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S E Mergenhagen

Publications and source records attributed to S E Mergenhagen.

At least 19 recordsLinked to original sources
PubMed1995

A 77-kDa protein associates with pp125FAK in mast cells and becomes tyrosine-phosphorylated by high affinity IgE receptor aggregation.

The focal adhesion kinase, pp125FAK, is a novel non-receptor protein tyrosine kinase expressed in different cells including mast cells. Here we report that a 77-kDa protein associates with pp125FAK in the mast cell analog, rat basophilic leukemia (RBL-2H3) cells. When pp125FAK immunoprecipitates were subjected to an in vitro kinase assay, there was prominent phosphorylation on tyrosine of pp125FAK and of a 77-kDa protein. By V8 protease digestion mapping and by immunoblotting with two different anti-pp125FAK antibodies, the 77-kDa protein was distinct from pp125FAK. This Fak Associated Protein or FAP was detected in RBL-2H3 cells but not in fibroblasts. The aggregation of the high affinity IgE receptor, Fc epsilon RI, induced the in vivo tyrosine phosphorylation of FAP. However, there was a marked decrease in the in vitro phosphorylation of FAP in the immunoprecipitates from Fc epsilon RI aggregated cells. Both of these Fc epsilon RI-mediated effects were enhanced by cell adhesion. There was strong association of FAP with non-tyrosine-phosphorylated pp125FAK. Thus this interaction does not appear to be mediated by the Src homology 2 domain. Together the data indicate that FAP associates with pp125FAK and suggest that FAP may play a role in Fc epsilon RI signaling.

3T3 Cells↗
PubMed1994

The aggregation of the high affinity IgE receptor induces tyrosine phosphorylation of paxillin, a focal adhesion protein.

Tyrosine phosphorylation of proteins is an essential component of high affinity IgE receptor (Fc epsilon RI) signaling and secretion. This signaling and secretion is also dependent on the organization of the cytoskeleton. Here we report that the aggregation of Fc epsilon RI on rat basophilic leukemia cells results in tyrosine phosphorylation of the cytoskeletal protein, paxillin. Tyrosine phosphorylation of paxillin is a relatively late event after Fc epsilon RI aggregation. Both the direct increase in intracellular Ca2+ with calcium ionophore and the activation of protein kinase C (PKC) with PMA induced tyrosine phosphorylation of paxillin. The optimal tyrosine phosphorylation of paxillin by Fc epsilon RI aggregation required PKC and extracellular Ca2+. However, there was also Fc epsilon RI-mediated tyrosine phosphorylation of paxillin independent of Ca2+ influx or PKC activation. By fluorescent microscopy, cell stimulation induced a redistribution of paxillin toward the periphery of the cells. Although Fc epsilon RI aggregation induced tyrosine phosphorylation of paxillin in nonadherent cells, adherence markedly enhanced this phosphorylation. Together, the data suggest a role for paxillin in Fc epsilon RI signaling.

Animals↗
PubMed1994

Adhesion molecules as regulators of mast-cell and basophil function.

Basophils and mast cells play a role both in immediate allergic reactions and in inflammation. Both types of cells have surface adhesion receptors that can mediate binding to other cells and to extracellular matrix glycoproteins. Here Majed Hamawy and colleagues discuss the importance of these adhesion molecules in regulating basophil and mast-cell functions.

Animals↗
PubMed1993

Mouse 3T3 fibroblasts induce rat basophilic leukemia (RBL-2H3) cells to acquire responsiveness to compound 48/80.

2H3 subline of rat basophilic leukemia (RBL-2H3) cells are mast cell analogs that lack responsiveness to nonimmunologic stimuli such as compound 48/80 and substance P. To determine if fibroblasts can influence this responsiveness, RBL-2H3 cells were cocultured with confluent monolayers of mouse 3T3 fibroblasts and assayed for secretagogue-induced histamine release. After 1 wk in coculture, RBL-2H3 cells began to respond to compound 48/80. Responsiveness reached a maximum at 2 wk in coculture and remained at this level for an additional 2 wk. Histamine release was specific, noncytotoxic, dose-dependent, and occurred even in the absence of extracellular Ca2+. No soluble factor from 3T3 cells was found that induced these alterations. Moreover, neither recombinant rat or mouse steel factor, at concentrations up to 250 ng/ml, was able to alter RBL-2H3 cell reactivity to compound 48/80. By 2 wk in coculture, RBL-2H3 cells also became responsive to substance P, although no changes in histamine content, Alcian blue+/safranin- staining or type of serine protease were detected. These results show that 3T3 fibroblasts cause an alteration in the functional repertoire of RBL-2H3 cells and that soluble steel factor cannot duplicate the effect.

