CD73 mRNA Research Articles

Establishment and characterization of porcine Sertoli cell line for the study of xenotransplantation

An understanding of the main mechanism that determines the ability of immune privilege related to Sertoli cells (SC) will provide clues for promoting a local tolerogenic environment. In this report, we established neonatal porcine SC line and evaluated their characteristics. SC line was established following the transfection of primary SC (NPSC) from the testis of neonatal pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. Immunohistochemistry and RT-PCR were performed to evaluate the character of immortalized SC lines. Our immortalized SC line (iPS) proliferated stably and had a phenotype similar to NPSC, as indicated by the immunoexpression of follicle stimulating hormone receptor (FSHR), and mRNA expression of androgen receptor (AR), and Wilms' tumor antigen (WT1). Interestingly, NPSC and iPS expressed mRNA of complement regulatory proteins (CRP) such as membrane cofactor protein (CD46), decay accelerating factor (DAF or CD55), and protectin (CD59), but CD59 mRNA expression was negligible in iPS. These results suggest that iPS, immortalized by the introduction of SV40 T, retain their original characteristics, except for the relatively low expression of CD59, and that they may be useful for future in vitro and in vivo studies of immune privilege mechanisms related to SC.

Adenosine stimulates connexin 43 expression and gap junctional communication in pituitary folliculostellate cells

Adenosine is known to stimulate interleukin (IL)-6 and vascular endothelial growth factor (VEGF) secretion from pituitary TtT/GF folliculostellate [corrected] (FS) cells indicating that it is an important paracrine regulator of anterior pituitary function. This study demonstrates that rodent anterior pituitary cell lines produce extracellular adenosine that is able to increase intercellular gap junction communication in FS cells. Ecto-5'-nucleotidase (CD73), the enzyme that generates adenosine from AMP, was demonstrated by immunocytochemistry in approximately 20% of anterior pituitary cells, and some of these cells colocalized with prolactin and growth hormone. CD73 mRNA and protein were detected in GH3 and MMQ (somatotroph-lactotroph lineages) and TtT/GF cells, and enzyme activity was demonstrated by the conversion of exogenously added fluorescent ethenoAMP to ethenoadenosine. Adenosine production, as measured by HPLC, was detected in GH3 (1 microM/h) and MMQ (3 microM/h) but not in TtT/GF cells. Adenosine (EC50: 0.5 microM) and NECA (universal adenosine receptor agonist; EC50 0.1 microM) stimulated connexin 43 (Cx43) mRNA and protein expression within 1-2 h in TtT/GF cells. Adenosine and NECA also stimulated gap junctional intercellular communication (as assessed by transmission of Alexa Fluor 488) by 6- to 8-fold in comparison with untreated TtT/GF cells. In cocultures of MMQ and TtT/GF cells, Cx43 expression in TtT/GF cells increased in proportion to the number of MMQ cells plated out. These data suggest that adenosine, formed locally in the anterior pituitary gland can stimulate gap junction communication in FS cells.

Effect of hypoxia/reoxygenation on CD73 (ecto-5′-nucleotidase) in mouse microvessel endothelial cell lines

Cerebral ischemia and post-ischemic reperfusion commonly result in significant brain damage. Brain microvessel endothelial cells, the key target cells and regulating sites, can secrete adenosine which plays an important neuroprotective role in the ischemic brain. A primary determinant of localized production of adenosine at tissue interfaces is ecto-5′-nucleotidase (CD73). In our experiments, we used bEnd.3 cells, immortalized mouse brain microvessel endothelial cell lines, as the target cells to study the effect of hypoxia and posthypoxic reoxygenation on CD73 in brain microvessel endothelial cells. CD73 activity in bEnd.3 cells exposed to hypoxia significantly increased in time-dependent way. The upregulation of CD73 mRNA and protein expression induced by hypoxia in bEnd.3 cells were detected by RT-PCR and Western blot. However, for reoxygenation studies, CD73 activity, mRNA and protein expression decreased at the initial stages, but increased at prolonged reoxygenation. Our results suggest that hypoxia can induce upregulation of CD73 expression in brain microvessel endothelial cells, which can be reversed by reoxygenation of short duration. But CD73 expression increased gradually with the duration of reoxygenation. Then, we infer that CD73 in brain microvessel endothelial cells plays a very important role through forming adenosine during brain ischemia and reperfusion.

