Alginic acid has anti-anaphylactic effects and inhibits inflammatory cytokine expression via suppression of nuclear factor-kappaB activation.

BACKGROUND: Alginic acid is comprised of complex polymerized polysaccharides, and can be chemically extracted from seaweed. Alginic acid has an inhibitory effect on histamine release, but its molecular mechanisms are not well understood. OBJECTIVE: To investigate the effect of alginic acid on the mast cell-mediated anaphylactic and inflammatory reaction using in vivo and in vitro models and elucidate its molecular mechanisms. MATERIALS AND METHOD: The effect of alginic acid on an allergy model was analysed by anaphylaxis, a histidine decarboxylase (HDC) assay, and a histamine assay. Cytokine production was analysed by means of ELISA. Cytokine expression was analysed by means of RT-PCR, and Western blotting. Transcription factor activity was analysed by a luciferase assay and a transcription factor-enzyme linked immunoassay. RESULTS: Alginic acid dose dependently inhibited compound 48/80-induced systemic anaphylaxis with doses of 0.25-1 g/kg 1 h (P<0.01, n=6) and significantly inhibited passive cutaneous anaphylaxis by 54.8%. Alginic acid (0.01-1 microg/mL) inhibited histamine release from serum and peritoneal mast cells (P<0.01). All these effects were stronger than those of disodium cromoglycate (DSCG), the reference drug tested. Alginic acid also inhibited HDC expression and activity on the phorbol myristate acetate (PMA)+A23187-stimulated human mast cell line, HMC-1 cells. Moreover, alginic acid significantly inhibited the production of PMA+A23187-induced inflammatory cytokines, IL-1beta and TNF-alpha, but not that of IL-6 or IL-8. In activated HMC-1 cells, the expression level of nuclear factor (NF)-kappaB/Rel A protein increased in the nucleus, whereas the level of NF-kappaB/Rel A in the nucleus was decreased by alginic acid treatment. In addition, alginic acid (0.01 microg/mL) decreased the PMA+A23187-induced luciferase activity and DNA-binding activity. CONCLUSION: The present results indicate that alginic acid has potent anti-anaphylactic and anti-inflammatory properties.

Flunarizine induces Nrf2-mediated transcriptional activation of heme oxygenase-1 in protection of auditory cells from cisplatin.

We investigated the cytoprotective mechanisms of flunarizine in cisplatin-induced death of auditory cells. Concomitant with an increase in viability, treatment with flunarizine resulted in a marked dissociation of Nrf2/Keap1 and subsequent intranuclear translocation of Nrf2, which was mediated by PI3K-Akt signaling. Overexpression of Nrf2 protected cells from cisplatin along with transcriptional activation of ARE to generate heme oxygenase-1 (HO-1). Pretreatment with flunarizine predominantly increased the transcriptional activity of HO-1 among Nrf2-driven transcripts, including HO-1, NQO1, GCLC, GCLM, GST micro-1, and GSTA4. Furthermore, both pharmacological inhibition and siRNA transfection of HO-1 completely abolished the flunarizine-mediated protection of HEI-OC1 cells and the primary rat (P2) organ of Corti explants from cisplatin. These results suggest that Nrf2-driven transcriptional activation of ARE through PI3K-Akt signaling augments the generation of HO-1, which may be a critically important determinant in cellular response toward cisplatin and the cytoprotective effect of flunarizine against cisplatin.

Aspects of gene polymorphisms in cerebral infarction: inflammatory cytokines.

During the last decade, a growing corpus of evidence has indicated an important role of inflammatory cytokines in the pathogenesis of cerebral lesion following stroke. Recent data suggest that genetics may in turn contribute to modulating the effects of inflammatory cytokines on cerebral infarction (CI). This paper reviews the physiologic characteristics of major inflammatory cytokines and recent research developments related to cell biology and pathobiology in CI. In particular, this review focuses on the genetic aspects of inflammatory cytokines and their implications in CI.

Activation of caspase-8 during N-(4-hydroxyphenyl)retinamide-induced apoptosis in Fas-defective hepatoma cells.

