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M Introna

Publications and source records attributed to M Introna.

At least 55 records · Page 3Linked to original sources
PubMed1997

TNF-alpha, unlike other pro- and anti-inflammatory cytokines, induces rapid release of the IL-1 type II decoy receptor in human myelomonocytic cells.

The present study was designed to investigate the effect of a series of cytokines on the release of the type II IL-1 decoy receptor, which represents a unique pathway of negative regulation of the IL-1 system. After 20 min, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-gamma, granulocyte-CSF, macrophage-CSF, and TGF-beta had little or no effect on IL-1 binding by human polymorphonuclear cells. In contrast granulocyte-macrophage-CSF and, to a greater extent, TNF markedly reduced IL-1 binding. The action of TNF was rapid, reaching 50% of its maximum (80%) at 2 min, and plateauing at 20 min with decrease in receptor number and no significant change in affinity. Loss of surface receptor was associated to rapid release of a 45-kDa IL-1-binding molecule identified as the decoy RII. TNF-induced release of the decoy RII was independent of protein synthesis and reactive oxygen intermediates. Monocytes showed a similar response to TNF, except for the size of the released molecule (approximately 60 kDa). TNF induced rapid release of its own receptors. In contrast IL-1beta affected neither its own receptors nor the TNF-R. TNF and, more efficiently, PMA caused release of the decoy RII in fibroblasts transfected with the full-length decoy RII or with a cytoplasmatic deletion mutant. TNF-induced decoy RII release represents an unidirectional pathway of communication in the interplay between the IL-1 and TNF system.

Cells, Cultured↗
PubMed1997

Characterization of the promoter for the human long pentraxin PTX3. Role of NF-kappaB in tumor necrosis factor-alpha and interleukin-1beta regulation.

The "long pentraxins" are an emerging family of genes that have conserved in their carboxy-terminal halves a pentraxin domain homologous to the prototypical acute phase protein pentraxins (C-reactive protein and serum amyloid P component) and acquired novel amino-terminal domains. In this report, a genomic fragment of 1371 nucleotides from the human "long pentraxin" gene PTX3 is characterized as a promoter on tumor necrosis factor-alpha (TNFalpha) and interleukin (IL)-1beta exposure in transfected 8387 human fibroblasts by chloramphenicol acetyltransferase and RNase protection assays. In the same cells, the PTX3 promoter does not respond to IL-6 stimulation. Furthermore, IL-1beta and TNFalpha responsiveness is not seen in the Hep 3B hepatoma cell line. The minimal promoter contains one NF-kappaB element which is shown to be necessary for induction and able to bind p50 homodimers and p65 heterodimers but not c-Rel. Mutants in this site lose the ability to bind NF-kappaB proteins and to respond to TNFalpha and IL-1beta in functional assays. Sp1- and AP-1 binding sites lying in proximity to the NF-kappaB site do not seem to play a major role for cytokine responsiveness. Finally, cotransfection experiments with expression vectors validate that the natural promoter contains a functional NF-kappaB site.

Animals↗
PubMed1997

IL-2-regulated expression of the monocyte chemotactic protein-1 receptor (CCR2) in human NK cells: characterization of a predominant 3.4-kilobase transcript containing CCR2B and CCR2A sequences.

NK cells migrate in response to C-C chemokines, including monocyte chemotactic protein-1 (MCP-1) and MCP-3. Increased migration was observed in IL-2-activated NK cells. It was therefore of interest to define the expression in resting and activated NK cells of the MCP-1 receptor (CCR2) for which two cDNAs (A and B) have been described. Specific oligonucleotides and reverse-transcriptase PCR revealed the presence in activated NK cells and mononuclear phagocytes of the fragments expected on the basis of the reported cDNAs. In addition, amplification with a common A/B- and an A-specific oligonucleotide yielded an unexpected, abundant, 1649-bp fragment. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated NK cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B, followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A-specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. The augmented migratory capacity of IL-2 activated vs resting NK cells was associated with increased CCR2 transcript levels.

