Histamine antagonizes tumor necrosis factor (TNF) signaling by stimulating TNF receptor shedding from the cell surface and Golgi storage pool

Tumor necrosis factor (TNF) activates pro-inflammatory functions of vascular endothelial cells (EC) through binding to receptor type 1 (TNFR1) molecules expressed on the cell surface. The majority of TNFR1 molecules are localized to the Golgi apparatus. Soluble forms of TNFR1 (as well as of TNFR2) can be shed from the EC surface and inhibit TNF actions. The relationships among cell surface, Golgi-associated, and shed forms of TNFR1 are unclear. Here we report that histamine causes transient loss of surface TNFR1, TNFR1 shedding, and mobilization of TNFR1 molecules from the Golgi in cultured human EC. The Golgi pool of TNFR1 serves both to replenish cell surface receptors and as a source of shed receptor. Histamine-induced shedding is blocked by TNF-alpha protease inhibitor, an inhibitor of TNF-alpha-converting enzyme, and through the H1 receptor via a MEK-1/p42 and p44 mitogen-activated protein kinase pathway. Cultured EC with histamine-induced surface receptor loss become transiently refractory to TNF. Histamine injection into human skin engrafted on immunodeficient mice similarly caused shedding of TNFR1 and diminished TNF-mediated induction of endothelial adhesion molecules. These results both clarify relationships among TNFR1 populations and reveal a novel anti-inflammatory activity of histamine.     

Role of LPS and receptor subtypes in the uptake of TNF by the murine lung

Tumor necrosis factor alpha (TNF) is an important mediator in lung injury. The kinetics of TNF uptake by the lung are not completely understood. In this study, we evaluated the role that lipopolysaccharide (LPS) and the two types of TNF receptor (p55 and p75) play in the uptake of circulating murine TNF by the murine lung. TNF radioactively labeled with 125I (I-mTNF) was administered intravenously (2 x 10(6) cpm/mouse) to mice with both receptors (wild-type) or to mice missing one (p55-/- or p75-/-) or both (p55-/- and p75-/-) TNF receptors. Blood to lung non-reversible sequestration (Ki) and reversible uptake (Vi) were measured with multiple-time regression analysis. Uptake by lung of I-mTNF in wild-type mice had reversible and non-reversible components. This uptake was decreased by intratracheal, but not by intravenous, LPS, suggesting modulation by local, rather than systemic, inflammation. The p75-/- deficient mice retained the Ki (saturable, non-reversible) component of TNF uptake, whereas p55-/- deficient mice retained the Vi (saturable, reversible) component of TNF uptake. Both Ki and Vi components of TNF uptake were absent in the lungs of p55-/- p75-/- deficient mice. These studies show that local inflammation inhibits the uptake of circulating I-mTNF by lung and that uptake consists of two distinguishable compartments: reversible uptake mediated by the p75 receptor and non-reversible sequestration mediated by the p55 receptor.     

Regulation of IL-1 and TNF receptor expression and function by endogenous macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) has a key role in regulation of innate and adaptive immunity and is implicated in sepsis, tumorigenesis, and autoimmune disease. MIF deficiency or immunoneutralization leads to protection against fatal endotoxic, exotoxic, and infective shock, and anti-inflammatory effects in other experimental models of inflammatory disease. We report a novel regulatory role of MIF in type 1 IL-1R and p55 TNFR expression and function. Compared with wild-type cells, MIF-deficient cells were hyporesponsive to IL-1- and TNF-induced MAPK activity, AP-1 activity, and cellular proliferation, while NF-kappaB function was preserved. Hyporesponsiveness of MIF-deficient cells was associated with down-regulation of cytokine receptor expression, which was restored by reconstitution of either an upstream kinase of MAPK, MAPK/ERK kinase, or MIF. These data suggest that endogenous MIF is required for cytokine activation of MAPK/AP-1 and cytokine receptor expression. This autocrine regulatory pathway defines an important amplifying role of endogenous MIF in cytokine-mediated immune and inflammatory diseases and provides further molecular evidence for the critical role of MIF in cellular activation.     