We observed that N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive and chemotherapeutic agent, effectively induced apoptosis in hepatoma cells. Interestingly, Fas-negative (Hep 3B and PLC/PRF/5) hepatoma cells were shown to be more susceptible to apoptosis induced by 4HPR than were Fas-positive (Hep G2 and SK-HEP-1) hepatoma cells. Thus, we explored the mechanisms underlying 4HPR-induced apoptosis in Fas-defective hepatoma cells. Hep 3B cells stably expressing the dominant-negative Fas-associated death domain (dnFADD) showed no alteration in 4HPR drug susceptibility, but when stably expressing E1B19K, Crm A, or dominant-negative FLICE (dnFLICE), Hep 3B cells were resistant, suggesting that 4HPR-induced apoptosis was mediated by caspase-8 activation. Furthermore, apoptosis could be completely blocked by Z-VAD-FMK (a general caspase inhibitor) or by IETD-CHO (a caspase-8 inhibitor), but was only partially blocked by Ac-DEVD-CMK (a caspase-3 inhibitor), by N-acetyl-L-cysteine (NAC) (an antioxidant), by N-acetyl-leucyl-leucyl-norleucinal (ALLN) (a calpain inhibitor I), or by Z-LEHD-FMK (a caspase-9 inhibitor). Time-sequence analysis of the induction of apoptosis by 4HPR revealed that an initial caspase-8 activation was followed by late mitochondrial cytochrome c release and minor caspase-9 activation, which suggested that caspase-8 activation is the primary upstream regulatory point. Activation of Bid or induction of proapoptotic Bax was not observed during apoptosis. In contrast, Bcl-xL expression was decreased during 4HPR-induced apoptosis. Taken together, these results indicate that 4HPR may be a potential chemotherapeutic drug, which is able to induce apoptosis in Fas-defective hepatoma cells through caspase-8 activation.

Sodium nitroprusside induces apoptosis of H9C2 cardiac muscle cells in a c-Jun N-terminal kinase-dependent manner.

Sodium nitroprusside (SNP) induces apoptosis in H9C2 cardiac muscle cells. Treatment with an exogenous NO donor SNP (2 mM) to H9C2 cells resulted in apoptotic morphological changes; a bright blue-fluorescent condensed nuclei and chromatin fragmentation by fluorescence microscope of Hoechst 33258-staining. The activity of caspase-3 like protease was increased during SNP-induced cell death. However, the activity of caspase-1 like protease was not affected by SNP. Pretreatment with Z-VAD-FMK (a pan-caspase inhibitor) or Ac-DEVD-CHO (a specific caspase-3 inhibitor) abrogated the SNP-induced cell death. SNP markedly activated three MAP kinases (JNK/SAPK, ERK and p38 MAP kinase) in the cardiac muscle cells. In this study, selective inhibition of the ERK or p38 MAPK pathway (by PD98059 or SB203580, respectively) had no effect on the extent of SNP-induced apoptosis in cardiac muscle cells. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of SNP-induced cell death. Taken together, we suggest that JNK/SAPK will be related to SNP-induced apoptosis of H9C2 cardiac muscle cells.

Dexamethasone suppresses tumor necrosis factor-alpha-induced apoptosis in osteoblasts: possible role for ceramide.

Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes, including apoptosis. Recently, it has been reported that tumor necrosis factor-alpha (TNF-alpha) activates the release of ceramide and that ceramide acts as a mediator for the TNF-alpha-induced stimulation of the binding affinity of nuclear factor-KB (NF-KB), a ubiquitous transcription factor of particular importance in immune and inflammatory responses. In this study we demonstrate that dexamethasone, which reduces the production of ceramide, significantly inhibits TNF-alpha-induced activation of NF-KB, c-Jun N-terminal kinase, also known as stress-activating protein kinase, caspase-3-like cysteine protease, redistribution of cytochrome c, and apoptosis in MC3T3E1 osteoblasts. Compared with TNF-alpha-induced JNK activation, ceramide elicits a more rapid activation of JNK within 30 min. C2-ceramide activates NF-KB and caspase-3 like protease to the same degree and with kinetics similar to those of TNF-alpha. This study provides evidence that the release of ceramide may be required as a second messenger in TNF-alpha-induced apoptosis. These results also suggest a regulatory role for dexamethasone in TNF-alpha-induced apoptosis via inhibition of ceramide release. Therefore, our in vitro results suggest that therapies targeted at the inhibition of ceramide release may abrogate inflammatory processes in TNF-alpha-related diseases, including rheumatoid arthritis and periodontitis.

High affinity IgG receptor activation of Src family kinases is required for modulation of the Shc-Grb2-Sos complex and the downstream activation of the nicotinamide adenine dinucleotide phosphate (reduced) oxidase.

