficking of Th1 cells into the lung, we transferred OVA-specific WT Th1 cells into STAT1+/+ and STAT1–/– mice by tail vein injection followed by daily aerosol challenges with OVA or PBS for 5–7 days. Trafficking of Ag-specific Th1 cells was robustly induced in WT mice by OVA but not PBS challenge. In STAT1–/– mice, trafficking of OVA-specific Th1 cells, following aerosol OVA challenge, was reduced into the BAL and the lung by 7- and 4-fold, respectively (Fig. 1a). The trafficking of Ag-specific Th1 cells was intact to the TLN, PLN, and the spleen. After adoptive transfer and aerosol OVA challenge, the total number of cells recovered from the BAL of STAT1–/– mice was reduced by 42% compared with cells recovered from the BAL of STAT1+/+ mice. The number of neutrophils in OVA-challenged STAT1–/– (OVA-STAT1–/–) and OVA-challenged STAT1+/+ (OVA-STAT1+/+) mice was similar as was the number of lymphocytes but there was a 46% decrease in the number of monocytes in the OVA-STAT1–/– mice (Fig. 1b). Interestingly, OVA-STAT1–/– mice that received Th1 cells had 13-fold more eosinophils in the BAL than OVA-STAT1+/+ mice that received Th1 cells (0.106 x 106 vs 0.008 x 106, p = 0.00001) (Fig. 1c). This was associated with evidence of mucous hypersecretion in OVA-STAT1–/– mice as indicated by positive PAS staining (Fig. 1d). Although the number of eosinophils was increased in the BAL of OVA-STAT1–/– mice following the adoptive transfer of Th1 cells and OVA challenge, this level of BAL eosinophilia was 10 times less than that observed after Th2 adoptive transfer and OVA challenge (Fig. 2b). As expected, the few cells recovered from the BAL of PBS-challenged STAT1+/+ and STAT1–/– mice following adoptive transfer of OVA-specific Th1 cells were almost entirely macrophages (>95%; data not shown).


Figure 1
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  		FIGURE 1. Trafficking of Ag-specific Th1 cells is dramatically decreased in STAT1–/–, but not WT and STAT6–/–, mice. OVA-specific DO11.10 Th1 cells were transferred i.v. into WT, STAT1–/–, and STAT6–/– mice followed by daily OVA challenges for 5–7 days. a, Ag-specific Th1 cells were identified using flow cytometry and anti-CD4 and anti-DO11.10 TCR (KJ1-26 mAb). b, BAL total cell count and differential. c, BAL eosinophil counts. d, PAS-stained formalin-fixed lung sections isolated from WT, STAT1–/–, and STAT6–/– mice (from left to right) after Th1 cell adoptive transfer and OVA challenge. OVA challenge resulted in a lymphocytic and neutrophilic inflammatory infiltrate in WT, STAT1–/–, and STAT6–/– mice. The cellular inflammatory response also included eosinophils in STAT1–/– mice. After adoptive transfer of OVA-specific Th1 cells followed by OVA challenge, PAS staining demonstrated mucous production in STAT1–/– mice (arrow), but not WT, and STAT6–/– mice (n = 4 mice in each group). Data are representative of at least two separate experiments and are presented as mean ± SEM. *, p < 0.02; **, p < 0.002; ***, p < 0.0002; ****, p < 0.00002 WT vs STAT1–/–.

 

Figure 2
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  		FIGURE 2. Trafficking of Ag-specific Th2 cells is intact in STAT1–/– mice: OVA-specific DO11.10 Th2 cells were transferred i.v. into WT and STAT1–/– mice followed by daily OVA challenges for 5–7 days. a, Ag-specific Th2 cells were identified using flow cytometry and anti-CD4 and anti-DO11.10 TCR (KJ1-26 mAb). b, BAL total cell count and differential. c, PAS-stained formalin-fixed lung sections isolated from WT, STAT1–/–, and STAT6–/– mice (from left to right) after Th2 cell adoptive transfer and OVA challenge. Data are representative of at least two separate experiments and are presented as mean ± SEM.