Health status of transgenic pigs expressing the human complement regulatory protein CD59

Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects. Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry. Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected. Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications.

774. Evaluation of Adenovirus Vectors Containing Serotype 35 Fibers for Gene Therapy of Cervical Cancer

Adenovirus vectors containing group B fibers (such as Ad5/35) are able to target tissues that are refractory to infection with commonly used Ad serotype 5 based vectors. To evaluate the utility of Ad5/35 vectors for gene transfer into tumor cells, we studied the expression of the Ad5 receptor, CAR, and the Ad5/35 receptor, CD46, on Papanicolaou (Pap) smears of normal, pre-malignant, and malignant cervical tissue (N= 45). We observed a significant correlation between the degree of malignancy and the level of CD46 expression, while in contrast, the level of CAR expression inversely correlated with the degree of malignancy. Using qRT-PCR, we showed that CAR mRNA levels were lower in malignant tissue biopsies than in histologically normal biopsies. However, there was no significant difference in CD46 mRNA levels in normal and malignant tissues, indicating that CD46 protein expression in tumors might be regulated at the post-transcriptional level. In an attempt to delineate reasons for low CAR expression in tumors, we analyzed the methylation status of CpG island present in the promoter of the CAR gene in DNA isolated from normal and malignant tumor tissue, but found the promoter unmethylated in both groups. To better predict the infectivity of Ad5/35 in tumor patients, we studied the binding of recombinant Ad fiber knob, which is the Ad capsid moiety that mediates attachment to receptors, to the surface of cells present in Pap smears. We found that binding of recombinant Ad5 and Ad35 fiber knobs mirrored the expression patterns of CAR and CD46. Finally, we cultured primary cells from cervical cancer biopsies and performed Ad5/35 transduction studies on primary cultures and single tumor cell clones derived from these cultures (N>40). Overall, the level of CAR and CD46 in cultured cells correlated well with transduction efficiency of Ad5 and Ad5/35 vectors expressing GFP. Importantly, all tumor cell cultures could be transduced at 100% efficiency with Ad5/35-GFP at low MOIs. Despite of the uniformly efficient tumor cell transduction, a conditionally replicating Ad5/35-based oncolytic vector expressing Ad E1A and TRAIL exhibited differences in tumor cell killing when analyzed in clonal tumor cell cultures. Mechanisms that confer resistance to viral oncolysis could include inefficient viral replication and/or execution of apoptosis. Genes whose expression or repression confer resistance are currently being studied using DNA array technology.

The expression of CD97EGF and its ligand CD55 on marginal epithelium is related to higher stage and depth of tumor invasion of gastric carcinomas

CD97EGF is a member of the EGF-TM7 family of class II seven-transmembrane (7TM), and its cellular ligand CD55 (also known as decay accelerating factor; DAF) protects host cells from complement attack. To determine whether the expression levels of these two molecules are correlated with the clinicopathological features of gastric carcinomas, a total of 35 gastric carcinomas and their corresponding margins and normal specimens were investigated by RT-PCR, Western blot analysis and immunohistochemistry. Transcript levels of CD97EGFand CD55 were higher in tumors than those in the margin and normal epithelial mucous tissues (P<0.05). However, the expression levels of CD97EGF and CD55 mRNA had no correlation with the clinicopathological features of gastric carcinoma patients. All three groups of specimens were immunoreactive for CD97EGF and the CD55 protein. Strong and specific immunoreactivities of CD97EGF were located in the mucosal epithelia of the marginal basal membrane. Expression of CD97(EGF) in the margins showed a marked difference between the depth of tumor invasion T1 and T2, 3 and 4, and stages I and II/III/IV of gastric carcinomas (P<0.05). The expression of CD55 protein was highly correlated with CD97EGF (R=0.6483, P<0.001). Western blot analysis confirmed the expression and distribution patterns of CD97EGF and CD55. Our findings suggest that CD97EGF may play a role in the development and invasion of gastric carcinomas by binding its cellular ligand CD55. Detection of the CD97EGF expression in the tumor margin could be referred to as the molecular edge of gastric carcinomas.