3T3 Cells↗
PubMed1993

Mast cells and their microenvironment: the influence of fibronectin and fibroblasts on the functional repertoire of rat basophilic leukemia cells.

To determine how the microenvironment in which mast cells are located may influence their function, we explored the effects of fibronectin and fibroblasts on histamine secretion in vitro from a mast cell model, the rat basophilic leukemia (RBL-2H3) cell line. RBL-2H3 cells bound specifically to fibronectin-coated surfaces. Binding was maximal by 1 hour, was not detectable at 0 degrees C or in the absence of Ca++, and was inhibited by preincubating the cells with a synthetic peptide containing the RGD sequence. Adherence to fibronectin stimulated RBL-2H3 cell spreading with a concomitant reorganization of the cytoskeleton and a repositioning of the cytoplasmic granules to the cell periphery. Although adherence to fibronectin did not by itself induce histamine release, when stimulated by either immunologic or non-immunologic means, fibronectin-adherent cells released dramatically more histamine than cells plated in wells coated with BSA only. Thus, RBL-2H3 cells bind specifically to fibronectin, and in so doing are stimulated to undergo changes in morphology and enhanced responsiveness to secretory stimuli. RBL-2H3 cells grown in coculture with 3T3 fibroblasts, but not RBL-2H3 cells grown alone, became responsive to the polymeric synthetic secretagogue Compound 48/80 and the neuropeptide Substance P. Maximum sensitivity to Compound 48/80 was attained by the second week in coculture. Histamine release was dose-dependent, noncytotoxic and occurred even in the absence of extracellular Ca++. Contact between the 2 cell types appeared to be a critical factor. RBL-2H3 cells, separated from 3T3 cells by a 0.45 micron filter, failed to secrete histamine in response to Compound 48/80.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The rat mast cell antigen AD1 (homologue to human CD63 or melanoma antigen ME491) is expressed in other cells in culture.

Previously we reported that the mAb AD1 recognized a heavily glycosylated 50- to 60-kDa protein (AD1 Ag) sterically close to the high-affinity IgE receptor on rat basophilic leukemia (RBL-2H3) cells. The N-terminal amino acid sequence of the AD1 Ag was nearly identical to that of human CD63 (melanoma-associated Ag ME491). In this study we cloned the cDNA of AD1 Ag from a rat basophilic leukemia 2H3 cDNA library. An open reading frame of 238 amino acids was identified that contained the N-terminal 43 amino acid sequence. No evidence of a signal peptide was found. However, four predominantly hydrophobic stretches of sequence were predicted to form membrane-spanning helices, and three putative N-glycosylation sites were identified. The AD1 Ag and CD63 were highly conserved between rat and human, suggesting that the sequence of this protein is important for its function. By immunostaining various rat tissues, the AD1 Ag was found localized to mast cells. However, it was located to lysosomes, secretory granules and the plasma membrane of RBL-2H3 cells and to lysosomes and plasma membrane of many other cultured cell lines. The AD1 Ag could be induced by placing cells in culture. Fibroblasts and hepatocytes freshly isolated from rat embryos stained very weakly for AD1 Ag; however, after 24 to 48 h in culture they were strongly positive. This increase in the expression of the AD1 Ag was accompanied by an increase in detectable RNA message. Therefore, AD1/ME491/CD63 Ag is a mast cell marker in tissue, but is also associated with other cells in culture.

Amino Acid Sequence↗
PubMed1992

Adherence of rat basophilic leukemia (RBL-2H3) cells to fibronectin-coated surfaces enhances secretion.