 
Ag-specific Th1 cell trafficking is intact in STAT6–/– mice

To determine whether STAT6 was also involved in the trafficking of Th1 cells into the lung, we transferred OVA-specific Th1 cells, generated from DO11.10 mice as described above, into STAT6+/+ and STAT6–/– mice by tail vein injection followed by aerosol challenges with OVA or PBS for 5–7 days. Ag-specific Th1 cells trafficked normally into the BAL, lung, TLN, PLN, and spleen of STAT6–/– mice after OVA challenge. These cells induced a neutrophilic and lymphocytic inflammatory response indistinguishable from that of WT mice (Fig. 1). These data demonstrate that resident STAT1 expression controlled the trafficking of Th1 cells, while resident STAT6 expression had no influence on Th1 cell trafficking.

Ag-specific Th2 cells traffic normally in STAT1–/– mice

To determine whether the regulatory role of STAT1 is specific to Th1 cells, we generated OVA-specific Th2 cells from DO11.10 mice in vitro using OVA peptide323–339, IL-4, and anti-IFN-{gamma} as previously described (21). The cells were harvested after 5–7 days in culture. We confirmed that these cells were functional Th2 cells by their preferential secretion of high levels of IL-4 and no IFN-{gamma}. Again, OVA-specific T cells were identified in tissue using the KJ1-26 clonotypic Ab. To determine whether STAT1 is involved in trafficking of Th2 cells into the lung, we transferred OVA-specific Th2 cells into STAT1+/+ and STAT1–/– mice by tail vein injection followed by five to seven daily aerosol challenges with OVA or PBS (25 min/day). Trafficking of Ag-specific Th2 cells was intact in OVA-STAT1–/– mice and OVA-STAT1+/+ mice into the BAL, lung, TLN, PLN, and spleen (Fig. 2a). After adoptive transfer and aerosol OVA challenge, there was no statistically significant difference between OVA-STAT1–/– and OVA-STAT1+/+ mice in the total BAL cell count and the BAL number of neutrophils, lymphocytes, monocytes, and eosinophils (Fig. 2b). There was evidence of mucous hypersecretion in WT and STAT1–/– mice but, as previously shown, there was no mucous hypersecretion in STAT6–/– mice (Fig. 2c). As expected, the few cells recovered from the BAL of PBS-challenged STAT1+/+ and STAT1–/– mice following adoptive transfer of OVA-specific Th2 cells were almost entirely macrophages (>95%; data not shown).

Ag-specific Th1 and Th2 cells proliferate normally in STAT1–/– and STAT6–/– mice, respectively

To ensure that the difference in the number of Ag-specific Th1 and Th2 cells in the BAL and lungs of STAT1–/– and STAT6–/– mice, respectively, was due to a recruitment difference and not a proliferation difference, we examined the proliferation of Ag-specific Th1and Th2 cells in WT, STAT1–/–, and STAT6–/– mice. To do this, OVA-specific Th1 and Th2 cells were stained with CFSE before adoptive transfer. After adoptive transfer of OVA-specific Th1 cells into STAT1–/– and WT mice and OVA-specific Th2 cells into STAT6–/– and WT mice via tail vein injection, mice received a daily OVA neb for 4 days. Twenty-four hours following the fourth OVA challenge, single-cell suspensions of the BAL and the lung were stained for the mouse D011.10 TCR and for CD4 and Ag-specific T cells identified as double-positive cells by flow cytometry. The extent of cellular proliferation was determined for Ag-specific T cells by also analyzing CFSE staining. The amount of CFSE is reduced by a factor of 2 with each cell division. The level of CFSE staining decreased from before to after OVA challenges to the same extent in WT and STAT1–/– mice (Fig. 3a, BAL, lung not shown) as well as WT and STAT6–/– mice (Fig. 3b, BAL, lung not shown), indicating normal proliferation of Ag-specific Th1 cells and Th2 cells in STAT1–/– and STAT6–/– mice, respectively. Therefore, the paucity of Ag-specific Th1 cells in the airways and the lung of STAT1–/– mice and Ag-specific Th2 cells in the airways and the lung of STAT6–/– mice is due to a trafficking defect and not a proliferation defect.


