Bone Marrow Retaining Colitogenic CD4+ T Cells May Be a Pathogenic Reservoir for Chronic Colitis
2006; Elsevier BV; Volume: 132; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2006.10.035
ISSN1528-0012
AutoresYasuhiro Nemoto, Takanori Kanai∥, Shin Makita, Ryuichi Okamoto, Teruji Totsuka, Kiyoshi Takeda, Mamoru Watanabe,
Tópico(s)Immunotherapy and Immune Responses
ResumoBackground & Aims: Although bone marrow (BM) is known as a primary lymphoid organ, it also is known to harbor memory T cells, suggesting that this compartment is a preferential site for migration and/or selective retention of memory T cells. We here report the existence and the potential ability to induce colitis of the colitogenic BM CD4+ memory T cells in murine colitis models. Methods: We isolated BM CD4+ T cells obtained from colitic severe combined immunodeficient mice induced by the adoptive transfer of CD4+CD45RBhigh T cells and colitic interleukin (IL)-10−/− mice that develop colitis spontaneously, and analyzed the surface phenotype, cytokine production, and potential activity to induce colitis. Furthermore, we assessed the role of IL-7 to maintain the colitogenic BM CD4+ T cells. Results: A high number of CD4+ T cells reside in the BM of colitic severe combined immunodeficient mice and diseased IL-10−/− mice, and they retain significant potential to induce type-1 T helper-mediated colitis in an IL-7–dependent manner. These resident BM CD4+ T cells have an effector memory (TEM; CD44highCD62L−IL-7Rhigh) phenotype and preferentially are attached to IL-7–producing BM cells. Furthermore, the accumulation of BM CD4+ TEM cells was decreased significantly in IL-7–deficient recipients reconstituted with the colitogenic lamina propria CD4+ TEM cells. Conclusions: Collectively, these findings suggest that BM-retaining colitogenic CD4+ memory T cells in colitic mice play a critical role as a reservoir for persisting lifelong colitis. Background & Aims: Although bone marrow (BM) is known as a primary lymphoid organ, it also is known to harbor memory T cells, suggesting that this compartment is a preferential site for migration and/or selective retention of memory T cells. We here report the existence and the potential ability to induce colitis of the colitogenic BM CD4+ memory T cells in murine colitis models. Methods: We isolated BM CD4+ T cells obtained from colitic severe combined immunodeficient mice induced by the adoptive transfer of CD4+CD45RBhigh T cells and colitic interleukin (IL)-10−/− mice that develop colitis spontaneously, and analyzed the surface phenotype, cytokine production, and potential activity to induce colitis. Furthermore, we assessed the role of IL-7 to maintain the colitogenic BM CD4+ T cells. Results: A high number of CD4+ T cells reside in the BM of colitic severe combined immunodeficient mice and diseased IL-10−/− mice, and they retain significant potential to induce type-1 T helper-mediated colitis in an IL-7–dependent manner. These resident BM CD4+ T cells have an effector memory (TEM; CD44highCD62L−IL-7Rhigh) phenotype and preferentially are attached to IL-7–producing BM cells. Furthermore, the accumulation of BM CD4+ TEM cells was decreased significantly in IL-7–deficient recipients reconstituted with the colitogenic lamina propria CD4+ TEM cells. Conclusions: Collectively, these findings suggest that BM-retaining colitogenic CD4+ memory T cells in colitic mice play a critical role as a reservoir for persisting lifelong colitis. It has long been known that T-cell precursors generated in the bone marrow (BM) migrate to the thymus, where T-cell development occurs. However, a fact often neglected is that under physiologic conditions, mature CD4+ and CD8+ T cells undergo extensive migration from the blood to the BM and vice versa. In both human beings and mice, T-cell receptor αβ+ cells constitute approximately 3%–8% of nucleated BM cells.1Zeng D. Hoffmann P. Lan F. Huie P. Higgins J. Strober S. Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation.Blood. 2002; 99: 1449-1457Crossref PubMed Scopus (62) Google Scholar, 2Di Rosa F. Pabst R. The bone marrow: a nest for migratory memory T cells.Trends Immunol. 2005; 26: 360-366Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar BM CD4+ and CD8+ T-cell populations contain a high proportion of cells displaying a memory phenotype, that is, expressing low levels of CD45RA in human beings3Mazo I.B. Honczarenko M. Leung H. Cavanagh L.L. Bonasio R. Weininger W. Engelke K. Xia L. McEver R.P. Koni P.A. Siberstein L.E. von Andrian U.H. Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.Immunity. 2005; 22: 259-270Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar and high levels of CD44 in mice.4Price P.W. Cerny J. Characterization of CD4+ T cells in mouse bone marrow I. Increased activated/memory phenotype and altered TCR Vβ repertoire.Eur J Immunol. 1999; 29: 1051-1056Crossref PubMed Google Scholar, 5Di Rosa F. Santoni A. Bone marrow CD8 T cells are in a different activation state than those in lymphoid periphery.Eur J Immunol. 2002; 32: 1873-1880Crossref PubMed Scopus (61) Google Scholar As early as 1974 it was documented that mouse CD4+ T cells migrate to the BM after priming, and it was proposed that BM CD4+ T cells contributed to the development of a memory antibody response in this organ.6Benner R. Meima F. van der Meulen G.M. van Muiswinkel W.B. Antibody formation in mouse bone marrow II. Evidence for a memory-dependent phenomenon.Immunology. 1974; 26: 247-255PubMed Google Scholar Recently, T cells persisting in extralymphoid organs such as the liver, lung, and skin have attracted increasing interest because it has been recognized that these T cells contribute considerably to the long-lived memory T-cell pool.7Masopust D. Vezys V. Marzo A.L. Lefrancois L. Preferential localization of effector memory cells in nonlymphoid tissue.Science. 2001; 291: 2413-2417Crossref PubMed Scopus (5) Google Scholar, 8Reinhardt R.L. Khoruts A. Merica R. Zell T. Jenkins M.K. Visualizing the generation of memory CD4 T cells in the whole body.Nature. 2001; 410: 101-105Crossref PubMed Scopus (852) Google Scholar In this context, BM has been shown to harbor a high number of antigen-specific CD8+ T cells for several months after resolution of acute infection.9Slifka M.K. Whitmire J.K. Ahmed R. Bone marrow contains virus-specific cytotoxic T lymphocytes.Blood. 1997; 90: 2103-2108PubMed Google Scholar For instance, adoptive transfer of BM cells from lymphochoriomeningitis virus–immune mice (>90 days after acute infection) to immunodeficient recipients provides antiviral protection, and thus CD8+ memory T cells from the BM are able to mount an effective secondary response.10Wherry E.J. Teichgraber V. Becker T.C. Masopust D. Kaech S.M. Antia R. von Andrian U.H. Ahmed R. Lineage relationship and protective immunity of memory CD8 T cell subsets.Nat Immunol. 