Rat basophilic leukemia (RBL-2H3) cells are a useful in vitro model for studies of mast cells and basophils. We examined the adherence of RBL-2H3 cells to different extracellular matrix proteins and the effect of such attachment on secretion. The cells bound to fibronectin-coated surfaces with maximum binding by 1 h at 37 degrees C. There was less attachment to laminin, collagen type I, and collagen type IV. There was no adherence to uncoated surfaces or in the absence of Ca2+. Binding to fibronectin was blocked by a synthetic peptide containing the sequence Arg-Gly-Asp. Therefore, the binding of RBL-2H3 cells to fibronectin may be mediated by surface molecules that belong to the integrin family. Adherence to fibronectin-coated surfaces resulted in cell spreading, a reorganization of the cytoskeletal elements, and a redistribution of the secretory granules. Attachment to fibronectin also dramatically enhanced high affinity IgE receptor-mediated histamine release. This enhancement was maximum by 1 h of adherence and lasted for at least 6 h. There was also enhanced secretion by the Ca2+ ionophore A23187. Thus, adherence to fibronectin can enhance both receptor and non-receptor-mediated release. Addition of soluble fibronectin to RBL-2H3 cells in suspension had no effect on secretion. Therefore, enhanced histamine release required cell attachment to immobilized fibronectin. These results suggest that secretion from mast cells/basophils may be modulated by their interaction with the extracellular matrix.

Animals↗
PubMed1992

Autocrine and paracrine regulation of astrocyte function by transforming growth factor-beta.

Recent evidence indicates that astrocytes have a wide range of functions, usually attributed to cells of the immune system, which are critical for maintaining a balanced homeostatic environment in the central nervous system (CNS). Moreover, these cells are known to participate in inflammatory events within the CNS by secreting cytokines such as transforming growth factor-beta (TGF-beta). In this study we have investigated the ability of TGF-beta to influence astrocyte functions. TGF-beta 1 mRNA is constitutively expressed by astrocytes in vitro, and when cultures are stimulated with exogenous TGF-beta 1 an increase in the expression of this mRNA can be shown, suggesting both autocrine and paracrine regulation. In in vitro assays, TGF-beta 1 is chemotactic for astrocytes in a dose-dependent fashion and inhibits astrocyte proliferation. These results indicating signal transduction by TGF-beta 1-prompted studies to explore receptor-ligand interactions on isolated astrocyte populations. In a receptor binding assay, we demonstrate that astrocytes appear to express three distinct TGF-beta receptor subtypes with nearly 10,000 receptors per cell. Thus, TGF-beta may play an important role in regulating astrocyte functions pivotal to the evolution of intracerebral immune responses including recruitment and activation of glial cells at local inflammatory sites within the CNS.

Animals↗
PubMed1992

Stimulation of human monocytes by endotoxin-associated protein: inhibition of programmed cell death (apoptosis) and potential significance in adjuvanticity.

Mononuclear phagocytes are essential for adjuvant activity and polyclonal immunoglobulin synthesis induced by endotoxin-associated protein (EP) from Salmonella spp. To define the mechanisms of EP-mediated immunostimulation, we evaluated monocyte functions central to adjuvanticity following exposure to Salmonella typhimurium EP. In this study, we show that EP promotes the survival of monocytes by blocking programmed cell death (apoptosis), enhancing the production of the immunostimulatory cytokine interleukin-1 (IL-1) and stimulating the increased expression of HLA-DR and IL-2 receptors, which are cell membrane proteins that facilitate antigen presentation and IL-2 regulation, respectively. These results indicate that, like lipopolysaccharide, EP is a potent activator of human monocytes and suggest that EP-induced immunostimulation may be mediated, in part, by enhanced monocyte survival, cytokine release, and receptor expression.

Bacterial Proteins↗
PubMed1992

Kupffer cells express type I TGF-beta receptors, migrate to TGF-beta and participate in streptococcal cell wall induced hepatic granuloma formation.