HIF‐dependent induction of apical CD55 coordinates epithelial clearance of neutrophils

Sites of inflammation are associated with dramatic shifts in tissue metabolism. Inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia-responsive genes. Given this association, we hypothesized that neutrophil (PMN) ligands expressed on epithelial cells may be regulated by hypoxia. Initial studies confirmed earlier results that epithelial hypoxia enhances PMN transepithelial migration and promotes apical clearance of PMN from the epithelial surface. A screen of known PMN ligands revealed a surprisingly stable expression pattern in hypoxia. However, this screen identified one gene, CD55, as a highly hypoxia-inducible molecule expressed on the apical membrane of mucosal epithelia. Subsequent studies verified the induction of CD55 mRNA and protein expression by hypoxia. Overexpression of CD55 by transfection in nonhypoxic epithelia resulted in a similar pattern of apical PMN clearance, and peptide mimetics corresponding to the PMN binding site on DAF blocked such apical clearance of PMN. Studies directed at understanding molecular pathways of hypoxia inducibility revealed that a approximately 200 bp region of the CD55 gene conferred hypoxia inducibility for CD55. These studies identified a functional binding site for the transcriptional regulator hypoxia-inducible factor (HIF). Taken together, these results identify HIF-dependent induction of epithelial CD55 in the resolution of ongoing inflammation through clearance of apical PMN.

In vitroCanine Distemper Virus Infection of Canine Lymphoid Cells: A Prelude to Oncolytic Therapy for Lymphoma

Measles virus (MV) causes the regression of human lymphoma xenografts. The purpose of this study was to determine if canine lymphoid cells could be infected in vitro with MV or canine distemper virus (CDV, the canine Morbillivirus equivalent of MV) and determine if in vitro viral infection leads to apoptotic cell death. Reverse transcriptase-PCR was used to examine the expression of both signal lymphocyte activation molecule (CD150) and membrane cofactor molecule (CD46) mRNA. An attenuated CDV expressing enhanced green fluorescent protein was used to infect canine cells in vitro. Both flow cytometry and reverse transcriptase-PCR was used to document CDV infection. Cell death was examined using a propidium iodide staining assay and Annexin V binding. Canine lymphoid cell lines and neoplastic B and T lymphocytes collected from dogs with spontaneous lymphoma expressed the Morbillivirus receptor CD150 mRNA. In contrast, only neoplastic lymphocytes expressed detectable levels of CD46 mRNA. Although MV did not infect canine cells, CDV efficiently infected between 40% and 70% of all three canine lymphoid lines tested. More importantly, CDV infected 50% to 90% of neoplastic lymphocytes isolated from dogs with both B and T cell lymphoma. Apoptosis of CDV-infected cell lines was documented. Attenuated CDV may be a useful treatment for canine lymphoma. As such, dogs with lymphoma may represent a biologically relevant large animal model to investigate the feasibility, safety, and efficacy of Morbillivirus therapy in a clinical setting with findings that may have direct applicability in the treatment of human non-Hodgkin's lymphoma.

Oncolytic Measles Virus Selectively Targets CD46 Overexpression on Myeloma Cells.