We used the U937 cell line to examine the modulation of adaptor protein interactions (Shc, Grb2, and Cbl) after high affinity IgG receptor (FcgammaRI) cross-linking, leading to the formation of the Grb2-Sos complex, the activation of Ras, and the regulation of the respiratory burst. Cross-linking of FcgammaRI induced the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitant with Ras activation, Sos underwent an electrophoretic mobility shift and the Sos-Grb2 association was increased (6-fold). The Grb2-Sos complex was present only in the membrane fraction and was augmented after FcgammaRI stimulation. Tyrosine-phosphorylated Shc, mainly the p52 isoform, was observed to transiently onload to the membrane Grb2-Sos complex on FcgammaRI stimulation. Cross-linking of FcgammaRI induces the tyrosine phosphorylation of Cbl, which forms a complex with Grb2 and Shc via the Cbl C terminus. Kinetic experiments confirm that Cbl-Grb2 is relatively stable, whereas Grb2-Sos, Grb2-Shc, and Cbl-Shc interactions are highly inducible. The Src family tyrosine kinase inhibitor, PP1, was shown to completely inhibit Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the FcgammaR-induced respiratory burst. Our results provide the first evidence that the upstream activation of Src kinases is required for the modulation of the Shc-Grb2 interaction and the myeloid NADPH oxidase response.

Role of Src in the modulation of multiple adaptor proteins in FcalphaRI oxidant signaling.

Cross-linking of Fc receptors for IgA, FcalphaR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcalphaR signals through the gamma subunit of FcepsilonRI in U937 cells differentiated with interferon gamma (IFNgamma). Our results provide the first evidence that FcalphaR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcalphaRI using anti-FcalphaRI induces the phosphorylation of the gamma subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcalphaRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcalphaRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcalphaR stimulation. These data indicate that the stimulation of FcalphaR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcalphaRI-induced production of superoxide anions and provide insight into the mechanism for FcalphaR-mediated activation of downstream oxidant signaling in myeloid cells.

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways.

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.

Effect of ionizing radiation on the differentiation of ROS 17/2.8 osteoblasts through free radicals.

Although the acceleration of bone regeneration by radiation has been reported, the mechanisms of action of radiation on bone are unclear. The present results indicate that ionizing radiation-stimulated differentiation could result from the generation of reactive oxygen species during radiation exposure. The free radical release is considered as the most important mechanism of bone effect by radiation treatment. In addition, we report that radiation induced transient activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation and the transcription factor, AP-1. The JNK and AP-1 activation is mediated with radiation-released free radicals in ROS 17/2.8 osteoblasts. These results indicate that ionizing radiation at a single dose of up to 5 Gray stimulates differentiation of ROS 17/2.8 osteoblasts via free radial release which may affect JNK/SAPK and AP-1 activities.

Differential interaction of Crkl with Cbl or C3G, Hef-1, and gamma subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (Fc gamma RI).

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.

Inhibition of nitric oxide synthesis by butanol fraction of the methanol extract of Ulmus davidiana in murine macrophages.

Since there is increasing evidence that nitric oxide (NO) plays a crucial role in the pathogenesis of inflammatory diseases, this study was undertaken to address whether the methanol (MeOH) extract and its fractions of the bark of Ulmus davidiana Planch (Ulmaceae) could modulate the expression of inducible NO synthase (iNOS) in thioglycollate-elicited murine peritoneal macrophages and murine macrophage cell line, RAW264.7 cells. Stimulation of the peritoneal macrophages and RAW264.7 cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) resulted in increased production of NO in the medium. However, the butanol (BuOH) fraction of the MeOH extract of U. davidiana barks showed marked inhibition of NO synthesis in a dose-dependent manner. The inhibition of NO synthesis was reflected in the decreased amount of iNOS protein, as determined by Western blotting. The BuOH fraction did not affect the viability of RAW264.7 cells, as assessed by methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay; rather, it reduced endogenous NO-induced apoptotic cell death via inhibition of NO synthesis in RAW264.7 cells. On the other hand, the BuOH fraction showed no inhibitory effect on the synthesis of NO by RAW264.7 cells, when iNOS was already expressed by the stimulation with IFN-gamma and LPS. Collectively, these results demonstrate that the BuOH fraction inhibits NO synthesis by inhibition of the induction of iNOS in murine macrophages.

The role of inducible 70-kDa heat shock protein in cell cycle control, differentiation, and apoptotic cell death of the human myeloid leukemic HL-60 cells.