Figure 3
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  		FIGURE 3. Ag-specific Th1 and Th2 cells proliferate normally in STAT1–/– and STAT6–/– mice, respectively. Level of CFSE staining was measured in Ag-specific CD4+ Th1 cells before their adoptive transfer and in Ag-specific CD4+ T cells isolated from the BAL after adoptive transfer and OVA challenge. a, CFSE staining of OVA-specific Th1 cells before (single graph on the right) and after (stacked graphs on the left) adoptive transfer of Th1 cells into WT and STAT1–/– mice followed by OVA challenge determined by flow cytometry. Graphs shaded gray on the left represent WT mice and graphs shaded white on the left represent STAT1–/– mice (n = 5 mice in each group, results from three mice in each group are represented here). b, CFSE staining of OVA-specific Th2 cells before (single graph on the right) and after (stacked graphs on the left) adoptive transfer of Th2 cells into WT and STAT6–/– mice followed by OVA challenge determined by flow cytometry. Graphs shaded gray on the left represent WT mice and graphs shaded white on the left represent STAT6–/– mice (n = 3 mice in each group).

 
Ag-specific Th1 cell trafficking into the lung is intact in T-bet–/– mice

Binding of IFN-{gamma} to the IFN-{gamma} receptor results in STAT1 activation. Activated STAT1 induces T-bet expression, which in turn leads to increased IFN-{gamma} secretion (31). To determine whether T-bet also plays a regulatory role in Th1 cell trafficking and whether the STAT1 control of Th1 cell trafficking is due to a decrease in T-bet expression, we transferred OVA-specific Th1 cells, generated from DO11.10 mice as described above, into WT (T-bet+/+) and T-bet–/– mice by tail vein injection followed by OVA aerosol challenges for 5–7 days. Ag-specific Th1 cells trafficked normally into the lung of T-bet–/– mice after OVA challenge. However, there was a 37% decrease in the number of Ag-specific Th1 cells in the BAL of T-bet–/– mice as compared with T-bet+/+ mice. There was no difference in the trafficking of Ag-specific Th1 cells into the TLN and the spleen. These data demonstrate that resident T-bet expression had no influence on the trafficking of Ag-specific Th1 cells into the lung but altered the trafficking of Ag-specific Th1 cells from the lung into the airways to a limited extent (Fig. 4a). After adoptive transfer and aerosol OVA challenge, there was a statistically significant decrease in the total BAL cell count (23%) and BAL monocyte count (48%) in T-bet–/– mice compared with T-bet+/+ mice. There was no statistically significant difference in the number of neutrophils, lymphocytes, and eosinophils in the BAL of T-bet–/– and T-bet+/+ mice (Fig. 4b). Consistent with lack of eosinophilia, T-bet–/– mice and T-bet+/+ mice did not show increased mucous hypersecretion by PAS staining (data not shown).


Figure 4
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  		FIGURE 4. Trafficking of Ag-specific Th1 cells into the lung is intact in T-bet–/– mice. OVA-specific DO11.10 Th1 cells were transferred i.v. into WT and T-bet–/– mice followed by daily OVA challenges for 5–7 days. a, Ag-specific Th1 cells were identified using flow cytometry and anti-CD4 and anti-DO11.10 TCR (KJ1-26 mAb). b, BAL total cell count and differential. Data are representative of two separate experiments and are presented as mean ± SEM. *, p < 0.05; **, p < 0.001. WT vs T-bet–/–.

 
PTX attenuates the trafficking of Ag-specific Th1 cells

Our previous work has shown that Th2 cell trafficking is mediated by PTX-sensitive chemoattractant receptors (32). PTX is an irreversible inhibitor of G{alpha}i-coupled chemoattractant receptors and inhibits chemokine-induced chemotaxis. To determine whether Ag-specific Th1-cell trafficking is mediated by chemoattractant receptors, Ag-specific Th1 cells were pretreated with PTX or vehicle for 24 h, washed, and adoptively transferred i.v. via the tail vein into WT mice that were then challenged with aerosolized OVA, once a day for 4 days. We found that pretreatment with PTX dramatically attenuated Ag-specific Th1 cell trafficking into the lung (0.115 x 106 vs 1.46 x 106, p = 0.0014) and the BAL (0.004 x 106 vs 0.228 x 106, p = 0.011; Fig. 5). These data indicate that Th1 cell trafficking into the lung and BAL in response to Ag is mediated by PTX-sensitive chemoattractant receptors.