TNF receptor I is required for induction of macrophage heat shock protein 70

Expression of heat shock proteins (HSP) is anadaptive response to cellular stress. Stress induces tumor necrosisfactor (TNF)- production. In turn, TNF- induces HSP70 expression.However, osmotic stress or ultraviolet radiation activates TNF-receptor I (TNFR-I) in the absence of TNF-. We postulated thatTNF- receptors are involved in the induction of HSP70 by cellularstress. Peritoneal M were isolated from wild-type (WT), TNF-knockout (KO), and TNFR (I or II) KO mice. Cells were culturedovernight and then heat stressed at 43 ± 0.5°C for 30 minfollowed by a 4-h recovery at 37°C. Cellular HSP70 expression wasinduced by heat stress or exposure to endotoxin [lipopolysaccharide(LPS)] as determined by immunoblotting. HSP70 expression induced byeither heat or LPS was markedly decreased in TNFR-I KO M, whereasTNFR-II KO M exhibited HSP70 expression comparable to that in WTmice. Expression of HSP70 after heat stress in TNF- KO M was alsosimilar to that in WT mice, suggesting that induction of HSP70 byTNFR-I occurs independently of TNF-. In addition, levels ofsteady-state HSP70 mRNA were similar by RT-PCR in WT and TNFR-I KO Mdespite differences in protein expression. Furthermore, the effect of TNFR-I appears to be cell specific, since HSP70 expression in splenocytes isolated from TNFR-I KO was similar to that in WT splenocytes. These studies demonstrate that TNFR-I is required for thesynthesis of HSP70 in stressed M by a TNF-independent mechanism andsupport an intracellular role for TNFR-I.

     

Stimulation ofin vitro proliferation of murine lymphocytes by synthetic oligodeoxynucleotides

To elucidate the properties of mitogenic nucleic acids, the ability of oligodeoxynucleotides to stimulate thein vitro proliferation of murine lymphocytes was investigated. The compounds tested were a series of oligodeoxynucleotides, synthesized with either phosphodiester or phosphorothioate chemistry and containing (dG) and (dC) alone or together. Among oligodeoxynucleotides tested, phosphorothioates were more active than phosphodiesters and stimulated thymidine incorporation under the same conditions as mitogenic non-mammalian DNA, Mitogenesis was unaffected by depletion of T cells, suggesting B cells as the predominant cell type stimulated. These results indicate that mitogenic nucleic acids need not have an extended polymeric structure and raise the possibility that antisense compounds have immunologic activity, at least in animal models.     

Differentiation in murine mastocytoma induced by macrophage gangliosides

In the membrane of mouse macrophages two gangliosides were detected which inhibit the division of murine mastocytoma P815 tumor cells. The two gangliosides were incorporated into the cytoplasmatic membrane of mastocytoma cells. The concentration necessary to achieve a complete inhibition of P815 tumor cell division is about 1 microM for both effective gangliosides. Macrophage ganglioside-induced inhibition of cell division is accompanied by morphological changes of the mastocytoma cells. While the cells are rounding, their diameter increases and serotonin and granules appear in the cytoplasm of the enlarged cells. Our findings suggest that macrophage gangliosides may differentiate mastocytoma cells into mast cells.     

EGF antisense oligodeoxynucleotides block murine odontogenesis in vitro

The initiation of odontogenesis depends on the site-specific proliferation of mandibular epithelium beginning at Day 11 in embryonic mice. We have previously reported that the local expression of epidermal growth factor mRNA in the murine mandible is developmentally regulated, expressed at Days 9 and 10 immediately prior to the initiation of tooth bud formation at Day 11. Exposure of Day 9 mandibular explants to antisense oligomers of epidermal growth factor blocks the initiation of odontogenesis. These results are the first demonstration of the involvement of epidermal growth factor in the inductive specification of a complex epithelial derivative.     

TNF-Induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.     

Carbohydrate sequences detected by murine monoclonal antibodies

The carbohydrate sequences of cell surface glycolipids change during differentiation and oncogenic transformation. To detect these structural changes, murine monoclonal antibodies have been produced in many different laboratories. Some of these antibodies are used to distinguish various cell types such as normal and transformed cells, while others are used to analyze developmentally regulated antigens. Recently, the structures of many of these carbohydrate antigens have been determined. The availability of these well-defined monoclonal antibodies will be useful for the study of the regulation and function of glycoconjugates.     

STAP-2 regulates c-Fms/M-CSF receptor signaling in murine macrophage Raw 264.7 cells

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. Our previous studies also revealed that STAP-2 binds to MyD88 and IKK-alpha/beta, and modulates NF-kappaB signaling in macrophages. In the present study, we examined physiological roles of the interaction between STAP-2 and c-Fms in Raw 264.7 macrophage cells. Our immunoprecipitation has revealed that c-Fms directly interacts with the PH domain of STAP-2 independently on M-CSF-stimulation. Ectopic expression of STAP-2 markedly suppressed M-CSF-induced tyrosine phosphorylation of c-Fms as well as activation of Akt and extracellular signal regulated kinase. In addition, Raw 264.7 cells over-expressing STAP-2 showed impaired migration in response to M-CSF and wound-healing process. Taken together, our findings demonstrate that STAP-2 directly binds to c-Fms and interferes with the PI3K signaling, which leads to macrophage motility, in Raw 264.7 cells.     

Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1beta. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.     

Insertional activation of N-myc by endogenous Moloney-like murine retrovirus sequences in macrophage cell lines derived from myeloma cell line-macrophage hybrids.

Hybrids formed from a myeloma cell line, NS1, and macrophages initially show myeloma properties but later, after loss of the parental macrophage genome and consequent loss of myeloma characteristics, express macrophage properties. Molecular studies demonstrated that macrophage properties in the hybridomas originate from the NS1 parental cells (M. Setoguchi, S. Yoshida, Y. Higuchi, S. Akizuki, and S. Yamamoto, Somatic Cell Mol. Genet. 14:427-438, 1988). In such hybrids, N-myc was activated by insertion of endogenous Moloney-like retrovirus sequences into mouse N-myc exon 3 when the hybrids gained macrophage properties. Interestingly, expression of N-myc took place in all aged hybrids. These results suggest that such unique insertional mutagenesis occurs in a regionally specific manner and that expression of N-myc may play a role in hematopoietic lineage conversion.     

Turnover and shedding of Ia antigens by murine spleen cells in culture.

Lactoperoxidase-catalyzed radioiodination was used to examine the metabolic fate of surface Ia antigens on murine spleen cells in culture. Ia antigens, detected predominately on splenic B lymphocytes, were lost from the cultured cells with biphasic kinetics: a 4 to 6 hr rapid phase, t 1/2 = 5 hr followed by slow release through 20 hr, t 1/2 = 30 hr. The rapid loss of Ia antigens observed was abolished by both harsh iodination conditions and nonphysiologic incubation conditions. The rapid decline in Ia activity was shown to be due to shedding of intact Ia antigens from the cell and to predominant release of IA subregion-coded proteins. Release of Ia antigens from the cell was accomplished by replacement at the cell surface, and thus reflected net membrane Ia turnover. Ia shedding was shown to be extremely temperature dependent, reflecting both a comparatively high activation enthalpy and entropy requirement for turnover.     

Infection of murine macrophage cell lines by Legionella pneumophila

Legionella pneumophila causes pneumonia by infecting alveolar macrophages. Although several model systems have been used for L. pneumophila virulence studies, no detailed comparisons have been made between them. An ideal in vitro virulence model should be cost-effective, easy to obtain in large amounts and as relevant as possible to the actual disease. We compared the MH-S cell line to human peripheral blood monocyte-derived macrophages and the J774A.1 cell line. We found that the interactions of L. pneumophila with MH-S at the cellular level resemble those of human primary monocyte-derived macrophages, suggesting that these cells provide a valuable model for this bacterial pathogen.     

The TNF receptor in TNF-mediated cytotoxicity

The in vitro cytotoxic capacity (if not every pleiotropic property) of tumor necrosis factor (TNF) begins by interaction with specific high affinity cell surface receptors. The characterization of receptors and ligand kinetics is reviewed in relationship to cytotoxicity. Decreased receptor number and affinity correlate with sensitivity within a given cell line. In L-M cells (a sensitive tumorigenic fibroblast), TNF induces a biphasic downregulation of receptors. Internalized ligand and receptors are largely cleared before the onset of cell death. Drugs affecting cytotoxicity may act primarily on an early 'association' stage (ligand receptor interaction, internalization or perhaps signal transduction) or on a later 'lytic' stage. Phorbol myristate acetate is an example of the former, while chloroquine, cholera toxin and dibutyryl cyclic AMP are examples of the latter.     

Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor.

The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4 beta-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct.     

Properties of primary murine stroma induced by macrophage colony-stimulating factor

The ability of purified human macrophage colony-stimulating factor (M-CSF) to accelerate the formation of stromal cells from murine bone marrow cells was investigated. The liquid culture of the marrow cells with M-CSF resulted in the formation of monolayers of macrophages on day 7. When the M-CSF was removed on that day and the residual adherent cells were cultured in the absence of M-CSF for an additional 7 days, many colonies appeared with cells that were morphologically distinguishable from M-CSF-derived macrophages. The appearance of the colonies was dependent on the concentration of M-CSF used at the beginning of the culture. Each colony was isolated as a single clone and analyzed. All clones were negative for esterase staining. These cells did not express M-CSF receptor mRNA and did not show a mitogenic response to M-CSF. On the contrary, these cells could be stimulated to proliferate by fibroblast growth factor and platelet-derived growth factor. The polymerase chain reaction analysis of these cells demonstrated constitutive expression of mRNA for M-CSF, stem cell factor, and interleukin (IL)-1, but not IL-3. Some clones expressed mRNA for granulocyte/M-CSF and IL-6. We also examined the ability of the cells to maintain murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in a coculture system. Most of the clones showed a significant increase in total HPP-CFC numbers after 2 weeks of coculture, although the extent of stimulation differed among clones. These results suggested that the colonies established by M-CSF were composed of functional stromal cells that were phenotypically different from macrophages. J. Cell. Physiol. 173:1–9, 1997. © 1997 Wiley-Liss, Inc.