Base Sequence↗
PubMed1997

MCP-1 and CCR2 in HIV infection: regulation of agonist and receptor expression.

Monocyte chemotactic protein-1 (MCP-1) interacts with the chemokine receptor CCR2. Two CCR2 cDNAs have been described. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated natural killer (NK) cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. We found that bacterial products and cytokines affect CCR2 expression. Interleukin-2 (IL-2) augmented CCR2 mRNA in monocytes and NK cells. The augmented migratory capacity of IL-2-activated versus resting NK cells was associated with increased CCR2 transcript levels. Lipopolysaccharide (LPS) and other microbial agents caused a rapid and drastic reduction of CCR2 mRNA levels. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced. These results suggest that regulation of receptor expression, in addition to agonist production, is probably a crucial point in the regulation of the chemokine system. Down-regulation of chemokine receptor expression may play a role in the modulation of HIV infection in macrophages by LPS. Levels of MCP-1 were markedly elevated in the cerebrospinal fluid (CSF) but not in blood of HIV-infected patients with cytomegalovirus (CMV) encephalitis. The CSF levels of MCP-1 in CMV encephalitis were markedly higher than those found in the CSF of HIV-infected patients with or without unrelated neurological diseases. IL-8, the prototype of C-X-C chemokines and RANTES and macrophage inflammatory protein-1 alpha (C-C chemokines) were not substantially increased in the liquor of CMV encephalitis patients. High levels of MCP-1 may underlie monocyte recruitment and tissue damage in CMV encephalitis and may represent a rapid and useful tool in the diagnostic armamentarium for neurological disorders associated with HIV.

AIDS Dementia Complex↗
PubMed1997

Expression of a long pentraxin, PTX3, by monocytes exposed to the mycobacterial cell wall component lipoarabinomannan.

PTX3 is a prototypic long pentraxin composed of a C-terminal domain similar to those of classical pentraxins (e.g., C reactive protein) and an unrelated N-terminal portion. PTX3 is expressed in a variety of cell types, notably mononuclear phagocytes and endothelial cells, after exposure to the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). The present study was designed to assess whether mycobacterial components were able to induce expression and production of PTX3. Mycobacterial lipoarabinomannan (LAM) induced expression of PTX3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside. Among mononuclear cells, monocytes were responsible for LAM-induced PTX3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced PTX3 expression. Pretreatment with actinomycin D abolished LAM-induced PTX3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced PTX3 expression was associated with the production of immunoreactive PTX3. IL-10 and IL-13 did not inhibit the induction of PTX3 by LAM. Under the same conditions, these anti-inflammatory cytokines inhibited MCP-1 expression. In contrast, gamma interferon inhibited LAM-induced PTX3 expression. Thus, in addition to IL-1, TNF, and lipopolysaccharide, mycobacterial cell wall components also induce expression and production of the long pentraxin PTX3. The significance of PTX3 in the immunobiology of mycobacterial infection and its relevance in relation to clinical involvement remain to be determined.

Antigens, Bacterial↗
PubMed1997

Early activation signals in endothelial cells. Stimulation by cytokines.

With limitation to the "proinflammatory program" induced in endothelial cells by exposure to interleukin-1, tumor necrosis factor, and interleukin-6, we review the available data on the signaling for these three cytokines, from receptor engagement to induction of gene transcription. Only a few molecular pathways have been characterized so far, and key issues in endothelial biology, such as endothelial specificity of gene expression and heterogeneity of different endothelial populations, remain largely unexplored.

Animals↗
PubMed1997

The A-myb transcription factor in neoplastic and normal B cells.

