Survival of satellite cells following exposure to the local anesthetic bupivacaine (Marcaine)

Summary Rat lumbrical muscles were incubated in a concentration of 10^-2 M bupivacaine for 5 or 15 min and examined after further incubation in the absence of the drug for periods totalling 1, 2, and 3h. Electron microscopy showed that muscle fibers and their component organelles and myonuclei underwent a series of irreversible degenerative changes. However, satellite cells retained their normal morphology under similar conditions. It is concluded that satellite cells are responsible for the rapid regeneration of muscles that follows degeneration induced by bupivacaine. The role of satellite cells in muscle regeneration is discussed.This investigation was supported by United States Public Health Service Research Grant NS 13296 from the National Institutes of Health. A supply of bupivacaine was kindly made available by Winthrop Laboratories, New York, N.Y., USA     

Selection using the alpha-1 integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells

Bone marrow-derived mesenchymal stem cells consist of a developmentally heterogeneous population of cells obtained from colony forming progenitors. As these colonies express the alpha-1 integrin (CD49a), here we single-cell FACS sorted CD49a+ cells from bone marrow in order to create clones and then compared their colony forming efficiency and multilineage differentiation capacity to the unsorted cells. Following selection, 40% of the sorted CD49a+ cells formed colonies, whereas parental cells failed to form colonies following limited dilution plating at 1 cell/well. Following ex vivo expansion, clones shared a similar morphology to the parental cell line, and also demonstrated enhanced proliferation. Further analysis by flow cytometry using a panel of multilineage markers demonstrated that the CD49a+ clones had enhanced expression of CD90 and CD105 compared to unsorted cells. Culturing cells in adipogenic, osteogenic or chondrogenic medium for 7, 10 and 15 days respectively and then analysing them by quantitative PCR demonstrated that CD49a+ clones readily underwent multlineage differentiation into fat, bone and cartilage compared to unsorted cells. These results thus support the use of CD49a selection for the enrichment of mesenchymal stem cells, and describes a strategy for selecting the most multipotential cells from a heterogeneous pool of bone marrow mononuclear stem cells.     

Low affinity binding of an LFA-3/IgG1 fusion protein to CD2+ T cells is independent of cell activation

Quantitative analysis of binding of the bivalent recombinant soluble fusion protein, LFA-3/IgG1, shows that the fusion protein binds to human CD2⁺ PBLs primarily through low affinity (K_D ～ 140 μM) but also through high avidity (90 nM) interactions. The concentration dependence for LFA-3/IgGl PBL binding took the form of two overlapping bell-shaped curves separated by a clear and reproducible minimum. This was accounted for in part by minor heterogeneity in the LFA-3/IgG 1 preparations, and potentially by the ability of the ligand to bind to both CD2 and Fc receptors (FcR), best evidenced by the distinct binding properties of the fusion protein to NK and T cells. The low affinity LFA-3/ IgG 1 binding to T cells is consistent with binding to CD2 only, and is in agreement with the low affinity reported for interactions between soluble forms of LFA-3 and CD2 by surface plasmon resonance technology. Moreover, as the low affinity determinations are similar for CD2 on resting and activated T cells, although the CD2 molecule has been reported to be altered to reveal new epitopes upon T cell activation, the binding data argue against multiple cell activation-dependent affinity states of CD2 for LFA-3 binding. This is distinct from that observed with other adhesion partners, and suggests that the different adhesion pathways utilize distinct mechanisms to mediate cell adhesion.     

Contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.     

The direct action of 1alpha,25(OH)2-vitamin D3 on purified mouse Langerhans cells

The action of vitamin D(3) on Langerhans cells (LCs) is not well understood. Using highly purified murine LCs (>95%), we investigated the direct action of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on their functions. 1,25(OH)(2)D(3) inhibited the expression of cell surface molecules including I-A(d), CD40, CD80, and CD86, leading to impaired ability of LCs to stimulate allogenic T cells in the mixed leukocyte reaction. Furthermore, this reagent inhibited chemotaxis of LCs to CCL21 and their survival. Interestingly, 1,25(OH)(2)D(3) reduced the IL-10 production by LCs, whereas the production of IL-6 and IL-12p40 upon activation by CD40 ligation was enhanced. With regard to inflammatory cytokines and chemokines, 1,25(OH)(2)D(3) upregulated the production of IL-1beta, CCL3, CCL4, and CCL5. The production of Th2-type chemokines, represented by CL17 and CCL22, was inhibited, whereas IFN-gamma-triggered production of Th1-type chemokines, represented by CXCL9, CXCL10, and CXCL11, was augmented. These data indicate that the mode of regulation of cytokine and chemokine production in association with 1,25(OH)(2)D(3) treatment seems to be another characteristic discriminating LCs from classical myeloid dendritic cells.     

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.     

Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis

Myocardial ischaemia (MI) results in extensive cardiomyocyte death and reactive oxygen species (ROS)‐induced damage in an organ with little or no regenerative capacity. Although the use of adult bone marrow mesenchymal stem cells (BMMSCs) has been proposed as a treatment option, the high cell numbers required for clinical use are difficult to achieve with this source of MSCs, and animal studies have produced inconsistent data. We recently demonstrated in small and large animal models of acute MI that the application of human term placenta‐derived multipotent cells (PDMCs), a foetal‐stage MSC, resulted in reversal of cardiac injury with therapeutic efficacy. However, the mechanisms involved are unclear, making it difficult to strategize for therapeutic improvements. We found that PDMCs significantly reduced cardiomyocyte apoptosis and ROS production through the paracrine factors GRO‐α, HGF and IL‐8. Moreover, culturing PDMCs on plates coated with laminin, an extracellular matrix (ECM) protein, resulted in significantly enhanced secretion of all three paracrine factors, which further reduced cardiomyocyte apoptosis. The enhancement of PDMC paracrine function by laminin was mediated through αvβ3 integrin, with involvement of the signalling pathways of JNK, for GRO‐α and IL‐8 secretion, and PI3K/AKT, for HGF secretion. Our results demonstrated the utility of PDMC therapy to reduce cardiomyocyte apoptosis through modulation of ECM proteins in in vitro culture systems as a strategy to enhance the therapeutic functions of stem cells.     

Variability in the expression of urokinase receptor (CD87) mutants on cells: relevance to cell adhesion

The urokinase-type plasminogen activator receptor (uPAR, CD87) is a glycosylphosphatidylinositol (GPI)-anchored protein, containing three homologous Ly-6 domains, that mediates integrin-independent cell adhesion by directly binding to extracellular matrix protein vitronectin (VN). To elucidate the structural requirements for the uPAR-dependent cell adhesion on VN, several glycolipid-anchored variants of uPAR were expressed in BAF3 cells, (mouse pre B-lymphocytes) followed by functional analysis. The individual domains of uPAR were expressed at very low levels, the two domain mutants were expressed to a higher level and the wild type uPAR was expressed highly. Point mutations in domain 2 of uPAR have been shown to diminish cellular binding of the ligand urokinase and we observed a lack of VN binding to this mutant. Flow cytometry with a number of monoclonal antibodies indicated that the domain-specific antigenic determinants in these mutants were well preserved. Only the cells expressing the intact uPAR with all three domains adhered strongly to a VN substrate, whereas none of the other transfected cells showed significant cell adhesion. Hence, any alterations in the domain structure of uPAR reduce its expression and only the intact receptor can sustain the direct cell adhesion on VN-rich matrices found at sites of inflammation and injury.     

Localization of an antibody to CD74 (MHC class II invariant chain) to human B cell lymphoma xenografts in nude mice

The tumor-specific localization of an anti-CD74 Ab, LL1, was demonstrated in nude mice bearing xenografts of human B-cell lymphoma. This Ab, conjugated to radionuclides emitting Auger electrons, including ¹²⁵I and ¹¹¹In, was previously reported to kill tumor cells in vitro effectively and specifically. The cytotoxic potency of this Ab is due to its uptake and catabolism at a very high level, which also affected the Ab biodistribution experiments. Thus, Ab localization to the tumor was only detected if a “residualizing” radiolabel was used, meaning a label that is trapped within cells, usually within lysosomes, after catabolism of the Ab to which it was conjugated. Similar results were obtained with three different residualizing labels: ¹¹¹In conjugated via the chelators benzyl diethylenetriaminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), or ¹³¹I-dilactitol-tyramine, a residualizing form of iodine. The Ab protein dose could be high, 0.5 mg/mouse, without causing a decrease in specific tumor uptake, probably reflecting the high capacity for uptake. Moreover, tumors of moderate size were found to cause rapid, specific removal of the Ab from the blood, also a result of catabolic processes. This induced blood clearance naturally affected the Ab localization experiments, but this factor could be circumvented by increasing the Ab protein dose. Using a different Ab, anti-(mature MHC class II), the ability of Ab to penetrate relatively large solid tumors was investigated. Complete saturation of antigenic sites was observed in tumors up to 0.3 g in size, but quite high Ab protein doses were required, 5.0 mg/mouse. These results provide a rationale for attempting therapy with radiolabeled LL1. Received: 4 November 1999 / Accepted: 19 January 2000     

Recombinant human (rh) stem cell factor and rhIL-4 stimulate differentiation and proliferation of CD3+ cells from umbilical cord blood and CD3+ cells enhance FcepsilonR1 expression on fetal liver-derived mast cells in the presence of rhIL-4