2003; 4: 225-234Crossref PubMed Scopus (1516) Google Scholar Primary T-cell responses to blood-borne antigens also can be initiated in the BM. This was shown initially in conditions of altered lymphocyte trafficking in splenectomized mice and then in individuals with normal lymphoid organs, for both CD4+ and CD8+ T-cell responses.11Feuerer M. Beckhove P. Garbi N. Mahnke Y. Limmer A. Hommel M. Hammerling G.J. Kyewski B. Hamann A. Umansky V. Schirrmacher V. Bone marrow as a priming site for T-cell responses to blood-borne antigen.Nat Med. 2003; 9: 1151-1157Crossref PubMed Scopus (273) Google Scholar Thus, the BM resembles a secondary lymphoid organ, although it lacks the organized T- and B-cell areas found in the spleen, lymph nodes, and Peyer's patches. Although accumulating evidence suggests that BM plays an important role in the communication with mature naive/memory T cells, there is no evidence for the role of BM memory CD4+ cells in chronic immune diseases, such as inflammatory bowel diseases (ulcerative colitis and Crohn's disease) and autoimmune diseases. Crohn's disease is characterized by chronic inflammation of the small and large intestine and structures apart from the bowel. Surgery does not cure Crohn's disease, and recurrence after surgery is the rule rather than the exception.12Munkholm P. Binder V. Clinical features and natural history of Crohn's disease.in: Kirsner J.B. Shorter R.B. Inflammatory bowel disease. Williams & Wilkins, Baltimore2004: 289-300Google Scholar There is also no correlation between recurrence of the disease and the dissection of regional lymph nodes and spleen.13Kameyama J. Sasaki I. Imamura M. Naito H. Sato T. Surgical treatment for Crohn's disease, with special reference to operative procedures and their relationship to recurrence.Tohuku J Exp Med. 1982; 137: 245-251Crossref PubMed Scopus (3) Google Scholar The evidence suggests that other sites might play a critical role in the recurrence of diseases as reservoirs of colitogenic memory CD4+ T cells. Furthermore, it is well known that interleukin (IL)-7 is important as a critical factor for the survival and homeostatic proliferation of memory CD4+ T cells, and that BM is a major site of IL-7 production.14Bradley L.M. Haynes L. Swain S.L. IL-7: maintaining T-cell memory and achieving homeostasis.Trends Immunol. 2005; 26: 172-176Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar We have shown previously that mucosal CD4+ T cells in colitic mice express IL-7Rα highly, and they are pathogenic cells responsible for chronic colitis.15Yamazaki M. Yajima T. Tanabe M. Fukui K. Okada E. Okamoto R. Oshima S. Nakamura T. Kanai T. Uehira M. Takeuchi T. Ishikawa H. Hibi T. Watanabe M. Mucosal T cells expressing high level of IL-7 receptor are potential targets for treatment of chronic colitis.J Immunol. 2003; 171: 1556-1563PubMed Google Scholar In vitro stimulation of these colitic lamina propria (LP) CD4+IL-7Rhigh T cells by IL-7, but not IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4+ T cells.16Okada E. Yamazaki M. Tanabe M. Takeuchi T. Nanno M. Oshima S. Okamoto R. Tsuchiya K. Nakamura T. Kanai T. Hibi T. Watanabe M. IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice.Am J Physiol Gastrointest Liver Physiol. 2005; 288: 745-754Crossref Scopus (37) Google Scholar These backgrounds prompted us to investigate the role of the resident BM memory CD4+ T cells in persisting lifelong colitis using a murine model of chronic colitis induced by the adoptive transfer of CD4+CD45RBhigh T cells. Female BALB/c, CB-17 severe combined immunodeficient (SCID), and C57BL/6 mice were purchased from Japan Clea (Tokyo, Japan). Female C57BL/6 Rag-2−/− mice were provided by Central Laboratories for Experimental Animals (Kawasaki, Japan). C57BL/6 Rag-1−/− mice and IL-7−/− mice were kindly provided by Dr. Zamoyska (National Institute for Medical Research, London, UK).17Seddon B. Tomlinson P. Zamoyska R. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells.Nat Immunol. 