The myb family of transcription factors has been strongly implicated in the regulation of cell growth and differentiation in the haematopoietic system. The v-myb oncogene, carried by avian defective retroviruses, causes leukaemias in the chicken and transforms haematopoietic cells in vitro. Its normal cellular equivalent c-myb, has been shown to promote the proliferation and block the differentiation of haematopoietic cells in several experimental models and is required for fetal haematopoiesis. Two other members of the family have been cloned more recently, A-myb and B-myb, which show sequence homology with c-myb in several domains, of which the DNA binding domain as well as other regulatory domains. Both have been shown to be transcription factors. B-myb is also involved in the control of proliferation and differentiation, but, unlike c-myb, it is expressed in many cell types. The third member of the family, A-myb, shows the most restricted pattern of expression, suggesting a very specific role for this transcription factor. A-myb is expressed in a subpopulation of normal B lymphocytes activated in vivo and localised in the germinal center of peripheral lymphoid organs and is not detected at significant levels in all other mature or immature haematopoietic populations studied, including bone marrow cells, T lymphocytes, granulocytes, monocytes, either at rest or after in vitro activation. These studies indicate that A-myb plays a role during a narrow window of normal B cell differentiation. A-myb expression has also been studied in a wide range of neoplastic B cells, representing the whole spectrum of B cell differentiation. A-myb is strongly expressed in Burkitt's lymphomas (BL) and slg+ B-acute lymphoblastic leukaemias (B-ALL) and not in all other leukaemias/lymphomas tested, with the exception of a subset of CLL (about 25% of cases). It is intriguing that the A-myb genome has been localised relatively close to the c-myc gene on chromosome 8, suggesting that the c-myc translocation in BL and B-ALL may affect A-myb transcription. Studies are in progress to investigate the functional relationship between A-myb and c-myc, particularly in the context of BL cells and to determine whether A-myb is deregulated in these cells.

Animals↗
PubMed1997

Gene transfer-mediated expression of physiological numbers of the type II decoy receptor in a myelomonocytic cellular context dampens the response to interleukin-1.

Available information suggests that the type II IL-1 receptor (RII) is a nonsignaling molecule which acts as a decoy for IL-1. The decoy function model for RII was supported by gene transfer experiments in fibroblasts and keratinocytes. Therefore, inhibition of IL-1 responsiveness after decoy RII gene transfer could reflect a non-physiological cellular context and receptor number. In the present study, constructs encoding RII or a cytoplasmic deletion mutant (delta 372-398) were transfected into U937 cells which express only low levels of RI detectable by RT-PCR. Gene transfer resulted in receptor numbers (approximately equal to 10(3)/cell) of the same order of magnitude as that found in normal myelomonocytic cells. Transfer of RII or a cytoplasmic deletion mutant into U937 did not increase responsiveness to IL-1, as assessed by IL-8 expression and production; it actually considerably dampened it. These results are consistent with the view that in a myelomonocytic cellular context, RII does not contribute to signaling and represents a unique pathway of negative regulation of the IL-1 system.

Cell Line↗
PubMed1997

Expression of monocyte chemotactic protein-3 in human monocytes and endothelial cells.

Monocyte chemotactic protein-3 (MCP-3) is a C-C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to investigate whether human endothelial cells (EC) and, by way of comparison, monocytes express MCP-3 and its regulation by pro- and anti-inflammatory cytokines. Unstimulated human umbilical vein EC and monocytes did not express MCP-3. Bacterial lipopolysaccharides (LPS), IL-1 and TNF induced low, but appreciable levels of MCP-3 transcripts. Interferon-gamma was a much weaker, but nevertheless active, inducer. MCP-3 transcripts were considerably less abundant than those for MCP-1. IL-4, IL-13 and IL-10 did not induce MCP-3 expression. These anti-inflammatory cytokines suppressed cytokine-induced MCP-3 expression in monocytes. In contrast, IL-4, IL-13 and IL-10 either did not affect or they amplified MCP-3 induction in EC. EC-produced MCP-3 may be important in the regulation of extravasation of receptor-expressing cells, including NK cells and eosinophils.

Cells, Cultured↗
PubMed1997

Redundant functions of B-Myb and c-Myb in differentiating myeloid cells.