Figure 5
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  		FIGURE 5. Trafficking of Ag-specific Th1 cells is mediated by chemoattractant receptors. OVA-specific Th1 cells were generated in vitro and pretreated with and without pertussis toxin (PTX) and then transferred into WT mice that then received four daily OVA challenges. Single-cell suspensions of the BAL, lung, thoracic lymph nodes, peripheral lymph nodes, and spleen were stained for CD4 and mouse DO11.10 TCR to determine the number of Ag-specific T cells by flow cytometry (n = 3 to 4 mice in each group). The trafficking of Ag-specific Th1 cells into BAL and lung was attenuated with pretreatment with PTX; *, p <0.02. PTX vs no PTX.

 
STAT1–/– mice have decreased levels of CXCL9, CXCL10, CXCL11, and CXCL16

Successful trafficking of Ag-specific Th1 cells requires coordinated secretion of multiple chemokines at sites of inflammation. It is believed that these chemokines interact with chemokine receptors that are expressed on Th1 cells and result in tissue recruitment. We hypothesized that the recruitment of Ag-specific Th1 cells can be explained by a STAT1-regulated pattern of chemokine secretion at sites of Th1 inflammation and the pattern of chemokine receptors expressed on tissue-infiltrating Th1 cells. In this regard, we found that after Th1 cell adoptive transfer and OVA aerosol challenge, STAT1–/– mice had decreased lung mRNA levels for CXCL9, CXCL10, CXCL11, and CXCL16 compared with WT mice (Fig. 6a). Similarly, CXCL9, CXCL10, CXCL11, and CXCL16 mRNA levels were decreased in the BAL cells isolated from STAT1–/– mice as compared with WT mice (data not shown).


Figure 6
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  		FIGURE 6. STAT1 deficiency alters the chemokine profile of the lung. OVA-specific Th1 cells were generated in vitro and transferred into WT and STAT1–/–, STAT6–/–, and T-bet–/– mice followed by 5–7 daily OVA challenges. Total lung RNA was subjected to Q-PCR analysis using specific primers for a variety of chemokines. a, Lung chemokine profile after adoptive transfer of OVA-specific Th1 cells into WT and STAT1–/– mice. b, Lung chemokine profile after adoptive transfer of OVA-specific Th1 cells into WT and STAT6–/– mice. c, Lung chemokine profile after adoptive transfer of OVA-specific Th1 cells into WT and T-bet–/– mice (n = 6–7 mice in each group). *, p < 0.05; **, p < 0.02; ***, p < 0.01. WT vs knockout mice. d, Staining of frozen lung samples with anti-CXCL9 Ab: WT lung stained with CXCL9 Ab then secondary Ab, showing staining of mononuclear cells with anti-CXCL9 Ab. There is no staining with CXCL9 Ab in the lung of CXCL9–/– mice and STAT1–/– mice. Arrow points to positive cytoplasmic staining with CXCL9 Ab in a lung mononuclear cell.

 
In contrast, the expression levels of these genes were unaffected when Th1 cells were transferred into STAT6–/– mice in which Th1 cell trafficking was intact (Fig. 6b). This differs from the expression level of CCL5, which was decreased in both STAT1–/– mice that have abnormal Th1 trafficking and STAT6–/– mice that have normal Th1 cell trafficking. Thus, the expression of CXCL9, CXCL10, CXCL11, and CXCL16 is preferentially regulated by STAT1 in vivo and correlated with Th1 cell trafficking. The expression levels of CXCL9, CXCL10, CXCL11, and CXCL16 in the lungs of T-bet–/– mice were normal (Fig. 6c), which correlated with the normal trafficking pattern observed into the lungs of T-bet–/– mice. Of note, the expression level of CCL17 was increased in STAT1–/– mice following the adoptive transfer of Ag-specific Th1 cells and Ag challenge, which may explain the increased number of eosinophils in STAT1–/– mice.

To determine whether lack of above RNA expression in STAT1–/– mice is associated also with a lack of protein expression, we performed immunohistochemistry staining on lung tissue using anti-CXCL9 Ab. We transferred OVA-specific Th1 cells, generated from DO11.10 mice as described above, into WT, CXCL9–/– mice and STAT1–/– mice followed by five daily OVA challenges. Twenty-four hours after the last challenge, lungs were harvested and frozen over liquid nitrogen. WT lungs stained with PBS followed by secondary Ab served as a negative control (data not shown). Mononuclear cells in WT lungs stained positive with CXCL9 Ab while there was no CXCL9 staining in CXCL9–/– and STAT1–/– lungs (Fig. 6d). This shows that in the absence of STAT1, CXCL9 protein expression is also abrogated.

CXCL10 replacement partially restores the trafficking of Ag-specific Th1 cells in STAT1–/– mice

We have observed that CXCL9, CXCL10, CXCL11, and CXCL16 expression levels are decreased in STAT1–/– mice and that the expression of these chemokines correlates with the trafficking of Th1 cells in vivo. To determine whether it is the decreased chemokine levels in the lung that causes the trafficking defect in STAT1–/– mice, we examined whether exogenous CXCL10 replacement could correct the trafficking defect seen in STAT1–/– mice. We adoptively transferred in vitro-generated OVA-specific, DO11.10, Th1 cells into WT and STAT1–/– mice. All mice received OVA aerosol challenges daily for 4 days. One-half of the WT mice and one-half of the STAT1–/– mice also received rCXCL10, intranasally, 2 h before each daily OVA aerosol challenge. Lungs and BAL were harvested 24 h after the fourth dose of CXCL10 and/or OVA, and the trafficking of OVA-specific Th1 cells was compared between the groups. Exogenous CXCL10 partially reconstituted the trafficking of Ag-specific Th1 cells into the lung and the BAL of STATI–/– mice. The addition of exogenous CXCL10 increased the number of Ag-specific Th1 cells in the lung of STAT1–/– mice to 72% of the WT (Fig. 7, left panel) and increased the number of Ag-specific Th1 cells in the BAL of STAT1–/– mice to 46% of the WT (Fig. 7, right panel). These data show that STAT1–/– mice are capable of recruiting Ag-specific Th1 cells once compensated for their altered chemokine expression.


Figure 7
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  		FIGURE 7. CXCL10 replacement partially restores the trafficking of Ag-specific Th1 cells in STAT1–/– mice. Ag-specific Th1 cells were generated in vitro and transferred into WT mice and STAT1–/– mice. One group of WT mice and one group of STAT1–/– mice received four daily OVA aerosol challenges. Another group of WT mice and another group of STAT1–/– mice were anesthetized with isoflurane and given rCXCL10, at 5 µg/50 µl of PBS, intranasally, 2 h before each daily OVA aerosol challenge for 4 days. Left panel, The number of KJ+ cells recruited into the lung in the WT vs STAT1–/– mice with and without CXCL10 replacement. **, p = 0.0006 WT vs STAT1–/–; *, p = 0.02 STAT1–/– with and without CXCL10. Right panel, The number of KJ+ cells recruited into the BAL in the WT and STAT1–/– mice with and without CXCL10 replacement. **, p = 0.00006 WT vs STAT1–/–; *, p = 0.008 STAT1–/– with and without CXCL10 (n = 8 mice in each group, in two separate experiments).

