ABSTRACT
BACKGROUND: Mesenchymal stem cells (MSCs) have multiple immunomodulatory properties and hold therapeutic potential for inflammatory diseases. However, the therapeutic and immunologic effects of human umbilical cord blood-derived MSCs (huMSCs) remain largely unexamined for asthma. OBJECTIVE: This study was to investigate the immunomodulatory properties of huMSCs in an ovalbumin (OVA)-induced murine asthma model. METHODS: Mice were injected intraperitoneally with OVA and an aluminium hydroxide adjuvant. huMSCs were administered via the tail vein (5×105 cells/100 uL) to female BALB/c mice prior to the initial OVA challenge. The effects of huMSCs were assessed by investigating airway hyperresponsiveness, histological changes, inflammatory cell numbers, serum allergen-specific antibodies, cytokine production in spleen, lung tissue, and bronchoalveolar lavage (BAL) fluid as well as expansion of regulatory T cells. RESULTS: Administration of huMSCs significantly reduced methacholine bronchial hyperresponsiveness and eosinophil counts in BAL cells. Similarly, there was a significant decrease in serum OVA-specific IgE and IgG1 levels along with Th2 cytokine production (IL-4, IL-5, and IL-13) in the lung and spleen tissues, whereas increased percentage of regulatory T cells was observed after treatment with huMSCs. CONCLUSIONS: Our results suggest that huMSC treatment reduces OVA-induced allergic inflammation, which could be mediated by regulatory T cells.
Subject(s)
Asthma/immunology , Asthma/metabolism , Fetal Blood/cytology , Immunomodulation , Mesenchymal Stem Cells/metabolism , Ovalbumin/immunology , Allergens/immunology , Animals , Cytokines/metabolism , Disease Models, Animal , Female , Humans , Immunization , Immunoglobulin E/blood , Immunoglobulin E/immunology , Inflammation Mediators/metabolism , Lymph Nodes/immunology , Methacholine Chloride/metabolism , Mice , Spleen/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolismABSTRACT
The phosphorylation pattern of numerous proteins in the soluble extracts of chick embryonic muscle cells changes dramatically during myogenesis. One of these proteins, the 63 kDa protein, whose phosphorylation state declines during the differentiation process, was identified as the myristoylated alanine-rich C kinase substrate (MARCKS), a major, specific substrate of protein kinase C (PKC). This decrease in the phosphorylation state of MARCKS was due to a decrease in the level of protein in the cytosol with a simultaneous increase in its level in the membrane fraction. Immunostaining of the cultured myoblasts also revealed that MARCKS translocated from the cytosol to the plasma membrane and to the peripheral region of nuclei as the mononucleated myoblasts fused to form multinucleated myotubes. Immunoprecipitation with an anti-PKC-theta antibody, but not with the antibodies against the other PKC isoforms, such as conventional PKC-alpha, novel PKC-delta, and novel PKC-epsilon, inhibited phosphorylation of MARCKS. Moreover, expression of PKC-theta was found to be down-regulated during the course of myogenic differentiation. In addition, treatment of the cells with PMA, which activates PKC-theta and hence increases the phosphorylation state of MARCKS, reversibly inhibited both MARCKS translocation and myoblast fusion. These results suggest that MARCKS is preferentially phosphorylated by PKC-theta in cultured myoblasts and that the down-regulation of PKC-theta; is partly responsible for MARCKS translocation during myogenesis. These results also suggest that PKC-theta-controlled MARCKS translocation is associated with, or a requisite event for, myoblast fusion.
Subject(s)
Cell Differentiation/physiology , Intracellular Signaling Peptides and Proteins , Isoenzymes/metabolism , Membrane Proteins , Muscle, Skeletal/embryology , Protein Kinase C/metabolism , Proteins/metabolism , Amino Acid Sequence , Animals , Brain/metabolism , Calcium/pharmacology , Cells, Cultured , Chick Embryo , Creatine Kinase/metabolism , Egtazic Acid/pharmacology , Immunohistochemistry , Kinetics , Molecular Sequence Data , Muscle, Skeletal/cytology , Myristoylated Alanine-Rich C Kinase Substrate , Peptide Fragments/chemistry , Phosphorylation , Proteins/chemistry , Substrate Specificity , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Two mRNA species with different sizes (3.8 kb and 2.8 kb) for the fyn proto-oncogene have been noticed during Northern hybridization analysis. However, the difference between the two mRNA species has not been resolved yet. By screening a phage expression library using the monoclonal antibody (mAb) B16-5 which recognizes Src homology 3 (SH3) domains of phospholipase C-gamma and Nck, we have cloned a cDNA encoding the larger species of fyn mRNA. The size of the clone was 3.5 kb and DNA sequencing analysis of the clone showed that it was fyn expressed mainly in T-cells, fyn (T), with an untranslated region 1 kb longer than the previously reported one. The 3'-end fragment of the clone hybridized only to the larger species (3.8 kb) of fyn mRNA but not to the smaller one (2.8 kb) on Northern blot analysis. Furthermore, an additional polyadenylation signal sequence was found at the end of this clone. These results indicate that the two mRNA species for fyn are produced by alternative polyadenylation.