Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.

Cytotoxic CD8+ T cells stimulate hematopoietic progenitors by promoting cytokine release from bone marrow mesenchymal stromal cells.

Cytotoxic CD8(+) T cells (CTLs) play a major role in host defense against intracellular pathogens, but a complete clearance of pathogens and return to homeostasis requires the regulated interplay of the innate and acquired immune systems. Here, we show that interferon γ (IFNγ) secreted by effector CTLs stimulates hematopoiesis at the level of early multipotent hematopoietic progenitor cells and induces myeloid differentiation. IFNγ did not primarily affect hematopoietic stem or progenitor cells directly. Instead, it promoted the release of hematopoietic cytokines, including interleukin 6 from bone marrow mesenchymal stromal cells (MSCs) in the hematopoietic stem cell niche, which in turn reduced the expression of the transcription factors Runx-1 and Cebpα in early hematopoietic progenitor cells and increased myeloid differentiation. Therefore, our study indicates that, during an acute viral infection, CTLs indirectly modulate early multipotent hematopoietic progenitors via MSCs in order to trigger the temporary activation of emergency myelopoiesis and promote clearance of the infection.

Exposure to food allergens through inflamed skin promotes intestinal food allergy through the thymic stromal lymphopoietin-basophil axis.

BACKGROUND Exposure to food allergens through a disrupted skin barrier has been recognized as a potential factor in the increasing prevalence of food allergy. OBJECTIVE We sought to test the immunologic mechanisms by which epicutaneous sensitization to food allergens predisposes to intestinal food allergy. METHODS Mice were epicutaneously sensitized with ovalbumin or peanut on an atopic dermatitis-like skin lesion, followed by intragastric antigen challenge. Antigen-specific serum IgE levels and T(H)2 cytokine responses were measured by ELISA. Expression of type 2 cytokines and mast cell proteases in the intestine were measured by using real-time PCR. Accumulation of basophils in the skin and mast cells in the intestine was examined by using flow cytometry. In vivo basophil depletion was achieved by using diphtheria toxin treatment of Baso-DTR mice. For cell-transfer studies, the basophil population was expanded in vivo by means of hydrodynamic tail vein injection of thymic stromal lymphopoietin (TSLP) cDNA plasmid. RESULTS Sensitization to food allergens through an atopic dermatitis-like skin lesion is associated with an expansion of TSLP-elicited basophils in the skin that promote antigen-specific T(H)2 cytokine responses, increased antigen-specific serum IgE levels, and accumulation of mast cells in the intestine, promoting the development of intestinal food allergy. Critically, disruption of TSLP responses or depletion of basophils reduced the susceptibility to intestinal food allergy, whereas transfer of TSLP-elicited basophils into intact skin promoted disease. CONCLUSION Epicutaneous sensitization on a disrupted skin barrier is associated with accumulation of TSLP-elicited basophils, which are necessary and sufficient to promote antigen-induced intestinal food allergy.

Support of hepatic regeneration by trophic factors from liver-derived mesenchymal stromal/stem cells

Mesenchymal stromal/stem cells (MSCs) have multilineage differentiation potential and as such are known to promote regeneration in response to tissue injury. However, accumulating evidence indicates that the regenerative capacity of MSCs is not via transdifferentiation but mediated by their production of trophic and other factors that promote endogenous regeneration pathways of the tissue cells. In this chapter, we provide a detailed description on how to obtain trophic factors secreted by cultured MSCs and how they can be used in small animal models. More specific, in vivo models to study the paracrine effects of MSCs on regeneration of the liver after surgical resection and/or ischemia and reperfusion injury are described.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Chronology of Lateglacial ice flow reorganization and deglaciation in the Gotthard Pass area, Central Swiss Alps, based on cosmogenic 10Be and in situ 14C

We reconstruct the timing of ice flow reconfiguration and deglaciation of the Central Alpine Gotthard Pass, Switzerland, using cosmogenic 10Be and in situ14C surface exposure dating. Combined with mapping of glacial erosional markers, exposure ages of bedrock surfaces reveal progressive glacier downwasting from the maximum LGM ice volume and a gradual reorganization of the paleoflow pattern with a southward migration of the ice divide. Exposure ages of ∼16–14 ka (snow corrected) give evidence for continuous early Lateglacial ice cover and indicate that the first deglaciation was contemporaneous with the decay of the large Gschnitz glacier system. In agreement with published ages from other Alpine passes, these data support the concept of large transection glaciers that persisted in the high Alps after the breakdown of the LGM ice masses in the foreland and possibly decayed as late as the onset of the Bølling warming. A younger group of ages around ∼12–13 ka records the timing of deglaciation following local glacier readvance during the Egesen stadial. Glacial erosional features and the distribution of exposure ages consistently imply that Egesen glaciers were of comparatively small volume and were following a topographically controlled paleoflow pattern. Dating of a boulder close to the pass elevation gives a minimum age of 11.1 ± 0.4 ka for final deglaciation by the end of the Younger Dryas. In situ14C data are overall in good agreement with the 10Be ages and confirm continuous exposure throughout the Holocene. However, in situ14C demonstrates that partial surface shielding, e.g. by snow, has to be incorporated in the exposure age calculations and the model of deglaciation.

Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells.

BACKGROUND Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family. METHODS Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry. RESULTS Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs. CONCLUSIONS We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.