Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium to arrive at a site of infection. By necessity, in the setting of multiple chemoattractants, the neutrophils must prioritize, favoring end target chemoattractants (e.g., fMLP and C5a) emanating from the site of infection over intermediary endogenous chemoattractants (e.g., IL-8 and LTB4) encountered en route to sites of infection. In this study, we propose a hierarchical model of two signaling pathways mediating the decision-making process of the neutrophils, which allows end target molecules to dominate over intermediary chemoattractants. In an under agarose assay, neutrophils predominantly migrated toward end target chemoattractants via p38 MAPK, whereas intermediary chemoattractant-induced migration was phosphoinositide 3-kinase (PI3K)/Akt dependent. When faced with competing gradients of end target and intermediary chemoattractants, Akt activation was significantly reduced within neutrophils, and the cells migrated preferentially toward end target chemoattractants even at 1/1,000th that of intermediary chemoattractants. End target molecules did not require chemotactic properties, since the p38 MAPK activator, LPS, also inhibited Akt and prevented migration to intermediary chemoattractants. p38 MAPK inhibitors not only reversed this hierarchy, such that neutrophils migrated preferentially toward intermediary chemoattractants, but also allowed neutrophils to be drawn out of a local end target chemoattractant environment and toward intermediary chemoattractants unexpectedly in an exaggerated (two- to fivefold) fashion. This was entirely related to significantly increased magnitude and duration of Akt activation. Finally, end target chemoattractant responses were predominantly Mac-1 dependent, whereas nondominant chemoattractants used primarily LFA-1. These data provide support for a two pathway signaling model wherein the end target chemoattractants activate p38 MAPK, which inhibits intermediary chemoattractant-induced PI3K/Akt pathway, establishing an intracellular signaling hierarchy.

The dermal papilla (DP) provide instructive signals required to activate epithelial progenitors and initiate hair follicle regeneration. DP cell numbers fluctuate over the hair cycle, and hair loss is associated with gradual depletion/atrophy of DP cells. How DP cell numbers are maintained in healthy follicles remains unclear. We performed in vivo fate mapping of adult hair follicle dermal sheath (DS) cells to determine their lineage relationship with DP and found that a subset of DS cells are retained following each hair cycle, exhibit self-renewal, and repopulate the DS and the DP with new cells. Ablating these hair follicle dermal stem cells and their progeny retarded hair regrowth and altered hair type specification, suggesting that they function to modulate normal DP function. This work identifies a bipotent stem cell within the adult hair follicle mesenchyme and has important implications toward restoration of hair growth after injury, disease, and aging.

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Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1 −/−) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-α and interleukin-1β) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1 −/− chimeric mice using bone marrow transplantation revealed that in mice with Lsp1 −/− endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1 −/− neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1 −/− animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1 −/− mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ “super-healer” strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not.

Hair follicle stem cells are regulated by intrafollicular and extrafollicular niche signals. Appropriate hair follicle regeneration relies on the coordinated release and integration of these signals. How immune cells, particularly cutaneous macrophages, influence the hair follicle stem cell niche and regeneration is not well understood. We took advantage of wound-induced hair growth (WIHG) to explore the relationship between wound macrophages and hair follicle regeneration. First, we showed that WIHG is dependent on CD11bF4/80 macrophages at 7-11 days after injury. Next, using CXCR1:CCR2 mice to capture the dynamic spectrum of macrophage phenotypes during wound healing, we showed that wound macrophages transition from a CXCR1 to a CXCR1 phenotype at the onset of WIHG. Finally, WIHG is abolished in mice deficient for CXCR1, delayed with pharmacological inhibition of transforming growth factor-β receptor type 1, and rescued with exogenous transforming growth factor-β1. Overall, we propose a model in which transforming growth factor-β1 and CXCR1 are critical for recruiting and maintaining the CCR2CXCR1Ly6CTNFα macrophages critical for stimulating WIHG.

1 The present study was designed to examine the possible role of neuronal nitric oxide synthase (nNOS) in regulation of leukocyte ± endothelial cell interactions in the absence of endothelial nitric oxide synthase (eNOS), using intravital microscopy of the cremasteric microcirculation of eNOS 7/7 mice. 2 Baseline leukocyte rolling and adhesion revealed no di erences between wild-type and eNOS 7/7 mice in either the cremasteric or intestinal microcirculations. 3 Superfusion with L-NAME (100 mM) caused a progressive and signi®cant increase in leukocyte adhesion in both wild-type and eNOS 7/7 mice, without detecting di erences between the two strains of mice. 4 Superfusion with 7-nitroindazole (100 mM), a selective inhibitor of nNOS, had no e ect on leukocyte adhesion in wild-type animals. However, it increased leukocyte adhesion signi®cantly in eNOS 7/7 mice, which was reversed by systemic L-arginine pre-administration. 5 Stimulation of the microvasculature with H 2 O 2 (100 mM) induced a transient elevation in leukocyte rolling in wild-type mice. Conversely, the e ect persisted during the entire 60 min of experimental protocol in eNOS 7/7 mice either with or without 7-nitroindazole. 6 Semi-quantitative analysis by RT ± PCR of the mRNA for nNOS levels in eNOS 7/7 and wildtype animals, showed increased expression of nNOS in both brain and skeletal muscle of eNOS 7/7 mice. 7 In conclusion, we have demonstrated that leukocyte-endothelial cell interactions are predominantly modulated by eNOS isoform in postcapillary venules of normal mice, whereas nNOS appears to assume the same role in eNOS 7/7 mice. Interestingly, unlike eNOS there was insu cient NO produced by nNOS to overcome leukocyte recruitment elicited by oxidative stress, suggesting that nNOS cannot completely compensate for eNOS.

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1’s meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.