 
The trafficking of CXCR3-deficient Ag-specific Th1 cells is impaired into the lung and BAL

We have shown that the expression level of CXCL9, CXCL10, CXCL11, and CXCL16 in the lung correlates with the recruitment of Ag-specific Th1 cells into the lung. To further establish the effect of this chemokine subset on the trafficking of Ag-specific Th1 cells, we asked whether Ag-specific Th1 cells deficient in CXCR3, the receptor for CXCL9, CXCL10, CXCL11, traffic normally to sites of allergen challenge. Ag-specific Th1 cells were generated in vitro from two groups of mice, both in the C57BL/6 background: OTII cells and CXCR3–/–OTII cells. OTII cells were transgenic for the TCR recognizing OVA and congenic for the Thy1.1 allele. The CXCR3–/–OTII cells were transgenic for the TCR recognizing OVA, deficient in CXCR3 and congenic for the Thy1.2 allele. Ag-specific Th1 cells were generated from naive CD62L+CD4+ T cells from age-matched OTII and CXCR3–/– OTII mice. After 6 days of growth in culture, cells were harvested and were shown to generate similar levels of IFN-{gamma}, thus having differentiated equally to a Th1 phenotype (data not shown). The cells were adoptively transferred into C57BL/6 mice carrying both the Thy1.1 and Thy1.2 alleles. The Thy1.1/Thy1.2 mice received either Ag-specific OTII Th1 cells or Ag-specific CXCR3–/–OTII Th1 cells. The recipient mice were given three daily, aerosolized OVA challenges. On the fourth day, the lung and the BAL were harvested and the number of Ag-specific Th1 cells was determined in each group. OVA-specific OTII Th1 cells were identified as Thy1.1+ while OVA-specific CXCR3–/–OTII Th1 cells were identified as Thy1.2+. Recipient cells were identified as Thy1.1+Thy1.2+. The trafficking of Ag-specific CXCR3–/–OTII Th1 cells was reduced by 2.6-fold into the lung (p value = 0.004, Fig. 8, left panel) and by 4.5-fold into the BAL (p value = 0.0004, Fig. 7, right panel), demonstrating that CXCR3 plays a role in Th1 cell trafficking into the lung and the BAL.


Figure 8
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  		FIGURE 8. The trafficking of CXCR3-deficient Ag-specific Th1 cells is impaired into the lung and BAL. Ag-specific Th1 cells were generated in vitro from OTII (Thy1.1+) and CXCR3–/– OTII (Thy1.2+) mice. The cells were adoptively transferred into separate C57BL/6 mice (Thy1.1+Thy1.2+). The recipient mice were given three daily aerosolized OVA challenges. On the fourth day, the lung and the BAL were harvested and the number of Ag-specific Th1 cells was determined in each group using Thy1.1 and Thy1.2 Ab and flow cytometry. Left panel, The number of Ag-specific Th1 cells in the lung (*, p = 0.004 OTII vs CXCR3–/–OTII); right panel, the number of Ag-specific Th1 cells in the BAL (*, p = 0.0004 OTII vs CXCR3–/–OTII) (n = 3–5 mice in each group, in two separate experiments).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we found quite clearly that Th1 and Th2 cell trafficking is differentially controlled in vivo. Here, we show that the trafficking of Ag-specific Th1 cells, adoptively transferred into STAT1–/– mice followed by aerosol Ag challenge, was dramatically decreased into the BAL and the lung while STAT6, which regulates the recruitment of Ag-specific Th2 cells (21), had no effect on Th1 cell trafficking. Moreover, we determined that the role of STAT1 in Th1 cell trafficking was specific to Th1 cells as Ag-specific Th2 cells traffic normally in STAT1–/– mice.

CD4+ T cells from STAT1–/– mice have been shown to differentiate to a Th1 phenotype and secrete IFN-{gamma} but to a lesser extent than the WT (33). Therefore, we used an adoptive transfer model to isolate the role of STAT1 in Th1 cell trafficking apart from its role in Th1 cell differentiation. Experiments using CFSE staining provided convincing evidence that the paucity of Ag-specific Th1 and Th2 cells in STAT1–/– and STAT6–/– mice, respectively, was not due to a defect in Th1 and Th2 proliferation in STAT1–/– and STAT6–/– mice but was due to a defect in the recruitment of Th1 and Th2 cells. Our study using PTX demonstrated that the trafficking of Th1 cells into the lung and the BAL is an active process that is dependent on chemoattractant receptors.