2003; 4: 680-686Crossref PubMed Scopus (387) Google Scholar IL-7−/− × Rag-1−/− mice and littermate IL-7+/+ × Rag-1−/− mice were generated in our laboratory. All mice were maintained under specific-pathogen–free conditions in the Animal Care Facility of the Tokyo Medical and Dental University. The Institutional Committee on Animal Research approved the experiments. The following monoclonal antibodies (mAbs) other than biotin-conjugated anti-mouse IL-7Rα (A7R34; Immuno-Biological Laboratories, Takasaki, Japan) were obtained from BD PharMingen (San Diego, CA) and used for purification of cell populations and flow-cytometric analysis: Fcγ (CD16/CD32)-blocking mAb (2.4G2), phycoerythrin (PE)-, peridinin chlorophyll protein, and phycoerythrin-phycoerythrin- 5′- disulfonatoindodicarbocyanine conjugated anti-mouse CD4 (RM4-5); fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD3 (145-2C11); PE- and allophycocyanin-conjugated anti-mouse CD44 (IM7); FITC- and PE-conjugated anti-mouse CD62L (MEL-14); FITC-conjugated anti-mouse CD69 (H1.2F3); PE-conjugated anti-mouse integrin α4β7 (DATK32); FITC-conjugated anti-mouse CD45RB (16A); FITC-conjugated hamster anti-mouse Bcl-2 (3F11); PE-conjugated streptavidin; biotin-conjugated rat IgG2; PE-conjugated mouse IgG; and PE-conjugated rat IgG. Flow cytometric 3-color analysis was performed as described.18Totsuka T. Kanai T. Iiyama R. Uraushihara K. Yamazaki M. Okamoto R. Hibi T. Tezuka K. Azuma M. Akiba Yagita H. Okumura K. Watanabe M. Ameliorating effect of anti-ICOS monoclonal antibody in a murine model of chronic colitis.Gastroenterology. 2003; 124: 410-421Abstract Full Text PDF PubMed Scopus (70) Google Scholar Colitis was induced in SCID/Rag-2−/− mice by the adoptive transfer of CD4+CD45RBhigh T cells as described.18Totsuka T. Kanai T. Iiyama R. Uraushihara K. Yamazaki M. Okamoto R. Hibi T. Tezuka K. Azuma M. Akiba Yagita H. Okumura K. Watanabe M. Ameliorating effect of anti-ICOS monoclonal antibody in a murine model of chronic colitis.Gastroenterology. 2003; 124: 410-421Abstract Full Text PDF PubMed Scopus (70) Google Scholar Colitic mice were killed at 6–8 weeks after transfer, and CD4+ T cells were isolated from BM, mesenteric lymph nodes (MLNs), and colonic LP. To measure cytokine production, 3 × 104 CD4+ T cells from MLN, LP, and BM were cultured in 200 μL of culture medium at 37°C in a humidified atmosphere containing 5% CO2 in 96-well plates (Costar, Cambridge, MA) precoated with 5 μg/mL hamster anti-mouse CD3ϵ mAb (145-2C11; BD PharMingen) and 2 μg/mL hamster anti-mouse CD28 mAb (37.51; BD PharMingen) in phosphate-buffered saline (PBS) overnight at 4°C. Culture supernatants were removed after 48 hours and assayed for cytokine production. Cytokine concentrations were determined by specific enzyme-linked immunosorbent assay (ELISA) as per the manufacturer's recommendation (R&D, Minneapolis, MN). Colitic SCID mice were killed and their cecums were removed. The cecums were opened and placed in 1 mL of PBS, and the cecal bacteria were expelled by mixing with a vortex, and residual cecal tissue was removed. After the addition of DNase (10 μg/mL), 1 mL of this bacterial suspension was added to 1 mL of glass beads.19Cong Y. Brandwein S.L. McCabe R.P. Lazenby A. Birkenmeier E.H. Sundberg J.P. Elson C.O. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease.J Exp Med. 1998; 187: 855-864Crossref PubMed Scopus (336) Google Scholar The cells were disrupted at 5000 revolutions per minute in a Mini-Bead Beater (BioSpec Products, Bartlesville, OK) for 3 minutes and then iced. The glass beads and unlysed cells were removed by centrifuging at 5000 × g for 5 minutes. The lysates were filter-processed in a similar manner. For antigen-presenting cells (APCs), spleen cells from normal BALB/c mice were prepared and treated with the appropriate concentration of cecal bacterial antigens (CBAs) as indicated at 2 × 107 cells/5 mL in a 15-mL tube overnight at 37°C. After washing twice, these