ABSTRACT
After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.
Subject(s)
5'-Nucleotidase/immunology , Adenosine/immunology , Cell Movement/immunology , Endothelium, Vascular/immunology , Lymph Nodes/immunology , T-Lymphocytes/immunology , 5'-Nucleotidase/genetics , 5'-Nucleotidase/metabolism , Adenosine/genetics , Adenosine/metabolism , Adenosine A2 Receptor Agonists , Adenosine Monophosphate/genetics , Adenosine Monophosphate/immunology , Adenosine Monophosphate/metabolism , Aminopyridines/pharmacology , Animals , Cell Movement/drug effects , Cell Movement/genetics , Dendritic Cells/enzymology , Dendritic Cells/immunology , Endothelium, Vascular/enzymology , Inflammation/enzymology , Inflammation/genetics , Inflammation/immunology , L-Selectin/immunology , L-Selectin/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Knockout , Receptor, Adenosine A2B/immunology , Receptor, Adenosine A2B/metabolism , T-Lymphocytes/enzymology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation/drug effects , Up-Regulation/genetics , Up-Regulation/immunology , Venules/enzymology , Venules/immunologyABSTRACT
Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.
