Homeostatic regulation of neutrophil production is thought to match neutrophil elimination to maintain approximately constant numbers in the blood. Here, we show that IL-17, a cytokine that regulates granulopoiesis through G-CSF, is made by gammadelta T cells and unconventional alphabeta T cells. These neutrophil-regulatory T cells (Tn) are expanded in mice that lack leukocyte adhesion molecules, which have neutrophilia and defective neutrophil trafficking. Normal neutrophils migrate to tissues, where they become apoptotic and are phagocytosed by macrophages and dendritic cells. This curbs phagocyte secretion of IL-23, a cytokine controlling IL-17 production by Tn cells. Adoptive transfer of wild-type, but not adhesion molecule-deficient, neutrophils into mice deficient in beta2 integrins transiently decreases neutrophilia and reduces levels of serum IL-17. Antibody blockade of the p40 subunit of IL-23 reduces neutrophil numbers in wild-type mice. These findings identify a major homeostatic mechanism for the regulation of neutrophil production in vivo.

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.Sphingosine 1-phosphate (S1P) 2 receptor agonists are likely to be the next generation of pharmacologic agents used to modulate immune system function. The prototype drug of this class is FTY720, which is highly efficacious in prolonging allograft survival and in ameliorating autoimmune disease in a variety of animal models (1-4). FTY720 is being tested in human trials for the indications renal transplantation and multiple sclerosis (5). Further, there is mounting evidence that S1P agonists are efficacious in animal models of atherosclerosis (6), renal ischemia-reperfusion injury (7), and acute lung injury (8).FTY720 is a sphingosine analog that, after activation by phosphorylation (to FTY720-P), disrupts lymphocyte trafficking by decreasing lymphocyte egress from lymph nodes and the thymus (9, 10). Although the precise mechanisms that underlie this phenomenon are uncertain, the profound lymphopenia that is the index of FTY720 action is dependent on agonist action at lymphocyte S1P 1 receptors. Since FTY720-P is also a potent agonist at the S1P 3 , S1P 4 , and S1P 5 receptors (11, 12), it remains unknown whether the multiple therapeutic benefits of the drug correlate with agonist activity at the S1P 1 receptor. The propensity for S1P 1 receptor responses to desensitize (13) and the similar behaviors of S1P 1 receptor null thymocytes and FTY720-treated mouse lymphocytes have led to the suggestion that FTY720-P is a functional antagonist (14). In this scenario, the drug exaggerates S1P tone to the extent that the lymphocyte S1P 1 receptor signaling is chronically down-regulated.The kinase(s) responsible for FTY720 activation is the gateway whereby S1P signaling can be accessed readily with a therapeutic agent. Knowledge of this enzyme is important specifically to guide S1P prodrug design and generally to gain insight into the normal role of S1P in immune function. The identity of the kinase is not known currently; two candidates are sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). These enzymes, which are expressed widely, catalyz...

Background-Mice

The initiating etiologic factor in Crohn's disease (CD) remains unclear. SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to human CD. We used bone marrow chimeras to determine if SAMP ileitis results from a primary immunological defect or from dysregulated mucosal immunity secondary to intrinsic, nonhematopoietic (e.g., epithelial) dysfunction. SAMP mice receiving wild-type (AKR) BM developed severe ileitis, whereas SAMP BM did not confer ileitis to WT recipients. WT lymphocytes from reconstituted SAMP mice resembled native SAMP populations in regard to surface phenotype and cytokine production. Ilea from native SAMP mice and SAMP recipients of wild-type BM displayed decreased epithelial barrier resistance ex vivo and increased epithelial permeability in vivo compared to native WT mice and AKR recipients of SAMP BM. This permeability defect preceded the development of ileal inflammation, was present in the absence of commensal bacteria, and was accompanied by altered ileal mRNA expression of the tight junction proteins claudin-2 and occludin. Our results provide evidence that the primary defect conferring ileitis in SAMP mice originates from a nonhematopoietic source. Generation of pathogenic lymphocytes is a consequence of this defect and does not reflect intrinsic proinflammatory leukocyte properties. Decreased barrier function suggests that defects in the epithelium may represent the primary source of SAMP ileitis susceptibility.

Chemokines, including CXCL1, participate in neutrophil recruitment by triggering the activation of integrins, which leads to arrest from rolling. The downstream signaling pathways which lead to integrin activation and neutophil arrest following G-protein-coupled receptor engagement are incompletely understood. To test whether G␣ i2 is involved, mouse neutrophils in their native whole blood were investigated in mouse cremaster postcapillary venules and in flow chambers coated with P-selectin, ICAM-1, and CXCL1. Gnai2 Ϫ/Ϫ neutrophils showed significantly reduced CXCL1-induced arrest in vitro and in vivo. Similar results were obtained with leukotriene B 4 (LTB 4 ). Lethally irradiated mice reconstituted with Gnai2 Ϫ/Ϫ bone marrow showed a similar defect in chemoattractant-induced arrest as that of Gnai2 Ϫ/Ϫ mice. In thioglycollate-induced peritonitis and lipopolysaccaride (LPS)-induced lung inflammation, chimeric mice lacking G␣ i2 in hematopoietic cells showed about 50% reduced neutrophil recruitment similar to that seen in Gnai2 Ϫ/Ϫ mice. These data show that neutrophil G␣ i2 is necessary for chemokineinduced arrest, which is relevant for neutrophil recruitment to sites of acute inflammation.(Blood.