APCs were treated with mitomycin-c before being added to T-cell cultures. BM, MLN, and LP CD4+ T cells obtained from normal mice and colitic CD4+CD45RBhigh T-cell–transferred SCID mice were cultured in the presence of APCs pretreated with cecal extract antigens in complete media. The culture supernatants were collected on day 3 of culture for interferon (IFN)-γ assay by ELISA. Colitic mice and age-matched normal BALB/c mice were given 1 mg of bromodeoxyuridine (BrdU) in PBS by intraperitoneal injection. Twenty-four hours later, mice were killed and the lymphocytes were prepared from BM, MLN, and colonic LP. Cells were first stained with PE-conjugated anti-CD4 mAbs for 2-color flow-cytometric analysis, or peridinin chlorophyll protein-conjugated anti-CD4 mAbs, APC-conjugated anti-CD44 mAbs, and PE-conjugated anti-CD62L mAbs for 4-color flow-cytometric analysis, and fixed and permeabilized with Cytofix-Cytoperm (BD PharMingen) solution according to the manufacturer's instructions. Cells were stained with FITC-conjugated anti-mouse BrdU (BD PharMingen) diluted in perm/wash buffer. A total of 1 × 106 cells from colitic mice induced by the adoptive transfer of CD4+CD45RBhigh T cells were stained for PE-conjugated anti-CD4 mAbs, and fixed and permeabilized with Cytofix-Cytoperm (BD PharMingen) solution according to the manufacturer's instructions. 7-AAD (10 μg/mL) and RNase (200 μg/mL) were added, and cells were incubated for 20 minutes at room temperature. Cells were acquired on a FACSCalibur (BD PharMingen) in their staining solution. Cell-cycle analysis of DNA histograms was performed with Cell Quest Software (BD PharMingen). Consecutive cryostat bone marrow sections (6 μm) were fixed and stained with the following rat antibodies: biotinylated CD4 (RM4-5) and polyclonal anti–IL-7 antibodies (R&D Laboratories). Alexa 594 goat anti-rat IgG, Alexa 488 goat anti-hamster IgG, and Alexa 488 rabbit anti-goat IgG (Molecular Probes, Eugene, OR) were used as second antibodies. All confocal microscopy was performed on a BioZERO BZ8000 (Keyence, Tokyo, Japan). To assess the in vivo potential of the residual BM CD4+ T cells in colitic SCID mice induced by the adoptive transfer of CD4+CD45RBhigh T cells to induce colitis, CD4+ T cells (1 × 105 cells/mouse) isolated from the BM, MLN, and LP of colitic mice or BM of age-matched normal BALB/c mice were injected into new SCID mice. In another set of experiments, BM CD4+ T cells (1 × 105 cells/mouse) isolated from colitic IL-10−/− mice (age, 20 wk) or age-matched normal C57BL/6 mice (1 × 105 cells/mouse) were injected into C57BL/6 RAG2−/− mice. To assess the role of commensal bacteria in the development of colitis and the retention of colitogenic BM CD4+ effector-memory T (TEM) cells, we used broad-spectrum antibiotics in another adoptive transfer experiment. CB-17 SCID mice were treated with or without ampicillin (1 g/L; Sigma, St. Louis, MO), vancomycin (500 mg/L; Abbott Labs, Abbott Park, Illinois), neomycin sulfate (1 g/L; Pharmacia/Upjohn, New York, NY), and metronidazole (1 g/L; Sidmak, Gujarat, India) in drinking water 4 weeks before beginning the adoptive transfer and during the course of the development of colitis based on a variation of the commensal depletion protocol of Fagarasan et al.20Fagarasan S. Muramatsu M. Suzuki K. Nagaoka H. Hiai H. Honjo T. Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora.Science. 2002; 298: 1424-1427Crossref PubMed Scopus (506) Google Scholar All recipient mice were weighed initially, then 3 times/wk after the transfer. They then were observed for clinical signs of illness as previously described.18Totsuka T. Kanai T. Iiyama R. Uraushihara K. Yamazaki M. Okamoto R. Hibi T. Tezuka K. Azuma M. Akiba Yagita H. Okumura K. Watanabe M. Ameliorating effect of anti-ICOS monoclonal antibody in a murine model of chronic colitis.Gastroenterology. 