Tracy L. Burcin

Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung

In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2-/- mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely, CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.

Sphingosine Kinase 2 Is Required for Modulation of Lymphocyte Traffic by FTY720

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.

Critical Role of Macrophage 12/15-Lipoxygenase for Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Mice lacking leukocyte type 12/15-lipoxygenase (12/15-LO) show reduced atherosclerosis in several models. 12/15-LO is expressed in a variety of cells, including vascular cells, adipocytes, macrophages, and cardiomyocytes. The purpose of this study was to determine which cellular source of 12/15-LO is important for atherosclerosis. Methods and Results—Bone marrow from 12/15-LO−/−/apoE−/− mice was transplanted into apoE−/− mice and vice versa. Deficiency of 12/15-LO in bone marrow cells protected apoE−/− mice fed a Western diet from atherosclerosis to the same extent as complete absence of 12/15-LO, although plasma 8,12-iso-iPF2&agr;-IV, a measure of lipid peroxidation, remained elevated. 12/15-LO−/−/apoE−/− mice regained the severity of atherosclerotic lesion typical of apoE−/− mice after replacement of their bone marrow cells with bone marrow from apoE−/− mice. Peritoneal macrophages obtained from wild-type but not 12/15-LO−/− mice caused endothelial activation in the presence of native LDL. Absence of 12/15-LO decreased the ability of macrophages to form foam cells when exposed to LDL. Conclusions—We conclude that macrophage 12/15-LO plays a dominant role in the development of atherosclerosis by promoting endothelial inflammation and foam cell formation.

The primary defect in experimental ileitis originates from a nonhematopoietic source

The initiating etiologic factor in Crohns 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.

Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

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.

IL-17A Inhibits the Expansion of IL-17A-Producing T Cells in Mice through “Short-Loop” Inhibition via IL-17 Receptor

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.

Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis

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.

IL-23 Is Required for Neutrophil Homeostasis in Normal and Neutrophilic Mice

IL-23 is secreted by macrophages and dendritic cells in response to microbial products and inflammatory cytokines. IL-23 is a heterodimer composed of the unique IL-23p19 subunit linked to the common p40 subunit that it shares with IL-12. IL-23 is implicated in autoimmune diseases, where it supports the expansion of IL-17A-producing CD4+ Th17 cells. IL-23 also regulates granulopoiesis in a neutrostat regulatory feedback loop through IL-17A-producing neutrophil regulatory (Tn) cells, most of which express γδ TCR. This homeostatic system is disrupted in mice lacking adhesion molecules like β2-integrins (Itgb2−/−) which have defective neutrophil trafficking and neutrophilia. To test the role of IL-23 in the homeostatic regulation of circulating neutrophil numbers, we measured blood neutrophil numbers in p40-deficient (IL12b−/−) mice and found them reduced compared with wild-type mice. IL12b−/−Itgb2−/− mice, lacking β2-integrins, IL-12, and IL-23 showed significantly blunted neutrophilia compared with Itgb2−/− mice. Treatment of both IL12b−/− and IL12b−/−Itgb2−/− mice with IL-23, but not IL-12, restored circulating neutrophil counts. Serum levels of IL-17A were readily detectable in Itgb2−/− mice, but not in IL12b−/−Itgb2−/− mice, suggesting that IL-17A production is reduced when IL-23 is absent. Similarly, tissue mRNA expression of IL-17A was reduced in IL12b−/−Itgb2−/−mice compared with Itgb2−/− controls. The total number of CD3+ IL-17A-producing Tn cells were significantly reduced in the spleen and lamina propria of IL12b−/−Itgb2−/− mice, with the largest reduction found in γδ+ T cells. Our results suggest a prominent role of IL-23 in the regulation of granulopoiesis and the prevalence of IL-17A-producing Tn cells.