Surface-Driven Collagen Self-Assembly Affects Early Osteogenic Stem Cell Signaling.

This study reports how extracellular matrix (ECM) ligand self-assembly on biomaterial surfaces and the resulting nanoscale architecture can drive stem cell behavior. To isolate the biological effects of surface wettability on protein deposition, folding, and ligand activity, a polydimethylsiloxane (PDMS)-based platform was developed and characterized with the ability to tune wettability of elastomeric substrates with otherwise equivalent topology, ligand loading, and mechanical properties. Using this platform, markedly different assembly of covalently bound type I collagen monomers was observed depending on wettability, with hydrophobic substrates yielding a relatively rough layer of collagen aggregates compared to a smooth collagen layer on more hydrophilic substrates. Cellular and molecular investigations with human bone marrow stromal cells revealed higher osteogenic differentiation and upregulation of focal adhesion-related components on the resulting smooth collagen layer coated substrates. The initial collagen assembly driven by the PDMS surface directly affected α1ß1 integrin/discoidin domain receptor 1 signaling, activation of the extracellular signal-regulated kinase/mitogen activated protein kinase pathway, and ultimately markers of osteogenic stem cell differentiation. We demonstrate for the first time that surface-driven ligand assembly on material surfaces, even on materials with otherwise identical starting topographies and mechanical properties, can dominate the biomaterial surface-driven cell response.

Dendritic cells: In vitro culture in two- and three-dimensional collagen systems and expression of collagen receptors in tumors and atherosclerotic microenvironments.

Dendritic cells (DCs) are immune cells found in the peripheral tissues where they sample the organism for infections or malignancies. There they take up antigens and migrate towards immunological organs to contact and activate T lymphocytes that specifically recognize the antigen presented by these antigen presenting cells. In the steady state there are several types of resident DCs present in various different organs. For example, in the mouse, splenic DC populations characterized by the co-expression of CD11c and CD8 surface markers are specialized in cross-presentation to CD8 T cells, while CD11c/SIRP-1α DCs seem to be dedicated to activating CD4 T cells. On the other hand, DCs have also been associated with the development of various diseases such as cancer, atherosclerosis, or inflammatory conditions. In such disease, DCs can participate by inducing angiogenesis or immunosuppression (tumors), promoting autoimmune responses, or exacerbating inflammation (atherosclerosis). This change in DC biology can be prompted by signals in the microenvironment. We have previously shown that the interaction of DCs with various extracellular matrix components modifies the immune properties and angiogenic potential of these cells. Building on those studies, herewith we analyzed the angiogenic profile of murine myeloid DCs upon interaction with 2D and 3D type-I collagen environments. As determined by PCR array technology and quantitative PCR analysis we observed that interaction with these collagen environments induced the expression of particular angiogenic molecules. In addition, DCs cultured on collagen environments specifically upregulated the expression of CXCL-1 and -2 chemokines. We were also able to establish DC cultures on type-IV collagen environments, a collagen type expressed in pathological conditions such as atherosclerosis. When we examined DC populations in atherosclerotic veins of Apolipoprotein E deficient mice we observed that they expressed adhesion molecules capable of interacting with collagen. Finally, to further investigate the interaction of DCs with collagen in other pathological conditions, we determined that both murine ovarian and breast cancer cells express several collagen molecules that can contribute to shape their particular tumor microenvironment. Consistently, tumor-associated DCs were shown to express adhesion molecules capable of interacting with collagen molecules as determined by flow cytometry analysis. Of particular relevance, tumor-associated DCs expressed high levels of CD305/LAIR-1, an immunosuppressive receptor. This suggests that signaling through this molecule upon interaction with collagen produced by tumor cells might help define the poorly immunogenic status of these cells in the tumor microenvironment. Overall, these studies demonstrate that through interaction with collagen proteins, DCs can be capable of modifying the microenvironments of inflammatory disease such as cancer or atherosclerosis.

α1ß1 integrin-mediated adhesion inhibits macrophage exit from a peripheral inflammatory lesion.

Integrins are adhesion molecules critical for the recruitment of leukocytes from blood into peripheral tissues. However, whether integrins are also involved in leukocyte exit from peripheral tissues via afferent lymphatics to the draining lymph node remains poorly understood. In this article, we show that adhesion by the collagen IV-binding integrin α1ß1 unexpectedly inhibited macrophage exit from inflamed skin. We monitored macrophages exiting mouse footpads using a newly developed in situ pulse labeling technique. Blockade of α1ß1 integrin or genetic deletion (Itga1(-/-)) increased macrophage exit efficiency. Chemotaxis assays through collagen IV showed more efficient migration of Itga1(-/-) macrophages relative to wild type. Given that macrophages are key orchestrators of inflammation, α1ß1 integrin adhesion may represent a mechanism for regulating inflammatory responses by controlling macrophage exit or persistence in inflamed tissues.

[Cloning of foot-and-mouth disease virus integrin receptor beta1 subunit and antibody production to its ligand-binding domain].

We produced beta1 gene which is about 2400 bp by reverse transcription polymerase chain reaction (RT-PCR) from bovine trachea, reclaimed and purified, then cloned the amplified fragment to pGEM-T easy vector, confirmed by sequencing. The immune-dominant epitope of beta1 gene was chosen by computer analysis and then syncretized ligand-binding domain from 346 bp to 843 bp of ecytoplasm with six histidine, expressed LBD protein massly in E. coli BL21 (DE3), and identified by SDS-PAGE. The fusion protein was purified with Ni-NTA affinity chromatography and immunized New Zealand rabbits preparing of its polyclonal antibody, the specific antibody titer was above 1:12,800 detected by indirect ELISA, the result of Western blot showed that this antibody could be recognized by LBD fusion protein.