We show in this report that the human myeloid leukemia cell line GFD8 is a useful model to compare the biological function of the structurally related c-Myb and B-Myb proto-oncogenes and to investigate the c-myb domains required for this function. GFD8 cells are dependent for growth on granulocyte-macrophage colony-stimulating factor and differentiate in response to phorbol myristate acetate (PMA). We have stably transfected this cell line with constructs constitutively expressing c-Myb or B-Myb. Deregulated expression of both c-Myb and B-Myb inhibited the differentiation observed in response to PMA and, in particular, the induction of the CD11b and CD11c antigens on the cell surface, and the induction of adherence. Furthermore, c-Myb and B-Myb enhanced expression of CD13 upon PMA treatment. Although deregulated Myb expression did not alter the growth factor dependence of the cells, it led to an increase in G2 relative to G1 arrest in cells induced to differentiate in response to PMA, whereas control vector-transfected cells were blocked mostly in G1. This decrease in G1 block took place despite normal induction of the cyclin-dependent kinase inhibitor protein p21 (CIP1/WAF1). Thus, GFD8 cells stably expressing the human B-Myb protein behaved in a manner indistinguishable from those stably expressing C-Myb for both differentiation and cell cycle parameters. In agreement with these findings and differently from most previous reports, transactivation assays show that B-myb can indeed act as a strong activator of transcription. Finally, we demonstrated that although the DNA-binding domain of c-myb is required for both the differentiation block and the shift in cell cycle after PMA treatment, phosphorylation by casein kinase II and mitogen-activated protein kinase at positions 11 and 12 or 532 of c-myb, respectively, are not. We conclude that c-Myb and B-Myb may activate a common cellular program in the GFD8 cell line involved in both differentiation and cell cycle control.

Antigens, Differentiation↗
PubMed1996

Inhibition of interleukin-1 responsiveness by type II receptor gene transfer: a surface "receptor" with anti-interleukin-1 function.

The hypothesis that the type II receptor (RII) acts as a decoy for interleukin-1 (IL-1) was tested by gene transfer in cells expressing only the type I receptor (8387 fibroblasts). RII-transfected cells showed defective responsiveness to IL-1 in terms of NFkappaB activation, cytokine gene expression and production. Blocking monoclonal antibodies against RII restored the capacity of RII-transfected cells to respond to IL-1 beta. Hence defective IL-1 responsiveness of RII-transfected cells requires surface expression of the molecule. RII-transfected cells showed normal responsiveness to TNF, which shares functional properties and elements in the signal transduction pathway with IL-1. Cells transfected with a deletion mutant of RII missing 26 of 29 amino acids of the cytoplasmic portion of the molecule showed impaired responsiveness to IL-2. Cells transfected with full-length or the cytoplasmic deletion mutant of RII released copious amounts of RII in the supernatant. However, transfected cells showed defective responsiveness to brief exposure to IL-1, in the absence of measurable released RII. These results indicate that impairment of the responsiveness to IL-1 following RII gene transfer was dependent upon surface expression of the molecule, specific for IL-1 and unaffected by truncation of the cytoplasmic portion. Thus, the type II "receptor" is a decoy surface molecule, regulated by antiinflammatory signals, whose only known function is to capture and block IL-1.

Base Sequence↗
PubMed1996

Regulated expression and release of the IL-1 decoy receptor in human mononuclear phagocytes.