2003; 124: 410-421Abstract Full Text PDF PubMed Scopus (70) Google Scholar To assess the role of IL-7 in the maintenance of BM CD4+ T cells, we further transferred LP CD4+ T cells (2 × 106 cells/mouse) isolated from colitic CD4+CD45RBhigh T-cell–transferred mice into IL-7−/− × Rag-1−/− and IL-7+/+ × Rag-1−/− mice. Mice were killed 5 days after transfer, and the spleen and BM cells were isolated and stained with PE-conjugated rat anti-CD3ϵ mAbs and FITC-conjugated rat anti-CD69 mAbs or isotype FITC-conjugated control antibody. Before staining for intracellular Bcl-2, cells (2 × 106) were stained with PE-conjugated rat anti-CD3 mAbs as described earlier. After washing, cells were fixed and permeabilized with Cytofix-Cytoperm (BD PharMingen) solution according to the manufacturer's instructions. Cells were stained with either FITC-conjugated hamster anti-mouse Bcl-2 or a control antibody diluted in perm/wash buffer. To further assess the proliferative responses of CD4+ T cells in IL-7+/+ × Rag-1−/− and IL-7−/− × Rag-1−/− recipients, LP CD4+ T cells from SCID mice with colitis induced by the adoptive transfer of CD4+CD45RBhigh T cells were labeled with carboxyfluoroscein succinimidyl ester (CFSE) (Molecular Probes) by incubating at 5 μmol/L in PBS, quenching with fetal calf serum, and washing with PBS 3 times. Cells were resuspended in PBS, and 3 × 106 total cells were transferred by intravenous injection into IL-7+/+ × Rag-1−/− and IL-7−/− × Rag-1−/− mice. In another set of experiments, we transferred with colitogenic BM CD4+ T cells from colitic CD4+CD45RBhigh T-cell–transferred Rag-2−/− mice into IL-7+/+ × Rag-1−/− and IL-7−/− × Rag-1−/− recipients to clarify whether these mice develop colitis. Mice were killed at 10 weeks after transfer. The results were expressed as the mean ± SD. Groups of data were compared by the Mann–Whitney U test. Differences were considered statistically significant when the P value was less than .05. To investigate the role of BM in consecutive immunopathology in immune-mediated diseases, we first compared the composition and phenotype of CD4+ T cells in BM, MLN, and colonic LP of colitic mice induced by the adoptive transfer of CD4+CD45RBhigh T cells into recipient CB-17 SCID mice and with those of age-matched normal BALB/c mice. CD3+CD4+ mature T cells were found to reside in BM, MLN, and LP (colitic mice: BM, 12.7 ± 4.4 × 105 per mouse; MLN, 7.01 ± 4.2 × 105; and LP, 187 ± 99 × 105; normal mice: BM, 16.6 ± 3.8 × 105; MLN, 99.6 ± 18 × 105; and LP, 4.17 ± 1.2 × 105). As shown in Figure 1, the BM CD4+ T cells, as well as MLN and LP CD4+ T cells, from the colitic mice, exclusively have a phenotype of CD44highCD62L− cells. Furthermore, these colitic BM CD4+ T cells expressed IL-7Rα highly, indicating that the colitic BM CD4+ T cells have a characteristic of TEM cells. In contrast, the BM CD4+ T cells from normal mice are composed of 3 subpopulations: CD44lowCD62L+ naive cells, CD44highCD62L+ central-memory T cells, and CD44highCD62L− TEM cells (Figure 1). CD69, which is associated with cell activation, was expressed by a significantly higher proportion of CD4+ T cells from colitic mice than from normal mice. Interestingly, BM CD4+ T cells from colitic mice expressed relatively, but not significantly, high levels of integrin α4β7, a homing receptor to the gut, as compared with BM CD4+ T cells from normal mice, but lower levels than did MLN CD4+ T cells from colitic mice. These data indicate that the integrin α4β7-expressing CD4+ memory T cells, which are instructed to express the molecule in MLN or Peyer's patches,21Johansson-Lindborm B. Svensson M. Wurbel M.A. Malissen B. Marquez G. Agace W. Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant.J Exp Med. 2003; 198: 963-969Crossref PubMed Scopus (399) Google Scholar, 22Mora J.R. Rosa Bonbo M. Manjunath N. Weninger W. Cavanagh L.L. Rosemblatt M. von Andrian U.H. Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells.Nature. 2003; 424: 88-93Crossref PubMed Scopus (920) Google Scholar migrate to the BM. We next examined whether the colitic BM CD4+ T cells retained the ability to produce type-1 T helper (Th1) cytokines as well as the colitic CD4+ T cells in other sites. The production of IFN-γ, tumor necrosis factor-α, and IL-2 by anti-CD3/CD28 mAb-stimulated BM CD4+ T cells from colitic mice was significantly higher than that by normal BM CD4+ T cells, but lower than those by anti-CD3/CD28 mAb-stimulated LP CD4+ T cells (Figure 2), indicating that the colitic BM CD4+ T cells could be primed to Th1-type cells, and sustained in the BM. To determine whether the BM CD4+ T cells from colitic mice express their pathogenic potential on stimulation with antigens derived from resident enteric bacteria, we examined in vitro IFN-γ secretion by normal and colitic BM, MLN, and LP CD4+ T cells stimulated with various concentrations of CBA. The results show that significantly higher levels of IFN-γ were produced by colitic BM CD4+ T cells in response to a high dose (1000 μg/mL) of CBA as compared with normal BM CD4+ T cells, but significantly lower than those by colitic LP CD4+ T cells, which responded to much lower concentrations (10, 100, 1000 μg/mL) of CBA (Figure 3). The similar result was obtained by paired samples of MLN (Figure 3) and splenic (data not shown) CD4+ T cells. These results indicated that the colitic BM CD4+ T cells have the potential to respond against bacterial antigens and thus have the possibility to be colitogenic similar to the colitic LP CD4+ T cells as we have shown previously.18Totsuka T. Kanai T. Iiyama R. Uraushihara K. Yamazaki M. Okamoto R. Hibi T. Tezuka K. Azuma M. Akiba Yagita H. Okumura K. Watanabe M. Ameliorating effect of anti-ICOS monoclonal antibody in a murine model of chronic colitis.Gastroenterology. 2003; 124: 410-421Abstract Full Text PDF PubMed Scopus (70) Google Scholar We next examined the distribution of IL-7–producing cells23Namen A.E. Lupton S. Hjerrild K. Wignall J. Mochizuki D.Y. Schmierer A. Mosley B. March C.J. Urdal D. Gillis S. Stimulation of B-cell progenitors by cloned murine interleukin-7.Nature. 1988; 333: 571-573Crossref PubMed Scopus (758) Google Scholar and their interaction with CD4+ T cells in the colitic BM. The IL-7–expressing cells were scattered throughout the BM as has been reported previously24Tokoyoda K. Egawa T. Sugiyama T. Choi B.-I.I. Nagasawa T. Cellular niches controlling B lymphocyte behavior within bone marrow during development.Immunity. 2004; 20: 707-714Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar and most CD4+ T cells were in close contact with the bodies of IL-7–expressing cells (Figure 4). In contrast, IL-7 was not expressed, and CD4+ T cells did not reside in the BM of IL-7−/− × Rag-1−/− mice used as a negative control (Figure 4). To examine the homeostatic proliferation of the colitic BM CD4+ T cells, 2 experimental approaches were used. First, we examined memory CD4+ T cells from each tissue for evidence of active cell division by DNA staining using 7AAD (Figure 5A). Cells actively synthesizing DNA could be identified by their increased DNA content, allowing us to identify tissues where active cell division was occurring. A larger percentage of CD4+ T cells was actively synthesizing DNA in both the colitic