Although commercially available OVA contains LPS, we believe that LPS was not a determining factor in our study. Adoptive transfer of OVA-specific Th1 cells followed by OVA challenge induced a robust Th1 inflammatory response in WT mice. In contrast, adoptive transfer of OVA-specific Th2 cells followed by OVA challenge, using the same source and concentration of OVA, induced a robust Th2 inflammatory response in WT mice. This indicates that LPS in the OVA preparation did not dictate the inflammatory response induced in the adoptive transfer model. Furthermore, STAT1–/– mice, receiving the same OVA, had a 4- to 7-fold decrease in the recruitment of Ag-specific Th1 cells into the lung and the BAL, respectively, compared with WT mice. Similarly, STAT6–/– mice receiving OVA failed to recruit Ag-specific Th2 cells (21).

In addition, we examined the role of T-bet, a transcription factor downstream of STAT1 and important for Th1 inflammation (23, 31, 34), on the trafficking of Ag-specific Th1 cells. In addition to CD4+ T cells, T-bet is expressed in CD8+ T cells, NKT cells, NK cells, dendritic cells, and macrophages (31, 35). The first wave of Ag-specific Th1 cells that arrives in the lung secretes IFN-{gamma}, which activates STAT1 in lung resident cells. We hypothesized that alveolar macrophages are a possible cellular source of STAT1-regulated chemokines important for Th1 cell trafficking. Because T-bet is expressed in macrophages, we asked whether the STAT1 signaling could be mediated through T-bet. T-bet deficiency did not affect the recruitment of Ag-specific Th1 cells into the lung but caused a modest decrease in the trafficking of Ag-specific Th1 cells from the lung into the airways. This is in sharp contrast to STAT1 deficiency, which causes a 7- and 4-fold decrease in the trafficking of Ag-specific Th1 cells into the BAL and the lung, respectively. The contrast observed between STAT1 and T-bet shows that STAT1 controls Th1 cell trafficking through a mechanism beyond its influence on T-bet expression.

In this study, we provide evidence that STAT1 controls the trafficking of Ag-specific Th1 cells by altering the expression of a distinct subset of chemokines at inflammatory sites. We observed that STAT1 deficiency abrogated the expression of CXCL9, CXCL10, and CXCL11 and decreased the expression of CXCL16 in the lungs. In addition, we have confirmed that the abrogation of gene expression is associated with an abrogation of protein expression using immunohistochemistry and anti-CXCL9 Ab. CXCL9, CXCL10, and CXCL11 are IFN-{gamma}-induced chemokines, at least partially regulated by STAT1 (36, 37, 38). Although the role of STAT1 in CXCL16 induction has not been delineated, TNF and IFN-{gamma} have been shown to induce CXCL16 synergistically (39).

The decrease in the expression of CXCL9, CXCL10, CXCL11, and CXCL16 in the lung specifically correlated with the trafficking defect observed in STAT1–/– mice in contrast to the expression level of CCL5, which decreased in both STAT1–/– mice that had abnormal Th1 cell trafficking and STAT6 mice that had normal Th1 cell trafficking. Furthermore, the recruitment of Ag-specific Th1 cells was normal in STAT6–/– mice and T-bet–/– mice, which had normal CXCL9, CXCL10, and CXCL11 levels. More importantly, the replacement of CXCL10 in STAT1–/– mice resulted in partial restoration of Ag-specific Th1 cell trafficking, demonstrating that, aside from altered chemokine expression, STAT1–/– mice are otherwise capable of recruiting Ag-specific Th1 cells. We also showed that Ag-specific Th1 cells that are deficient in CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, have impaired trafficking into the lung and the BAL. Collectively, these studies establish the relative contribution of the STAT1-chemokine-CXCR3 axis to trafficking of Ag-specific Th1 cells.