Several studies have suggested a role for heat shock proteins (hsps) during development and differentiation. However, relatively little is known about the role of hsp70 in controlling human hematopoietic cell differentiation and death. Here, we show that constitutive expression of human inducible 70-kDa heat shock protein (hsp70) promotes differentiation of HL-60 cells and prevents apoptosis that occurred after terminal differentiation or directly by apoptotic agents. After treatment with phorbol 12-myristate 13-acetate (PMA), hsp70-overexpressing cells (HL-60/hsp70) underwent rapid growth arrest and plastic adherence and expressed more CD14 than parental HL-60 or empty vector-transformed cells (HL-60/puro). HL-60/hsp70 cells also rapidly differentiated into granulocytes by addition of all-trans-retinoic acid, as assessed by phenotypic changes after staining with Wright-Giemsa. After differentiation into monocyte/macrophage-like cells or granulocytes, hsp70-overexpressing cells showed little evidence for apoptosis and had a prolonged survival, indicating that the survival-enhancing properties of hsp70 counteract programmed cell death that accompanies terminal differentiation. HL-60/hsp70 cells also showed more resistance than parental cells against apoptotic agents such as sodium nitroprusside, a NO-generating agent, or Taxol, a microtubule stabilizing agent. Further, heat shock of parental HL-60 cells at 42 degrees C for 3 h increased hsp70 levels, promoted plastic adherence (< 6 h) of the cells in respond to PMA, and protected cells from SNP or Taxol. Taken together, these studies demonstrate that hsp70 plays a crucial role in the differentiation of myeloid cells, participating in cell cycle controls and phenotypic changes, with protecting effects on apoptosis induced by different pathways.

Effect of stem cell factor, interleukin-6, nitric oxide and transforming growth factor-beta on the osteoclast differentiation induced by 1 alpha,25-(OH)2D3 in primary murine bone marrow cultures.

Osteotropic hormones and cytokines are involved in the differentiation of osteoclast progenitors from haematopoietic stem cells to multinucleated osteoclasts which mediate bone resorption. Stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta are implicated in the regulation of bone resorption by osteoclast. We test whether stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta affect the generation of osteoclast-like multi-nucleated cells induced by 1 alpha,25-(OH)2D3. 1 alpha,25-(OH)2D3 increase the generation of osteoclast-like cells retaining osteoclast characteristics including multinuclearity and positive staining for tartrate-resistant acid phosphatase. Combined treatment of stem cell factor with interleukin-6 synergistically potentiates the ability of 1 alpha,25-(OH)2D3 to generate tartrate-resistant acid phosphatase-positive multinucleated cells. However, either stem cell factor or interleukin-6 alone does not induce the generation of tartrate-resistant acid phosphatase-positive multinucleated cells. Transforming growth factor-beta produces a biphasic effect on osteoclast generation induced by 1 alpha,25-(OH)2D3. Transforming growth factor-beta stimulates osteoclast generation at low concentration (0.1 ng/ml) whereas it suppresses the formation of osteoclast-like cell at higher concentration (1 ng/ml). Sodium nitroprusside, a donor of nitric oxide, almost completely inhibits the generation of 1 alpha,25-(OH)2D3-induced osteoclast at high concentration (100 microM), but it significantly enhances the osteoclast generation at low concentrations (3 microM). These results suggest that stem cell factor, interleukin-6, transforming growth factor-beta, and nitric oxide interact with 1 alpha,25-(OH)2D3 to modulate the differentiation of hematopoietic precursors toward committed osteoclast precursors.

Nitric oxide inhibits c-Jun N-terminal kinase 2 (JNK2) via S-nitrosylation.

S-nitrosylation by S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, suppresses the phosphotransferase activity of cJun N-terminal kinase 2 (JNK2)/stress activated protein kinase (SAPK) in dose- and time-dependent manners in vitro. JNK2 activity is significantly decreased at 10 microM of GSNO, which is dramatically reversed by adding 10 mM of DTT. Reduced form of glutathione protects the GSNO-induced suppression of JNK2 activation in a dose-dependent fashion. However, GSNO-treated Sek1 does not affect the JNK2 activity of phosphotransferation toward c-Jun N-terminal1-79 protein. These results indicate that NO may exert a regulatory role of JNK2 activity by S-nitrosylation of the protein in apoptotic signaling pathway. Suppression of JNK2 phosphotransferase activity by NO is also supported by the observation that NO plays an important anti-apoptotic roles in heptocytes, splenocytes, eosinophils and B lymphocytes.

Cyclic adenosine monophosphate inhibits nitric oxide-induced apoptosis in human leukemic HL-60 cells.