The aim of this study was to investigate the expression and release of the IL-1 type II decoy receptor (R) in mononuclear phagocytes, which play a central role in immune and chronic inflammatory reactions. Human monocytes expressed both type I and type II R transcripts, the latter being two- to threefold more represented. By cross-linking and Ab blocking, the predominant surface IL-1-binding molecule was the decoy RII. IL-4, IL-13, and dexamethasone induced RI and RII transcripts and augmented the number of IL-1-binding sites with no modification of Kd values. The induced surface receptor was identified as the decoy RII. These stimuli induced the release of a soluble R with a m.w. of approximately 60 kDa, of which N-glycosylation contributed 22 kDa compared with 45 kDa released from polymorphonuclear leukocytes, of which N-glycosylation contributed 15 kDa. IL-13 and dexamethasone induced a release of 24 ng/ml/2 x 10(7) cells (from 8.7 to 43.2 ng/ml) and 25.6 ng/ml/2 x 10(7) cells (from 9.7 to 36.8 ng/ml) of decoy RII in 18 h, respectively (six donors). Thus, for instance, IL-13-treated (18 h) cells expressed 3.5 x 10(3) sites/cell and released 12 x 10(3) decoy RII/cell. The released decoy RII from monocytes bound IL-1apha and IL-1 receptor antagonist 30- and 2-fold less avidly than IL-1beta, respectively. In vitro-matured, monocyte-derived macrophages showed higher levels of surface expression and release of the IL-1 decoy RII. The results show that, on exposure to diverse molecules with anti-inflammatory properties, mononuclear phagocytes express and release copious amounts of a novel version of the soluble IL-1 decoy RII.

Dexamethasone↗
PubMed1996

Inhibition of monocyte chemotaxis to C-C chemokines by antisense oligonucleotide for cytosolic phospholipase A2.

Monocyte chemotactic protein (MCP)-1, a member of the C-C (or beta) branch of the chemokine superfamily, at chemotactic concentrations, induced a rapid release of [3H]arachidonic acid but not of [14C]oleic acid from prelabeled human monocytes. This effect was associated with an increase in the intensity of the immunoreactive band corresponding to the phosphorylated form of cytosolic phospholipase A2, (cPLA2). To address the role of cPLA2 in the induction of monocyte chemotaxis, cells were treated with a specific antisense oligonucleotide. Monocytes cultured in the presence of 10 microM antisense oligonucleotide for 48 h showed a marked decrease (57 +/- 5%; n = 4) of cPLA2 expression, as evaluated by Western blot analysis and a nearly complete inhibition (81.8 +/- 4.2%; n = 3) of [3H]arachidonic acid release in MCP-1-stimulated cells. Monocyte chemotaxis in response to MCP-l also was inhibited in a concentration-dependent manner by cPLA2 antisense oligonucleotide (IC50 = 1.9 +/- 1.1 microM; n = 3), with complete inhibition observed between 3 and 10 microM. No inhibition of chemotactic response was observed in monocytes treated with a control oligonucleotide. Monocyte migration in response to MCP-3, RANTES (regulated on activation normal T cells expressed and secreted), and MIP-1 alpha/LD78 also was inhibited (>70%) in antisense oligonucleotide-treated cells. On the contrary, the chemotactic response elicited by formyl-methionyl-leucyl-phenylalanine and C5a, two "classical" chemotactic agonists, was minimally affected (<20%) by antisense oligonucleotide treatment. These data show that cPLA2 plays a major role in [3H]arachidonic acid release by MCP-1 in human monocytes and provide direct evidence for the involvement of cPLA2 in C-C chemokine-induced monocyte chemotaxis.

Arachidonic Acid↗
PubMed1996

Reactive oxygen intermediates cause rapid release of the interleukin-1 decoy receptor from human myelomonocytic cells.

Free radicals play an important role in inflammation. We found that reactive oxygen intermediates (ROI) inhibit interleukin-1beta (IL-1beta) binding on human myelomonocytes. Production of superoxide anion (O2-) by Xanthine (X) and Xanthine-Oxidase (XO) or NADPH caused a reduction (48% +/- 15% in 25 experiments) in the IL-1beta binding of polymorphonuclear cells (PMN) and monocytes that was inhibited by superoxide dismutase (SOD). Hydrogen peroxide (H2O2) was only active on monocytes and this effect was prevented by catalase. O2(-)-induced loss of IL-1beta binding on PMN reached half maximum at 5 minutes and peaked after 30 minutes. The reduction of IL-1beta binding was due to reduction of IL-1beta receptors (R) on PMN surface without any change in affinity. ROI-induced reduction of surface IL-1R was not caused by receptor internalization, but rather by the release of a soluble form (45 kD) of the type II decoy R. The action of ROI on IL-1 binding was selective because major histocompatibility complex class I, CD18 and CD16 were unaffected. The O2(-)-induced release of IL-1 decoy R was not affected by protein synthesis inhibitors, but was partially blocked by protease inhibitors. Release of the IL-1 type II decoy R might represent one mechanism by which ROI antagonize and limit the proinflammatory effects of IL-1.