Multiple myeloma remains incurable and novel therapeutics are urgently needed. We have shown that live attenuated Edmonston strain measles virus (MV-Edm) is potently oncolytic in multiple myeloma xenograft models and can efficiently infect CD138 sorted primary myeloma cells inducing extensive intercellular fusion which leads to cell death (Blood, 2001, v98, p2002). In contrast, the virus causes minimal damage to normal PHA-stimulated peripheral blood lymphocytes, fibroblasts and smooth muscle cells. To facilitate non-invasive imaging of sites of viral gene expression, we introduced the human thyroidal sodium iodide symporter (NIS) gene into the viral genome (MV-NIS). MV-NIS infected myeloma cells efficiently concentrate radioiodine which provides a basis for noninvasive imaging and destruction of infected tumors using beta emitters such as 131I (Blood, 2004, v103, p1641). Clinical translation of MV-NIS in patients with refractory myeloma is planned. However, the mechanism underlying the tumor selectivity of MV-Edm has not previously been defined. MV-Edm uses either of two cellular receptors for cell entry and cell fusion; CD46, a regulator of complement activation, is expressed on all nucleated cells, wheras SLAM (signaling lymphocyte activation molecule) is expressed only on activated B lymphocytes, T lymphocytes, and monocytes, and is not present on myeloma plasma cells. Using a panel of CHO cell transfectants, we demonstrated that MV-Edm -mediated cell killing increases exponentially above a threshold CD46 receptor density (Can Res, 2004, v64, p4919). We subsequently obtained bone marrow aspirates from 18 myeloma patients and performed multi-parameter flow cytometry to determine the relative expression levels of CD46 on the myeloma plasma cells versus the rest of the hematopoietic cells in the bone marrow. The myeloma cells expressed significantly higher levels (5-fold) of CD46 receptors (MFI=294.9 + 204.4, means ± SD) on their cell surfaces compared to hematopoietic cells of the erythroid, myeloid and lymphoid lineages (MFI=58.6 ± 9.0). Analysis of the myeloma array data generated by the Shaughnessy lab indicated that, in comparison to plasma cells from healthy volunteers (2997 ± 1053, n=30, means ± SD), primary myeloma cells express 2-fold elevated levels of CD46 mRNA (6592 ± 4029, n=72, p=0.001, Wilcoxon signed rank test). Taken together, these findings provide strong support for the hypothesis that selective destruction of myeloma cells by oncolytic measles viruses is a direct consequence of their overexpression of the cellular receptor CD46.

Complement inhibitor membrane cofactor protein (MCP; CD46) is constitutively shed from cancer cell membranes in vesicles and converted by a metalloproteinase to a functionally active soluble form.

Human cell-surface protein CD46 protects cells from complement damage, regulates immune functions through signaling and acts as a receptor for certain pathogenic microbes. Multiple molecular weight isoforms of membrane bound CD46 are produced by alternative splicing of the CD46 mRNA in an area coding for the serine/threonine/proline-rich region or for the cytoplasmic tail. We demonstrate that CD46 becomes proteolytically modified on cell membranes. We observed that tumor cells liberated intact 60-65 kDa forms of CD46 into the cell culture medium on the surface of vesicles with a diameter of 200 nm. Furthermore, soluble CD46 (55-60 kDa) containing the glycosylated STP-region but lacking the hydrophobic transmembrane sequence and cytoplasmic domains was released from tumor cell membranes. The use of selective inhibitors indicated that CD46 release is due to specific cleavage by a metalloproteinase. Exposure of the cells to hydrogen peroxide (H2O2) or their detachment from the pericellular matrix increased the shedding of soluble CD46. Both vesicular and soluble forms of CD46 remained functional and promoted C3b cleavage by factor I. The results show that the functional activity of CD46 is not restricted to the tumor cell membranes but can be liberated in vesicles and by a metalloproteinase.

Increased CD59 protein expression predicts a PSA relapse in patients after radical prostatectomy.

Human protectin (CD59) is a regulator of complement activation that inhibits complement-mediated cell lysis, and thus might confer immune resistance to tumor cells. CD59 expression has been described in a variety of human malignancies, including breast cancer. Since a comprehensive investigation of CD59 expression in prostate cancer has not been conducted yet, we aimed to determine the significance of CD59 expression in prostate cancer. Eighty-six primary adenocarcinomas of the prostate were immunostained using a monoclonal CD59 antibody (clone MEM-43) and a standard detection system. The immunoreactivity of the tumor was evaluated as low versus high for statistical analysis. Additionally, CD59 mRNA levels were determined by real-time PCR in matched (tumor/normal) microdissected tissues from 26 cases. Cytoplasmic CD59 immunoreactivity was found in epithelia of prostate cancer, prostatic intraepithelial neoplasia, benign hyperplasia, atrophic, and normal glands. High rates of CD59 expression were noted in 36% of prostate cancer cases and were significantly associated with tumor pT stage (P = 0.043), Gleason grade (P = 0.013) and earlier biochemical (PSA) relapse in Kaplan-Meier analysis (P = 0.0013). On RNA level, we found an upregulation in 19.2% (five cases), although the general rate of CD59 transcript was significantly lower in tumor tissue (P = 0.03). CD59 protein is strongly expressed in 36% of adenocarcinomas of the prostate and and is associated with disease progression and adverse patient prognosis.

BHV-1 infection and inflammatory cytokines amplify the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood mononuclear cells in vitro.

Bovine herpesvirus-1 (BHV-1) has been reported to increase the susceptibility of cattle to respiratory disease caused by Mannheimia (Pasteurella) haemolytica A1. The principal virulence factor of M. haemolytica is a leukotoxin (LKT) that can specifically kill ruminant leukocytes following its binding to the beta2-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 (LFA-1)). In this study, we investigated the effects of experimental infection of bovine peripheral blood mononuclear cells (MNCs) with BHV-1 in vitro, on the subsequent interaction of these cells with the M. haemolytica LKT. We found that BHV-1 infection increased LFA-1 expression (as assessed by flow cytometry), and subsequently enhanced LKT binding and cytotoxicity to bovine MNCs. We also found that BHV-1 infection increased CD18, IL-1beta, and IFN-gamma mRNA expression by MNCs. As previously reported for bovine polymorphonuclear neutrophils (PMNs), MNCs increased their expression of LFA-1, and their LKT binding and cytotoxicity, following exposure to IL-1beta, TNF-alpha, and IFN-gamma. These findings suggest that BHV-1 infection, and the resulting release of inflammatory cytokines, can stimulate expression of LFA-1 in bovine MNCs, thus enhancing the binding and biological effects of LKT. If such a mechanism occurs in vivo it might explain, in part, the increased susceptibility of BHV-1 infected cattle to bovine pasteurellosis.

Expression of CD97 and CD55 in human medullary thyroid carcinomas

The heterodimeric CD97 protein is a member of the EGF-TM7 family of class II seven-transmembrane (7TM) receptors of 75-90 kDa and structurally related to the secretin receptor family. CD97 is expressed on leucocytes, lymphocytes and in cells of the hematopoietic system. The precise role for CD97 is still unknown. The ubiquitously expressed CD55 (also known as decay accelerating factor, DAF) protects host cells from complement attack. In addition, CD55 is a bacterial/viral receptor and was identified as a ligand for CD97. Employing computer aided UV-laser microdissection CD97 and CD55 were investigated in C-cells of non-neoplastic thyroid specimens (n=3) and in medullary thyroid carcinomas (n=54) by multiplex RT-PCR. Frozen sections of all tissues were investigated by immunohistochemistry. All non-malignant thyroid specimens expressed CD97 mRNA weakly and were devoid of immunoreactive CD97 protein. Transcripts for CD97 were detected in all 54 MTC tissue specimens and CD97 gene activity directly correlated with the histopathological stage of the MTC. CD97 transcriptional activity was high in advanced stages of MTC such as pT3/4. pT1/2 tumors with exclusive intrathyroidal growth revealed weak CD97 expression. CD55 gene expression was significantly lower in normal C-cells than in tumor tissues and all MTC displayed strong and specific CD55 immunostaining. We did not observe a correlation between the expression of CD55 mRNA or protein, respectively, and pTNM classification. In summary, in the present study we have identified CD97 as a novel marker expressed in dedifferentiated neoplastic human thyroid C-cells. CD97 and CD55 may facilitate adhesion of C-cell carcinoma to surrounding surfaces which would result in rapid tumor cell spread.

Analysis of the level of mRNA expression of the membrane regulators of complement, CD59, CD55 and CD46, in breast cancer.

We have examined the relative mRNA expression of the complement (C) regulatory proteins CD59, CD55 and CD46 in RNA isolated from 50 primary breast cancer specimens using a semiquantitative RT-PCR approach. Having normalized the mRNA expression levels of the C regulators relative to actin, we subsequently correlated their expression with estrogen receptor (ER) and various clinical, pathologic and biochemical features of the disease. CD59 and CD46 were detected in all clinical biopsies, while CD55 mRNA was detected in the majority of samples. The comparative levels of expression between the 3 regulators analyzed, using Spearman rank correlation test, revealed a significant association (p = 0.01; r = 0.36) between CD46 and CD59. CD46 exhibited the most striking pattern of association, with increased levels of expression being associated with ER-positive samples and lower levels of expression associated with a loss of differentiation and epidermal growth factor receptor positivity. Application of Spearman rank correlation test revealed CD46 expression was significantly associated with expression of ER at the level of protein (p = 0.031; r = 0.31) and mRNA (p < 0.001; r = 0.52). CD46 expression also correlated with insulin-like growth factor receptor-positive samples using Spearman rank correlation test (p = 0.016; r = 0.34), but negatively associated with tumor samples either exhibiting histologic grade 3 when compared to grades 1 or 2 or displaying elevated levels of inflammatory cell infiltrate. Immunohistochemical analysis of a limited series (n = 8) of paraffin-embedded breast cancers indicated that the level of CD46 protein expression directly associates with that of the mRNA and, where prominent, is localized in the tumor epithelial cell population, including at the plasma membrane. These data provide new information on expression of these important regulators in breast cancer and suggest that CD46 should be evaluated as a novel prognostic indicator.

Characteristics of CD59 up-regulation induced in porcine endothelial cells by alphaGal ligation and its association with protection from complement.

Activation of endothelial cells may result in proinflammatory and procoagulant changes, or in changes that protect the endothelial cells (EC) from injurious insults. Stimulation of porcine EC with human anti-porcine antibodies, or lectins from Bandeiraea simplicifolia that bind terminal Galalpha(1-3)Gal (abbreviated alphaGal), can induce EC protection from cytotoxicity by human complement. These EC also exhibit up-regulation of CD59 protein and mRNA expression. Porcine CD59 has been reported to protect porcine cells from human complement. Therefore we investigated the specificity requirements and other characteristics of the induced CD59 up-regulation, as well as the role of up-regulated CD59 in lectin-induced protection of EC from human complement. Aortic EC were incubated in vitro with alphaGal-binding lectins B. simplicifolia lectin I isolectin B4 (IB4) and B. simplicifolia lectin I (BS-I) and CD59 expression was assessed by flow cytometry and enzyme linked immunosorbent assay (ELISA). Binding requirement was studied using disaccharides containing either alphagalactosyl or betagalactosyl moieties to inhibit CD59 up-regulation. Protection from complement killing was assessed after incubation of EC with human serum as a source of anti-porcine antibodies and complement. The role of CD59 in lectin-induced protection was studied in the presence of an anti-pig CD59 antibody and after removal of CD59 using phosphatidylinositol (PI)-specific phospholipase C (PI-PLC). We found that induction of CD59 up-regulation required specific binding of the lectin to terminal alphaGal and was not induced either by soluble factors that may be released from EC by stimulation with the lectin or by TNF-alpha, IFN-gamma, or IL-1alpha. Unstimulated or BS-I-treated EC showed little or no expression of decay accelerator factor (DAF). Removal of membrane-associated CD59 (and other proteins that are associated with the membrane through PI linkage) with PI-PLC from EC that had been exposed to lectin restored their complement sensitivity to various degrees, depending on the extent of lectin-induced protection. Cytotoxicity was completely restored in cells that exhibited partial protection induced with lectin at low doses or for a short period of time. However, EC that were fully resistant to complement did not regain sensitivity to complement after removal of CD59. Changes in CD59 expression did not modify the degree of C9 binding. Induction of CD59 expression required specific binding of the lectin to terminal alphaGal and was not induced by soluble factors that may be released from EC by lectin stimulation. Increased CD59 expression may contribute to this form of protection from complement; however, mechanisms other than CD59 up-regulation appear to be essential for the development of full protection.

Thyroid hormone upregulates ecto-5′-nucleotidase/CD73 in C6 rat glioma cells

Thyroid hormones have profound effects on the central nervous system, such as proliferation, secretion of growth factors and gene expression regulation. Ecto-NTPDases and ecto-5′-nucleotidase can control the extracellular ATP/adenosine levels, which have been described as proliferation factors. Here, we investigated the influence of T 3 on the enzyme cascade which catalyzes interconversion of purine nucleotides in rat C6 glioma cells. Exposure of C6 cells to T 3 caused a dose dependent increase of 30% in the AMP hydrolysis up to 0.25 nM, which was suppressed by actinomycin. No significant alteration was observed on ATP/ADP hydrolysis and T 4 at higher concentrations (10–1000 nM) promoted an increase in AMP hydrolysis that was not dose dependent. T 3 treatment also increased the expression of CD73 mRNA. Besides the importance of the ecto-5′-NT in the cell proliferation and differentiation, its overexpression can enhance extracellular adenosine levels, which could also be an important proliferation signal.

Effect of IL-1β, TNF-agr; and IL-4 on complement regulatory protein mRNA expression in human articular chondrocytes

Most of the cells possess complement regulatory proteins (CRPs) that protect them against complement-mediated damage. In our previous work we revealed that human articular chondrocytes express CPRs. Moreover, increase of CRPs expression after treatment of chondrocytes with proinflammatory cytokines IL-1beta and TNF-alpha has been demonstrated by ELISA technique. Chondroprotective cytokine IL-4 stimulated expression of CD46 only. In this work RT-PCR technique was used to evaluate the expression of mRNA of cell surface CPRs in cultured isolated articular human chondrocytes after treatment with IL-1beta, TNF-alpha and IL-4. Chondrocytes stimulated with IL-1beta and TNF-alpha showed augmented levels of CD46, CD55 and CD59 mRNA. Treatment with IL-4, however, increased only the level of CD46 mRNA. These results confirm and extend our previous observations. CD35 mRNA was not found. Expression of complement regulatory proteins on chondrocytes and its upregulation by cytokines stimulating matrix degradation could be important for the protection of these cells against complement-mediated lysis, which might be caused by immunocomplexes deposited in articular cartilage in inflammatory joint diseases.

IFN-gamma-induced apoptosis and microbicidal activity in monocytes harboring the intracellular bacterium Coxiella burnetii require membrane TNF and homotypic cell adherence.

IFN-gamma is critical for the protection against intracellular bacteria through activation of the antimicrobial machinery of phagocytes. Coxiella burnetii, the etiological agent of Q fever, is a strictly intracellular bacterium that inhabits monocytes/macrophages. We previously showed that IFN-gamma induced C. burnetii killing by promoting the apoptosis of infected monocytes. We show in this study that IFN-gamma-induced apoptosis of infected monocytes was characterized by a time- and dose-dependent activation of caspase-3. IFN-gamma-mediated caspase-3 activation and C. burnetii killing depend on the expression of membrane TNF. Indeed, TNF was transiently expressed on the cell surface of infected monocytes a few hours after IFN-gamma treatment. In addition, anti-TNF Abs inhibited IFN-gamma-mediated caspase-3 activation whereas soluble TNF had no effect on infected cells. Concomitantly, IFN-gamma induced homotypic adherence of C. burnetii-infected monocytes. The latter required the interaction of beta(2) integrins with CD54. When adherence was disrupted by pipetting, by a combination of Abs specific for CD11b, CD18, and CD54, or by an antisense oligonucleotide targeting CD18 mRNA, both cell apoptosis and bacterial killing induced by IFN-gamma were inhibited. Thus, adherence via CD54/beta(2) integrins together with membrane TNF are required to eliminate C. burnetii-infected cells through cell contact-dependent apoptosis. Our results reveal a new component of the antimicrobial arsenal mobilized by IFN-gamma against infection by intracellular bacteria.

