InVivoMAb anti-mouse E-selectin (CD62E)

Immune protection relies on the capacity of neutrophils to infiltrate challenged tissues. Naive tissues, in contrast, are believed to remain free of these cells and protected from their toxic cargo. Here, we show that neutrophils are endowed with the capacity to infiltrate multiple tissues in the steady-state, a process that follows tissue-specific dynamics. By focusing in two particular tissues, the intestine and the lungs, we find that neutrophils infiltrating the intestine are engulfed by resident macrophages, resulting in repression of Il23 transcription, reduced G-CSF in plasma, and reinforced activity of distant bone marrow niches. In contrast, diurnal accumulation of neutrophils within the pulmonary vasculature influenced circadian transcription in the lungs. Neutrophil-influenced transcripts in this organ were associated with carcinogenesis and migration. Consistently, we found that neutrophils dictated the diurnal patterns of lung invasion by melanoma cells. Homeostatic infiltration of tissues unveils a facet of neutrophil biology that supports organ function, but can also instigate pathological states.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-alpha, interleukin 1beta, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (Selp(KI)). Selp(KI) (/) (KI) mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-alpha or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of Selp(KI) (/) (KI) mice. However, TNF-alpha did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in Selp(KI) (/-) mice. Higher basal P-selectin in Selp(KI) (/) (KI) mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.

Endothelial E- and P-selectins mediate lymphocyte trafficking in inflammatory processes by interacting with lymphocyte selectin ligands. These are differentially expressed among different T cell subsets and function alone or in cooperation to mediate T cell adhesion. In this study, we characterize the expression and functionality of E-selectin ligands in Th type 17 lymphocytes (Th17 cells) and report that CD43 functions as a Th17 cell E-selectin ligand in vitro that mediates Th17 cell rolling on the vascular endothelium and recruitment in vivo. We demonstrate Th17 cells express CD44, P-selectin glycoprotein ligand (PSGL)-1, and CD43. Few PSGL-1(-/-)CD43(-/-) Th17 cells accumulated on E-selectin under shear flow conditions compared with wild-type cells. CD43(-/-) Th17 cell accumulation on E-selectin was impaired as compared with wild-type and PSGL-1(-/-), and similar to that observed for PSGL-1(-/-)CD43(-/-) Th17 cells, indicating that CD43 alone is a dominant ligand for E-selectin. Notably, this finding is Th17 cell subset specific because CD43 requires cooperation with PSGL-1 in Th1 cells for binding to E-selectin. In vivo, Th17 cell recruitment into the air pouch was reduced in CD43(-/-) mice in response to CCL20 or TNF-alpha, and intravital microscopy studies demonstrated that CD43(-/-) Th17 cells had impaired rolling on TNF-alpha-treated microvessels. Furthermore, CD43(-/-) mice were protected from experimental autoimmune encephalomyelitis and had impaired recruitment of Th17 cells in the spinal cord. Our findings demonstrate that CD43 is a major E-selectin ligand in Th17 cells that functions independent of PSGL-1, and they suggest that CD43 may hold promise as a therapeutic target to modulate Th17 cell recruitment.

Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid beta2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo.