A prominent feature of sensitizing environmental compounds that cause allergic contact dermatitis is the rapid induction of an innate inflammatory response that seems to provide danger signals for efficient T cell priming. We generated mouse models of mast cell deficiency, mast cell-specific gene inactivation, and mast cell reporter mice for intravital imaging and showed that these adjuvant effects of contact allergens are mediated by mast cells and histamine. Mast cell deficiency resulted in impaired emigration of skin DCs to the lymph node and contact hypersensitivity was dramatically reduced in the absence of mast cells. In addition, mast cell-specific inactivation of the Il10 gene did not reveal any role for mast cell-derived IL-10 in the regulation of contact allergy. Collectively, we demonstrate that mast cells are essential promoters of contact hypersensitivity, thereby highlighting their potential to promote immune responses to antigens entering via the skin.

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Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker. IntroductionNeutrophils are the most abundant and, arguably, the most important leukocyte in the vertebrate immune system. Under normal conditions, human bone marrow (BM) produces ϳ 10 11 neutrophils per day. 1 In "danger situations" such as peripheral infections, the constant release of neutrophils can be dramatically increased within hours, a process termed danger or stress mobilization. 2 The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) is central to the danger mobilization of neutrophils in both humans and mice. 3 However, although recombinant G-CSF has been used in clinical hematology for 20 years, the molecular mechanisms by which it mobilizes neutrophils are still not well understood.Neutrophils are restrained in the BM by the binding of their chemokine receptor Cxcr4 to the chemokine Cxcl12, which is expressed in a membrane-associated fashion by BM stromal cells. 4 There is evidence that G-CSF breaks the Cxcr4-Cxcl12 bond by activating neutrophil proteases, 5 thereby releasing neutrophils from the BM into the bloodstream. 3,6 However, several findings cannot be explained by the Cxcr4-Cxcl12 breakage concept alone. First, G-CSF can mobilize neutrophils in protease-deficient mice, arguing against the need for protease activation for this process. 7,8 Second, because neutrophil-specific deletion of Cxcr4 in mice results in much higher numbers of circulating neutrophils compared with wild-type animals, factors other than Cxcr4 must be involved in steering neutrophils into the BM blood sinuses (unless the process is passive). Finally, the specific Cxcr4-antagonist AMD3100 a...

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Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an IntroductionIn the adult bone marrow (BM), the hematopoietic function is supported by the proliferation and differentiation of a finite number of transplantable hematopoietic stem cells and progenitors (HSCs/Ps) that have self-renewal and multipotential differentiation capacity. Endosteal and neighboring vascular/perivascular niches have been proposed as anatomic sites that control HSC/P activity, providing signals that regulate the stem cell pool size, survival, and migration. [1][2][3][4] The definition of the cell components of the BM hematopoietic microenvironment (HM) is an area of debate. Endothelial cells, 2 mesenchymal stem cells (MSCs), 5 and osteoblasts and osteogenic progenitors (OB/Ps) 1,4,6 represent major cell components of the BM HM.A poorly studied mechanism of intercellular communication (IC) in the HM is mediated by gap junction (GJ) channels. GJ channels are formed by a large family of proteins called connexins (Cxs). Each neighboring cell contributes cell-to-cell channels with 1 hemichannel that dock head-to-head. GJ channels have the ability to transfer ions and low-molecular weight secondary messengers from one cell to another depending on concentration gradients and Cx-dependent selective permeability. 7 Connexin-43 (Cx43), one member of this protein family, is highly expressed by adult BM stromal cells, 8 osteoblasts, 9 endothelial cells, 2 and MSCs, 5 and is also expressed by HSCs. 10 The presence of a functional Cx43-dependence of GJIC between osteoblasts and stromal cells, as well as between stromal cells and HSCs, has been confirmed in vitro. 11 After HSC transplantation, HSCs must first home to the BM, and then localize and anchor in suitable microenvironments within the BM, a process known as lodgment. 12 This process involves the migration of HSC/Ps through different layers of cell components in the HM, including endothelial cells and mesenchymal-origin cells, which act as extrinsic HSC/P migration regulators, 13 and is directed by chemokine gradients. Myeloablation through irradiation has been postulated to modify the functional ability of the BM HM to allow HSC homing and retention through clearance of HSC niches. 14 The effect of irradiation on the BM HM is not well known, but diverse studies have shown that it is not innocuous 15 and includes the up-regulation of expression and secretion of Cxcl12 16 and the up-regulation of the expression of Cx43 in the BM. 17 It has been proposed that Cx43 hemichannels are important in reinforcing cell-to-cell migration and that Cx43-dependent GJ are needed in the process of neuronal migration during neocortex form...

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Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.