Mechanism of host cell protection from complement in murine cytomegalovirus (CMV) infection: identification of a CMV-responsive element in the CD46 promoter region.

Mouse cells ubiquitously express CRRY, which is a functional orthologue of human decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46), and thus protects cells from homologous complement. NIH3T3 cells expressed minute levels of mouse CD46 (mCD46) mRNA but barely produced mCD46 protein. mCD46 message and protein levels were markedly increased during mouse cytomegalovirus (mCMV) infection. Consistently, mCD46-expressing cells became resistant to mouse complement; primary-cultured fibroblasts from mCD46 gene-disrupted mice showed no increase in protection, resulting in complement-dependent cytolysis. Thus, the marked up-regulation of mCD46 in mouse fibroblast cells/cell lines by mCMV infection participates in host cell protection from complement. By mCD46 promoter deletion assay, the region necessary for induction of the promoter activity by mCMV infection was shown to be restricted to a sequence of 19 bp, which was homologous to the corresponding portion in human CD46, and the promoter regions of early-inducible human CMV UL36 and human herpesvirus 6 UL29. The results were confirmed by mutation analysis of this 19-bp region. We designated this sequence as the CMV-responsive element (CMVRE). Electrophoretic mobility shift assay demonstrated the existence of a CMVRE-binding factor, expression of which was significantly increased after mCMV infection. Thus, mCMV up-regulates the gene expression of mCD46 via CMVRE and CMVRE-binding factor, resulting in mCD46 protein expression on mCMV-infected cells. Since both the membrane and soluble mCD46retained complement regulatory activity, mCD46 induced by mCMV infection may act as a regulator of systemic complement activation. This represents a unique strategy of mCMV survival in host cells with sufficient replication by circumventing host complement attack.

EFFECT OF STRENGTH TRAINING ON CYTOKINE MRNA LEVELS IN SKELETAL MUSCLE OF DIABETIC HISPANIC ELDERS

There is evidence that the cytokines tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, interleukin (IL)-1β, and insulin-like growth factor-I (IGF-I) have roles in muscle regenerative and anabolic processes. PURPOSE: To examine the effects of progressive strength training (ST) on mRNA levels of TNF-α, TGF-β1, IL-1β, and the two muscle IGF-I isoforms, liver-like IGF-I (L.IGF-I) and the mechanically sensitive IGF isoform, MGF, in skeletal muscle of older individuals with type 2 diabetes (T2D). METHODS: Thirty men (n = 15) and women (n = 15), aged 67.0 ± 7.3 yrs with T2D were randominzed to 16 weeks of ST (EX) or to their usual diabetic care only (CON). All subjects underwent biopsy of the vastus lateralis at baseline. A post-intervention biopsy was performed 48 hrs after 1RM testing in EX and CON subjects. Cytokine and growth factor mRNA levels were determined by semi-quantitative real-time PCR. Leukocyte infiltration was monitored via the β2 integrin CD18, a pan-leukocyte marker. RESULTS: ST increased type I and II muscle fiber cross-sectional areas (CSA)(P < 0.05), fat free mass measured by DXA (P < 0.001), and strength (P < 0.001). TNF-α, TGF-β1, L.IGF-I, MGF and CD18 mRNA levels increased with ST (P < 0.05). All cytokines were significantly correlated with one another and all correlated with CD18. There were no associations between L.IGF-I or MGF and any cytokines. Increased TNF-α, TGF-β1, IL-1β, and CD18 mRNA levels correlated with the increase in muscle fiber CSA (P < 0.05), but L.IGF-I and MGF mRNA did not. CONCLUSION: The ST-induced hypertrophy of type I and II fibers appears to be related to increases in gene expression of cytokines known to be associated with muscle injury and regeneration (TNF-α, TGF-β1 and IL-β1), but not with increased gene expression of either IGF-I isoform. The increase in CD18, and its correlation with increased cytokine mRNA levels suggests that leukocytes recruited into skeletal muscle contribute directly or indirectly to the increased cytokine gene expression. Supported by the Brookdale Foundation, ARS contract 58-1950-9-001, and NIH GCRC grant M01 RR000054.