We previously reported that rhIL-4 induced apoptosis and rhIL-6 mediated protection of human mast cells derived from cord blood mononuclear cells. Based on the result, we attempted to obtain the phenotypes and differentiation of CD3+ cells from cord blood by investigating their cell surface markers in the presence of rhSCF plus rhIL-4. The effect of co-cultured CD3+ cells on fetal liver mast cells (FLMCs) was also determined. Phenotypes from cord blood-derived cells were analyzed by flow cytometry and cell numbers were determined. Fetal liver mast cells were cultured with cord blood-derived cells (mainly CD3+) in the presence of rhSCF and/or rhIL-4 and were analyzed to determine cell number and expression of Kit+ and FcepsilonR1. The percentage of CD3+ cells from cord blood-derived cells on day 0 was about 41 +/- 13.5%, following monocytes and granulocytes. CD3+ cells increased in number (1.5-fold) and purity (90%), whereas other cell types did not survive. More than 60% of CD3+ cells from cord blood at day 0 were CD4(-)CD8-. These double-negative cells dramatically decreased by 1 week of culture, while CD4+CD8+ cells increased in number and purity through 3 weeks of culture, and then decreased as greater numbers of single-positive T cells emerged. We also found that FcepsilonR expression on FLMC increased in the presence of rhIL-4, but was not affected by the T cells that developed from cord blood mononuclear cells. The results indicate that IL-4, a Th2 type cytokine, together with rhSCF, can induce T cell proliferations, differentiation, and maturation from cord blood progenitor cells.     

C3d and Epstein-Barr Virus (CR2/CD21 Ligands) Stimulate Cells of an HTLV-I Line,MT-2

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with ³²P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.     

No effects of pulsed electromagnetic fields on expression of cell adhesion molecules (integrin, CD44) and matrix metalloproteinase-2/9 in osteosarcoma cell lines

Pulsed electromagnetic fields (PEMF) could enhance the cytocidal effects of chemotherapeutic drugs on malignant tumor cell lines, but metastasis effects of PEMF on tumor cells have not been investigated. We investigated the effects of PEMF exposure on the expression levels of some metastasis-related molecules, including integrin α subunits (α1, α2, α3, α4, α5, α6, αv), integrin β subunits (β1, β2, β3, β4), CD44, and matrix metalloproteinase-2/9 (MMP-2/9) in four human osteosarcoma cell lines (HOS, MG-63, SAOS-2, NY) and two mouse osteosarcoma cell lines (DOS, LM8) by using FACScan analysis, gelatin zymography, and Western blot analysis. Our results indicate that PEMF exposure has no effect on the expression of some molecules that are associated with tumor cell invasion and metastasis, and therefore suggest that PEMF exposure may be safely applied to chemotherapy for osteosarcoma.     

MAP kinases p38 and JNK are activated by the steroid hormone 1alpha,25(OH)2-vitamin D3 in the C2C12 muscle cell line

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1alpha,25(OH)2-vitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1alpha,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen-activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1Alpha,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c-Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced-p38 activation. Of relevance, 1alpha,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen-activated protein kinase activating protein kinase 2 (MAPKAP-kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation-dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1Alpha,25(OH)2D3 also promotes the phosphorylation of c-jun N-terminal protein kinases (JNK 1/2), the response is fast (0.5-1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1alpha,25(OH)2D3 (1 nM). The relative contribution of ERK-1/2, p38, and JNK-1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established.     

Indoleamine 2, 3-dioxygenase （IDO） is essential for dendritic cell activation and chemotactic responsiveness to chemokines

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs),and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC functions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflammatory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regulated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.     

1alpha,25-Dihydroxyvitamin D3-3beta-(2)-bromoacetate,an affinity labeling derivative of 1alpha,25-dihydroxyvitamin D3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells

In this report we describe that 1,25(OH)(2)D(3)-3-BE, a VDR-affinity labeling analog of 1,25(OH)(2)D(3), showed strong and dose-dependent growth-inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF-7 breast cancer, PC-3, and LNCaP prostate cancer and PZ-HPV-7 immortalized normal prostate cell-lines. Furthermore, 10(-6) M of 1,25(OH)(2)D(3)-3-BE induced apoptosis specifically in LNCaP and PC-3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)(2)D(3)-3-BE) was not due to probable degradation (hydrolysis) of 1,25(OH)(2)D(3)-3-BE or random interaction of this molecule with cellular proteins. Tissue- or cell-specific action of 1,25(OH)(2)D(3) and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)(2)D(3) and its analogs in various cell-lines potentially limits their application as anticancer agents. We showed that 1,25(OH)(2)D(3)-3-BE displayed similar growth-inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen-independent PC-3 cell-lines. Therefore, these results raise the possibility that 1,25(OH)(2)D(3)-3-BE or similar VDR-cross linking analogs of 1,25(OH)(2)D(3) might be considered for further development as potential candidates for prostate cancer.