and normal BM than in any other tissues (Figure 5A). Although the difference was slight, it was reproducible over 3 independent experiments. Second, colitic mice were injected with BrdU to provide evidence of recent DNA synthesis. To accurately examine the differences in cell proliferation in different tissues, it was necessary to give a short pulse of BrdU because longer treatment with BrdU might obscure the differences among the various tissues, probably because of the migration of dividing cells among the tissues. Mice thus were killed 24 hours after the injection of BrdU, and BrdU incorporation was measured in the CD4+ T cells obtained from BM, MLN, and LP (Figure 5B). Significantly higher percentages of memory T cells were synthesizing DNA in the colitic BM, MLN, and LP as compared with those in the paired normal BM, MLN, and LP. Because we compared dissimilar subsets in this setting because normal BM contains all subsets, such as naive, central memory, and TEM CD4+ T cells, yet in contrast colitic BM CD4+ T cells are constituted of TEM cells exclusively (Figure 1), we next compared colitic BM and LP CD4+CD44highCD62L− TEM cells with the paired normal TEM cells. As shown in Figure 5C, DNA synthesis in colitic BM and LP CD4+CD44highCD62L− TEM cells was increased significantly as compared with that in the paired normal gated TEM cells (Figure 5C). Based on the earlier-described results, we hypothesized that the colitic BM retaining CD4+ TEM cells is a pathogenic reservoir for persisting lifelong colitis. To prove this, we performed an adoptive transfer experiment by transferring colitic BM, MLN, and LP CD4+ TEM cells obtained from CD4+CD45RBhigh-transferred SCID mice and normal BM CD4+ T cells into new SCID mice (Figure 6A). As shown in Figure 6B, mice transferred with the colitic BM, MLN, and LP CD4+ T cells manifested progressive weight loss at 8 weeks after transfer. These mice had diarrhea with increased mucus in the stool, anorectal prolapse, and hunched posture by 4–6 weeks. In contrast, mice transferred with normal BM CD4+ T cells appeared healthy, showing a gradual increase of body weight and no diarrhea during the period of observation (Figure 6B and C). At 8 weeks after transfer, colitic BM CD4+ T-cell–transferred mice, but not mice transferred with normal BM CD4+ T cells, had enlarged colons with greatly thickened walls (Figure 6D). The assessment of colitis by clinical scores showed a clear difference between mice transferred with colitic BM CD4+ T cells and mice transferred with normal BM CD4+ T cells (Figure 6C). In addition, the clinical scores of mice transferred with colitic BM CD4+ T cells were comparable with those of mice transferred with colitic MLN or LP CD4+ T cells. Histologic examination showed prominent epithelial hyperplasia with glandular elongation and massive infiltration of mononuclear cells in LP of the colon from colitic BM CD4+ T-cell–transferred mice as well as colons from the colitic MLN or LP CD4+ T-cell–transferred mice (Figure 6E). In contrast, pathologic findings were not observed in the LP of the colon from mice transferred with normal BM CD4+ T cells (Figure 6E). This difference also was confirmed by histologic scoring of multiple colon sections (Figure 6F). A further quantitative evaluation of CD4+ T-cell accumulation was made by isolating CD3+CD4+ T cells. Few CD3+CD4+ T cells were recovered from the colonic LP in the normal BM CD4+ T-cell–transferred mice as compared with the mice transferred with the colitic BM, MLN, or LP CD4+ T cells (Figure 6G). Somewhat unexpectedly, the number of CD4+ T cells recovered from the BM of normal BM CD4+ T-cell–transferred mice
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