Increased expression of CD16, CD69, and very late antigen-1 on blood monocytes in active sarcoidosis.

BACKGROUND: Different types of immune cells are involved in the formation of granulomas, a hallmark of pulmonary sarcoidosis. Proinflammatory monocytes are activated circulating monocytes thought to be related to the initial events of granuloma formation. We tested the hypothesis that peripheral blood monocytes in patients with active pulmonary sarcoidosis have an activated phenotype and, secondly, that measuring this activation status can provide a new tool for monitoring disease activity. METHODS: Blood was collected of 23 steroid-naive patients presenting with pulmonary sarcoidosis and 10 healthy control subjects. Expression of CD16 (Fc-gamma type III receptor), CD69 (a general activation marker of cells of the hematopoietic lineage), and the integrin very late antigen (VLA)-1 (on interaction with extracellular matrix compounds mediates cell adhesion) was measured by flow cytometry. RESULTS: Percentages of monocytes expressing CD16, CD69, and VLA-1 in patients vs control subjects were 56.2 +/- 4.1% vs 12.2 +/- 2.4% (p < 0.0001), 87.3 +/- 2.1% vs 8.6 +/- 3.3% (p < 0.0001), and 66.5 +/- 3.6% vs 11.2 +/- 2.3% (p < 0.0001), respectively. Moreover, the CD69+VLA-1+ monocyte subset, abundantly present at disease presentation, was found to decrease to normal levels during follow-up with disease remission. CONCLUSIONS: Peripheral blood monocytes from patients with pulmonary sarcoidosis show a highly activated phenotype. Phenotyping circulating monocytes might be a promising tool for monitoring sarcoidosis disease activity but needs further investigation.

Transcriptional and epigenetic regulation of the integrin collagen receptor locus ITGA1-PELO-ITGA2.

The integrin collagen receptor locus on human chromosome 5q11.2 includes the integrin genes ITGA1 and ITGA2, and the cell cycle regulation gene PELO, embedded within ITGA1 intron 1. ITGA1 contains a CArG box that is bound by serum response factor (SRF), while PELO contains two Sp1 binding elements. A comparison of mRNA levels in megakaryocytic (MK) and non-megakaryocytic (non-MK) cell lines and an analysis of the transcriptional activity of promoter-LUC reporter gene constructs in transfected cells revealed that ITGA1 is selectively suppressed in the MK lineage. Sodium bisulfite genomic sequencing established that a CpG-rich ITGA1 promoter region (-209/+115) is fully methylated at 19 CpG sites in MK cells that do not express alpha1beta1, but completely demethylated in expressing cells. In vitro methylation of ITGA1 suppresses transcription, while treatment of megakaryocytic cells with 5-aza-2'-deoxycytidine, but not Trichostatin A, resulted in de novo expression of ITGA1. During thrombopoietin-induced in vitro differentiation of primary human cord blood mononuclear cells into megakaryocytes, we observed rapid, progressive CpG methylation of ITGA1, but not PELO or ITGA2. Thus, selective CpG methylation of the ITGA1 promoter is a specific feature of alpha1beta1 regulation that coincides with the initiation of megakaryocyte differentiation.

The alpha 1 beta 1 and alpha E beta 7 integrins define a subset of dendritic cells in peripheral lymph nodes with unique adhesive and antigen uptake properties.

Dendritic cells (DCs) are a heterogeneous population of APCs with critical roles in T cell activation and immune regulation. We report in this study the identification and characterization of a novel subset of DCs resident in skin-draining peripheral lymph nodes of normal mice. This subset of CD11c(high)CD40(high)CD8alpha(intermediate (int)) DCs expresses the collagen-binding integrin, alpha1beta1, and the E-cadherin-binding integrin, alphaEbeta7. Although alpha1beta1 and alphaEbeta7 are also expressed on CD11c(high)CD40(int)CD8alpha(high) lymphoid DCs, CD11c(high)CD40(high)CD8alpha(int) DCs demonstrate preferential integrin-mediated adhesion to collagen and fibronectin. This DC subset most likely acquires expression of these integrins in peripheral lymph node, as this subset is not found in the spleen or mesenteric lymph node, and recent DC migrants from the skin lack expression of alpha1beta1 and alphaEbeta7 integrins. Resident CD40(high) DCs express alpha1beta1 integrin and colocalize with collagen in lymph nodes. When compared with CD11c(high)CD40(high)CD8alpha(int) DCs lacking expression of these integrins, the alpha1beta1+alphaEbeta7+DC subset exhibits more efficient formation of Ag-independent conjugates with T cells, and a decreased ability to acquire soluble Ag. Thus, the alpha1beta1 and alphaEbeta7 integrins define a unique population of peripheral lymph node-derived DCs with altered functional properties and adhesive potential that localizes these cells to sites in lymph nodes where Ag presentation to T cells occurs.

Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells.

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1)beta(1) parallels that of viral-specific effector CD8(+) T cells (defined by tetramer and IFN-gamma staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of alpha(1)beta(1) blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.