MLN and PLN lymphocytes were isolated 3 days after intraperitoneal administration of OVA, and the ability of OT-1 cells and endogenous CD8+ lymphocytes to migrate to CCL25 (250 nM) was determined. is usually continually exposed to a large array of foreign antigens and must respond appropriately to maintain mucosal integrity while at the same time mounting effective immune responses to potential pathogens. The recruitment of T lymphocytes to intestinal effector Rabbit Polyclonal to HLA-DOB sites is usually thought to play a critical role in this process. Following activation in secondary lymphoid organs, T lymphocytes gain the ability to migrate from Trimebutine the blood to tertiary effector tissues such as the intestine and skin (1). Subsets of previously activated T lymphocytes display selective tissue tropism for these sites, a process that is controlled by specific combinations of cell adhesion molecules (1, 2). Previously activated T lymphocytes homing to the intestine express high levels of 47 integrin, whose ligand, MAdCAM-1, is usually expressed on postcapillary venules in the intestinal lamina propria. Indeed, 7 integrin appears to be critical for the entry of previously activated T lymphocytes into the intestinal lamina propria and epithelium (3, 4). In addition to cell adhesion molecules, accumulating evidence exists for an involvement of Trimebutine chemokines and their receptors in the recruitment of activated lymphocyte subsets to effector tissues (5). For example, the CC chemokine receptor 4 (CCR4) and the CCR10 ligand, CCL27, were recently shown to contribute to lymphocyte recruitment to inflamed skin (6, 7). The chemokine receptor CCR9 is usually selectively and functionally expressed on human small-intestinal lymphocytes (8), and its ligand, CCL25, is usually constitutively expressed by murine and human small-intestinal epithelial cells (9, 10), indicating a potential role for this chemokine receptor/chemokine pair in lymphocyte localization to the small intestine. However, examination of CD8+ lymphocyte numbers in the small-intestinal epithelium of CCR9C/C mice has yielded conflicting results (11, 12). Furthermore, small-intestinal epithelial cells constitutively express a number of chemokines with activity for T lymphocytes, including CXCL12, CCL28, and CX3CL1, indicating a potential role for other chemokines in this process (13C15). Thus the in vivo role of CCL25 and CCR9 in T lymphocyte recruitment to the small intestine remains unclear. In the current study we have examined expression and regulation of CCR9 on murine CD8+ lymphocytes in vivo and decided the role of CCL25 in the recruitment of recently activated CD8+ lymphocytes Trimebutine to the small-intestinal mucosa. Methods Mice. C57BL/6J-Ly5.1 mice were obtained from Charles River Laboratories (Wilmington, Massachusetts, USA). OT-1 mice were kindly provided by A. Mowat (University of Glasgow, United Kingdom). All mice were maintained at the animal facility at the Department of Microbiology, Immunology and Glycobiology, Lund University, and were used between 8 and 14 weeks of age. Antibodies and reagents. Anti-CD8 (53-5.8), anti-Ly5.2 (104), anti-CD62L (Mel-14), anti-CD44 (IM7), anti-CD4 (RM4-5), and anti-CD69 (H1.2F3) antibodies Trimebutine and streptavidin-allophycocyanin were from Pharmingen (San Diego, California, USA). Anti-E (M290) and anti-7 (FIB-504) antibodies were kindly provided by C. Parker (Dana-Farber Cancer Institute, Boston, Massachusetts, USA). Hybridomas producing anti-CD8 (YTS 169-4), anti-CD4 (GK1.5), anti-B220 (RA3.6B2), antiCFcRII/III (2.4G2), and antiCMHC-II (M5/114) antibodies were from American Type Culture Collection (Rockville, Maryland, USA). Goat anti-rabbit Ig was from Jackson ImmunoResearch Laboratories Inc. (West Grove, Pennsylvania, USA), polyclonal rabbit antiCmouse CCR9 antibody was from G. Mrquez (K629; ref. 16), 7-amino-actinomycin D (7AAD) was from Sigma-Aldrich (Steinheim, Germany), and recombinant murine CCL25 was from R&D Systems Europe (Abingdon, United Kingdom). Murine stromal cell-derivedCfactor 1 was a kind gift from I. Clark-Lewis (University of British Columbia, Vancouver, British Co-lumbia, Canada). Adoptive transfers. We injected 2 106 to 3 106 CD8+ OT-1 cells intravenously into C57BL/6J-Ly5.1 mice. Two to three days later, we injected mice intraperitoneally with 5 mg ovalbumin (OVA, grade VI; Sigma-Aldrich) with or without 100 g LPS (= 19) and about 55% of peripheral lymph node (PLN) (mean 53.0%, SD.