Cells, Cultured↗
PubMed1996

Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites.

Pentraxins, which include C reactive protein (CRP) and serum amyloid P component (SAP), are prototypic acute phase reactants that serve as indicators of inflammatory reactions. Here we report genomic and cDNA cloning of mouse ptx3 (mptx3), a member of the pentraxin gene family and characterize its extrahepatic expression in vitro and in vivo. mptx3 is organized into three exons on chromosome 3: the first (43 aa) and second exon (175 aa) code for the signal peptide and for a protein portion with no high similarity to known sequences the third (203 aa) for a domain related to classical pentraxins, which contains the "pentraxin family signature." Analysis of the N terminal portion predicts a predominantly alpha helical structure, while the pentraxin domain of ptx3 is accommodated comfortably in the tertiary structure fold of SAP. Normal and transformed fibroblasts, undifferentiated and differentiated myoblasts, normal endothelial cells, and mononuclear phagocytes express mptx3 mRNA and release the protein in vitro on exposure to interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF)alpha. mptx3 was induced by bacterial lipopolysaccharide in vivo in a variety of organs and, most strongly, in the vascular endothelium of skeletal muscle and heart. Thus, mptx3 shows a distinct pattern of in vivo expression indicative of a significant role in cardiovascular and inflammatory pathology.

3T3 Cells↗
PubMed1996

Expression of A-myb, but not c-myb and B-myb, is restricted to Burkitt's lymphoma, sIg+ B-acute lymphoblastic leukemia, and a subset of chronic lymphocytic leukemias.

The A-myb gene encodes a transcription factor that is related both functionally and structurally to the v-myb oncogene. Following our observations that A-myb is expressed in a restricted subset of normal mature human B lymphocytes, with the phenotype CD38+, CD39-, slgM-, we have now investigated the pattern of A-myb expression in neoplastic B cells representating the whole spectrum of B-cell differentiation and compared it to that of c-myb and B-myb. In a panel of 32 B-cell lines, A-myb was very strongly expressed in most Burkitt's lymphoma (BL) cell lines, but weak or negative in 2 pre-B acute lymphoblastic leukemia (ALL), 4 non-Hodgkin's lymphoma (NHL), 6 Epstein-Barr virus-immortalized lymphoblastoid cell lines, and 6 myeloma lines. Protein expression paralleled that of the RNA. We have also investigated A-myb expression in 49 fresh cases of B leukemias. Among 24 ALL, 6 were of the null and 11 of the common type and all these were negative for A-myb expression; on the other hand, all 7 B-ALL cases (slg+), as well as one fresh BL case with bone marrow infiltration, expressed A-myb. A-myb was undetectable in 4 prolymphocytic leukemias (PLL) but was strongly expressed in 5/20 (25%) of chronic lymphocytic leukemia (CLL) samples. In the latter A-myb did not correlate with phenotype or clinical stage. Finally, we have studied the progression of one case of CLL into Richter's syndrome and have found that the Richter's cells expressed about 25-fold less A-myb RNA than the CLL cells from the same patient. The pattern of c-myb and B-myb was clearly distinct from that of A-myb. C-myb and B-myb were expressed in all neoplastic groups, except in CLL cells. Thus, A-myb expression, unlike that of c-myb and B-myb, is restricted to a subset of B-cell neoplasias (in particular BL and slg+B-ALL) representative of a specific stage of B-cell differentiation. This expression may in part reflect expression of A-myb by the normal germinal center B cells that are the normal counterpart of these transformed B cells. The data presented strongly support a role for this transcription factor in B-cell differentiation and perhaps in B-cell transformation in some neoplasias.

B-Lymphocytes↗