Intraperitoneal injection of Group A streptococcal cell wall (SCW) fragments into female Lewis rats results in the induction of an acute hepatic inflammation that progresses to granulomatous lesions. Kupffer cells have been shown to rapidly clear circulating SCW which triggers production of TGF-beta. In this study, we examined Kupffer cells for the expression of TGF-beta receptors to determine if these cells might be modulated in an autocrine/paracrine fashion by TGF-beta during SCW-hepatic inflammation. By receptor crosslinking and subsequent SDS-PAGE analysis we demonstrate that Kupffer cells express Type I TGF-beta receptors, but not Types II and III. Scatchard analysis indicated a receptor density of approximately 1100 receptors per cell. Functionally, TGF-beta was found to be chemotactic for Kupffer cells in vitro and this chemotactic response was higher in cells isolated from rats 1-21 days post SCW-injection. Although TGF-beta 1 mRNA is constitutively expressed by Kupffer cells, in vitro stimulation of the cultures with purified TGF-beta augments the expression of TGF-beta 1 mRNA and protein synthesis suggesting autocrine/paracrine regulation. These results indicate that TGF beta secreted by Kupffer cells during SCW-induced hepatic inflammation may amplify its own expression and regulate Kupffer cell functions relevant to the formation of granulomatous lesions within the liver.

Animals↗
PubMed1991

Inhibitors of reactive oxygen intermediates suppress bacterial cell wall-induced arthritis.

Peritoneal and peripheral blood monocyte-macrophages from inbred Lewis (LEW) rats generate higher levels of reactive oxygen intermediates (ROI) in response to group A streptococcal cell walls (SCW) than do similar populations of cells from histocompatible Fischer rats. This differential sensitivity of the phagocytes to SCW is reflected in differences in susceptibility of the two strains to the development of arthritis in response to SCW. After systemic administration of the SCW, LEW rats develop acute and chronic erosive polyarthritis, whereas the Fischer rats are arthritis resistant. Inasmuch as these data suggested that the SCW-induced release of inflammatory cell products such as ROI might be an important contributory factor in the pathogenesis of arthritis in the LEW rats, the animals were injected with SCW and treated with ROI inhibitors. A single intraarticular injection of superoxide dismutase or catalase significantly reduced the SCW-induced inflammatory response and evolution of erosive arthritis in the treated animals (articular index 3.6 +/- 0.36 for SCW only vs 1.4 +/- 0.3 for SCW + SOD; p less than 0.001; n = 6). These data indicate that ROI play a pivotal role in synovitis and, furthermore, that suppression of these inflammatory mediators modulates both acute and chronic SCW-induced inflammation of the joint.

Animals↗
PubMed1991

Macrophage- and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome.

The multifunctional cytokine, transforming growth factor beta (TGF-beta), was identified by immunocytochemistry in the brain tissues of four patients with acquired immune deficiency syndrome (AIDS), but not in control brain tissue. The TGF-beta staining was localized to cells of monocytic lineage as well as astrocytes, especially in areas of brain pathology. In addition, the brain tissues from the AIDS patients contained transcripts for human immunodeficiency virus 1 (HIV-1) by in situ hybridization, suggesting a correlation between the presence of HIV-1 in the brain and the expression of TGF-beta. However, the expression of TGF-beta was not limited to HIV-1-positive cells, raising the possibility of alternative mechanisms for the induction of TGF-beta in these AIDS patients' brains. To investigate these mechanisms, purified human monocytes were infected in vitro with HIV-1 and were shown to secrete increased levels of TGF-beta. In addition, HIV-1-infected monocytes released a factor(s) capable of triggering cultured astrocytes that are not infected with HIV-1 to secrete TGF-beta. The release of TGF-beta, which is an extremely potent chemotactic factor, may contribute to the recruitment of HIV-1-infected monocytic cells, enabling viral spread to and within the central nervous system (CNS). Moreover, TGF-beta augments cytokine production, including cytokines known to be neurotoxic. The identification of TGF-beta within the CNS implicates this cytokine in the immunopathologic processes responsible for AIDS-related CNS dysfunction.

AIDS Dementia Complex↗
PubMed1990

Inhibition of phospholipase activity in human monocytes by IFN-gamma blocks endogenous prostaglandin E2-dependent collagenase production.