IL-23 and IL-17A regulate granulopoiesis through G-CSF, the main granulopoietic cytokine. IL-23 is secreted by activated macrophages and dendritic cells and promotes the expansion of three subsets of IL-17A-expressing neutrophil-regulatory T (Tn) cells; CD4−CD8−αβlow, CD4+CD8−αβ+ (Th17), and γδ+ T cells. In this study, we investigate the effects of IL-17A on circulating neutrophil levels using IL-17R-deficient (Il17ra−/−) mice and Il17ra−/−Itgb2−/− mice that lack both IL-17R and all four β2 integrins. IL-17R deficiency conferred a reduction in neutrophil numbers and G-CSF levels, as did Ab blockade against IL-17A in wild-type mice. Bone marrow transplantation revealed that IL-17R expression on nonhemopoietic cells had the greatest effects on regulating blood neutrophil counts. Although circulating neutrophil numbers were reduced, IL-17A expression, secretion, and the number of IL-17A-producing Tn cells were elevated in Il17ra−/− and Il17ra−/−Itgb2−/− mice, suggesting a negative feedback effect through IL-17R. The negative regulation of IL-17A-producing T cells and IL-17A and IL-17F gene expression through the interactions of IL-17A or IL-17F with IL-17R was confirmed in splenocyte cultures in vitro. We conclude that IL-17A regulates blood neutrophil counts by inducing G-CSF production mainly in nonhemopoietic cells. IL-17A controls the expansion of IL-17A-producing Tn cell populations through IL-17R.

L-selectin ligands might be relevant for inflammatory cell trafficking into the small intestine in a spontaneous model of chronic ileitis (i.e., SAMP1/YitFc mice). Immunoblockade of peripheral node addressin or mucosal addressin cell adhesion molecule 1 failed to ameliorate ileitis, whereas P-selectin glycoprotein ligand 1 (PSGL-1) neutralization attenuated both the adoptively transferred and spontaneous disease. PSGL-1 was detected in venules of mesenteric lymph node and small intestine by immunohistochemistry and confirmed by real-time reverse transcription polymerase chain reaction and flow cytometry. In addition, reconstitution of wild-type mice with PSGL-1−/− bone marrow demonstrated that PSGL-1 messenger RNA and PSGL-1 protein expression remained on endothelium, localized within mesenteric lymph node and small intestine. Endothelial PSGL-1 bound P-selectin–IgG and its blockade or genetic deletion altered the recruitment of lymphocytes to the small intestine, as revealed by intravital microscopy and homing studies. Endothelial expression of PSGL-1 adds a new dimension to the various cellular interactions involved in small intestinal recruitment. Thus, the multiple roles of PSGL-1 may explain why targeting this single adhesion molecule results in attenuation of chronic murine ileitis, a disease previously resistant to antiadhesion molecule strategies.

SAMP1/YitFc mice develop discontinuous, transmural inflammatory lesions in the terminal ileum, similar to what is found in human Crohn disease. Compared with the mesenteric lymph nodes (MLNs) of AKR control mice, SAMP1/YitFc MLNs contain a 4.3-fold expansion in total B cell number and a 2.5-fold increased percentage of CD4(+) T cells expressing the alpha(E)beta(7) integrin. Although alpha(E)beta(7)(+)CD4(+) T cells possess a regulatory phenotype (CD25(+), L-selectin(lo), and CD45RB(lo)), express IL-10, and suppress effector T cell proliferation in vitro, they cannot prevent ileitis development in SCID mice adoptively transferred with effector CD4(+) T cells, although the CD4(+)CD25(+) subset, which overlaps with the alpha(E)beta(7)(+)CD4(+) subset, prevents colitis. The alpha(E)beta(7)(+)CD4(+) T cells express high levels of ICOS, a costimulatory molecule that augments B cell function, suggesting their involvement in the increase in B cells, IgA(+) cells, and soluble IgA found within the MLNs and ileum of SAMP1/YitFc mice. MLN B cell numbers correlate with ileitis severity in SAMP1/YitFc mice, and cotransfer of SAMP1/YitFc MLN B cells along with CD4(+) T cells increases ileitis severity in SCID mice compared with transfer of CD4(+) T cells alone. SAMP1/YitFc B cells prevent alpha(E)beta(7)(+)CD4(+) T cells from suppressing effector T cell proliferation. We conclude that SAMP1/YitFc MLN B cells contribute to the development of SAMP1/YitFc ileitis.