We observed that STAT1–/– mice developed BAL and lung eosinophilia after adoptive transfer of OVA-specific Th1 cells followed by aerosol OVA challenge. Two chemokine patterns may explain the eosinophilia observed in STAT1–/– mice. First, CCL17 was expressed at higher levels in the lung of STAT1–/– mice in the setting of Th1 inflammation and eosinophils express CCR4, the receptor for CCL17 (40). Therefore, the airway eosinophilia seen in STAT1–/– mice may be the result of increased levels of CCL17 in the lungs of STAT1–/– mice. STAT1 has been shown to induce suppressor of cytokine signaling protein 1 and suppressor of cytokine signaling 1 down-regulates the phosphorylation of STAT6 by IL-4 (41, 42). Therefore, in the absence of STAT1, STAT6 response to IL-4 is exaggerated and this in turn could explain the increased expression of the IL-4-inducible chemokine CCL17 seen in STAT1–/– mice (43). In addition, the lack of CXCL9 in the BAL and the lung of STAT1–/– mice may have contributed to the tissue and BAL eosinophilia observed in these mice as recent reports have identified CXCL9 as an inhibitor of eosinophil recruitment into the lungs (44). Of note, the development of BAL and tissue eosinophilia in STAT1–/– mice after Th1 cell transfer was associated with airway mucous production. As IL-13 signaling through STAT6 controls mucous hypersecretion in the airways (45), this may also reflect an overactive STAT6 pathway in the absence of STAT1 signaling.

T-bet–/–CD4+ Th1 cells express diminished levels of IFN-{gamma} (23) and, when adoptively transferred, show impaired trafficking in vivo (46, 47) at least partly due to their lower CXCR3 expression levels (47). We were interested in studying the role of T-bet in Th1 cell cell trafficking independent of its role in Th1 cell differentiation. Therefore, we adoptively transferred Ag-specific T-bet+/+ cells into WT and T-bet–/– mice and demonstrated that resident T-bet expression does not affect Th1 cell trafficking into the lung and only has a modest effect on Th1 cell trafficking from the lung into the airways. Our work shows that STAT1 and T-bet differ in many aspects. First, tissue STAT1 has a significantly more profound effect on the trafficking of Ag-specific Th1 cells. This difference in trafficking is consistent with the different chemokine patterns seen in STAT1–/– mice and T-bet–/– mice. Although STAT1–/– mice have decreased CXCL9, CXCL10, CXCL11, and CXCL16 levels, T-bet–/– mice have normal levels of these chemokines, possibly due to their ability to respond to IFN-{gamma} secreted by adoptively transferred effector Th1 cells. T-bet appears to affect Th1 cell trafficking by regulating the expression of CXCR3 on Th1 cells (47) but STAT1 influences Th1 cell trafficking through the regulation of chemokine expression in the tissue. Second, eosinophil numbers did not increase in T-bet–/– mice following the adoptive transfer of Ag-specific Th1 cells and Ag challenge and there was no associated mucous hypersecretion or CCL17 increase in T-bet–/– mice (data not shown). This suggests that the cross-regulation between STAT1 and STAT6 is not mediated through T-bet.

In summary, we show that tissue STAT1 regulates the trafficking of Ag-specific Th1 cells in vivo through the induction of a distinct subset of chemokines. Our study also demonstrates that Th1 and Th2 cell trafficking are differentially regulated in vivo by STAT1 and STAT6, respectively. These findings reinforce the notion that differential control of Th1 and Th2 cell trafficking contributes to the distinct effector functions of Th1 and Th2 cells and that it may be possible to differentially manipulate Th1 and Th2 cell trafficking for therapeutic purposes.


   Acknowledgments
 
We thank Dr. David Levy for STAT1–/– mice, Dr. Laurie Glimcher for T-bet–/– mice, Dr. Craig Gerard for CXCR3–/– mice, Dr. Joshua Farber for CXCL9–/– mice, and Andrew Carafone for his technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01 AI40618-08 (to A.D.L.), R01 AI40618-08S1 (to A.D.L., Z.M.), and K08 AI049957 (to C.M.F.). Back

2 Address correspondence and reprint requests to Dr. Andrew D. Luster, Division of Rheumatology, Allergy and Immunology, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Building 149-8301, 13th Street, Charlestown, MA 02129. E-mail address: luster.andrew{at}mgh.harvard.edu Back

3 Abbreviations used in this paper: WT, wild type; BAL, bronchoalveolar lavage; PTX, pertussis toxin; TLN, thoracic lymph node; PLN, peripheral lymph node; PAS, periodic acid Schiff. Back

Received for publication November 23, 2005. Accepted for publication January 25, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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