Previous studies from our laboratory have demonstrated that exposure of human monocytes to a stimulant, such as Con A, results in the production of the enzyme collagenase through PGE2-dependent pathway. Inasmuch as rIFN-gamma has been shown to modulate monocyte/macrophage PG synthesis, we examined the effect of rIFN-gamma on the activation sequence leading to collagenase production. The addition of rIFN-gamma (10 to 1000 U/ml) to Con A-stimulated monocytes resulted in a dose-dependent inhibition of PGE2 and collagenase synthesis. The suppression of collagenase production by rIFN-gamma was related to its ability to reduce PGE2 levels as demonstrated by the restoration of collagenase activity by the addition of PGE2. HPLC analysis of the arachidonic acid (AA) metabolites released by monocytes showed that rIFN-gamma caused a reduction in the release of AA and products of the cyclooxygenase and lipoxygenase pathways. These data indicated that rIFN-gamma decreased eicosanoid production by inhibiting the release of AA from phospholipids. This conclusion was supported by the reduction in membrane bound phospholipase activity in rIFN-gamma-treated monocytes. Moreover, the inhibition by rIFN-gamma of PGE2 and collagenase was reversed by the addition of phospholipase A2. Our findings demonstrate that rIFN-gamma inhibits phospholipase activity in activated monocytes and as a result blocks PGE2-dependent collagenase synthesis.

Cells, Cultured↗
PubMed1989

Inflammatory and immunomodulatory roles of TGF-beta.

Transforming growth factors (TGFs) are small polypeptides that were initially defined by their ability to induce transformation of non-neoplastic cells in culture. However, it has become increasingly clear that TGFs are not restricted in function to promoting cell growth. One type of transforming growth factor, TGF-beta, is a multifunctional molecule which has unique and potent effects on many target cells and tissues. In this article, Sharon Wahl, Nancy McCartney-Francis and Stephan Mergenhagen focus on the evolving role of TGF-beta in regulating inflammation, immune responses and tissue repair.

Animals↗
PubMed1988

Inhibition of allergic reactions with monoclonal antibody to the high affinity IgE receptor.

A mAb that reacts with the high affinity IgE-R on the rat basophilic leukemia cells (RBL-2H3) was used to inhibit allergic reactions. In vitro, the intact mAb BA3 and its Fab fragment inhibited radiolabeled IgE binding to the RBL-2H3 cells. The mAb binds to the IgE-R with a higher affinity than does IgE. Whereas the intact mAb released histamine from the RBL-2H3 cells, the Fab was inactive. The addition of the Fab fragments to RBL-2H3 inhibited the IgE-mediated histamine release reaction. The Fab fragments also inhibited in vivo passive cutaneous reactions in rats when injected intradermally either before or after IgE. The injection of the mAb Fab i.v. before the injection of the IgE into the skin sites also inhibited reactions, although it was less effective. The results demonstrate that anti-R antibodies can be used as a model for inhibiting immediate hypersensitivity reactions.

Animals↗
PubMed1988

Granulocyte-macrophage colony-stimulating factor regulation in murine T cells and its relation to cyclosporin A.

The coordinated expression of the hematopoietic growth factors, interleukin 3 (IL3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in activated T cells suggests common synthetic pathways. However, cyclosporin A (CsA) appears to differentially effect the synthesis of these two lymphokines. When the supernatants from the concanavalin A-stimulated thymoma EL-4 or the T-cell hybrid 2B4 were assayed on the GM-CSF/IL3-dependent PT-18 and on the IL3-dependent DA-1 cell lines, IL3 activity could not be detected following CsA treatment, but substantial growth activity, which was identified as GM-CSF, was observed on the PT-18 cell line. CsA did not affect the kinetics of GM-CSF release, but inhibited the release of IL3 over a period of 40 h after the cells were stimulated and treated with CsA. In addition, CsA could be added up to 1 h after stimulation of the cells without affecting GM-CSF activity but inhibiting completely the IL3 activity. To substantiate the inability of CsA to inhibit GM-CSF activity, GM-CSF gene expression was evaluated. By Northern analysis GM-CSF mRNA was not inhibited by doses of CsA up to 1 microgram/ml. The data reveal that CsA can dissociate between the production of IL3 and GM-CSF, suggesting that these two CSFs are regulated by different mechanisms.

Animals↗