Previously we reported that phorbol ester, a protein kinase C (PKC) activator, exhibits a unique pattern of potentiation of nitric oxide (NO)-related apoptosis in HL-60 human promyelocytic leukemia cells. Here we show that elevation of intracellular cAMP could protect HL-60 cells from NO- or NO plus PMA-induced DNA damage. Exposure of cells to sodium nitroprusside (SNP; 0.5 to 4 mM), a NO-generating agent, induced apoptotic cell death as monitored by morphological means, gel electrophoresis, and in situ TdT-apoptosis assay. However, concomitant incubation of the cells with DB-cAMP markedly inhibited SNP-induced apoptotic cell death in a dose-dependent manner. Similar results were obtained with other commonly used cAMP analogs such as CPT-cAMP and 8-C1-cAMP and the intracellular cAMP-elevating agent such as forskolin. In contrast, pretreatment of HL-60 cells with H89 or KT5720, which are known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of cAMP analogs and forskolin on SNP-induced apoptosis. Synergism between SNP and phorbol ester to induce apoptosis was also inhibited by prior treatment of HL-60 cells with DB-cAMP or forskolin. The effect of DB-cAMP in maintaining cell viability was not associated with the onset of G0/G1 cell cycle arrest. In addition, neither dimethyl sulfoxide nor retinoic acid (which produce granulocyte differentiation) could produce cAMP effect. Under the same conditions, DB-cAMP also inhibited NO- or NO plus phorbol ester-induced apoptosis in another transformed cell line, U-937 cells. Taken together, these findings suggest that exposure of HL-60 cells to cAMP analogs renders them more resistant to NO-induced DNA damage and further suggest the existence of specific down-modulatory mechanisms related to NO-induced apoptotic DNA fragmentation.

CBL-GRB2 interaction in myeloid immunoreceptor tyrosine activation motif signaling.

In this study, we provide the first evidence for role of the CBL adapter protein interaction in Fc gammaRI receptor signal transduction. We study the Fc gammaRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-gamma-differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, Fc gammaRIgamma of Fc gammaRI, providing direct evidence that CBL functions in myeloid ITAM signaling. Fc gammaRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon Fc gammaRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461-670) binds to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671-906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways for myeloid Fc gammaRI signaling.

Enhancing and priming of macrophages for superoxide anion production by taxol.

Taxol, an anticancer drug, has been known not only to block cell division by stabilizing microtubules but also to activate murine macrophages to express TNF-alpha, interleukin-1, and to produce nitric oxide (NO). We therefore reasoned that taxol could activate murine macrophages to generate reactive oxygen intermediates, such as superoxide anion (O2-), which are responsible for intracellular killing of pathogenic microbes. Treatment of RAW264.7 cells, murine macrophage cell line, with taxol increased phorbol ester-induced O2- production in a dose dependent manner (approximately 2 fold). In addition, taxol rapidly (< 1 hr) primed RAW264.7 cells to enhance O2- release stimulated with PMA. Taxol also enhanced stimulation of O2- production by FMLP, but not by Con A. This effect was abolished by prior treatment with both superoxide dismutase (SOD) and N-acetyl-L-cystein, a free radical scavenger. To investigate the mechanism of taxol-induced macrophage stimulation, we evaluated the ability of colchicine, a drug that inhibit tubulin polymerization, and cAMP analogues, which is known to depolymerize microtubule. Taxol-induced O2- production was inhibited by the treatment with both colchicine and DB-cAMP. Taken together, these results demonstrated that taxol provides two signals, "priming" and "enhancing", to generate superoxide anion via the stabilization of microtubules in murine RAW264.7 cells

Nitric oxide is a regulator of bone remodelling.

Nitric oxide (NO) is known to be implicated in the metabolism of bone, especially as a mediator of cytokine effects on the remodelling of bone tissue. In this study we examine whether NO affects the osteoblast activation or the osteoclast differentiation of primary mouse osteoblast-like and osteosarcoma ROS 17/2.8 cell lines. Primary osteoblast and ROS 17/2.8 cells released NO upon stimulation of interleukin-1 beta, tumour necrosis factor-alpha, and interferon-gamma. Sodium nitroprusside, a donor of nitric oxide, increased the activity of alkaline phosphatase in ROS 17/2.8 cells as well as the number of calcified nodule formations in primary mouse osteoblast-like cells. Sodium nitroprusside also completely inhibited 1 alpha, 25-(OH)2D3-induced osteoclast generation in a high concentration (100 microM). However, a low concentration of sodium nitroprusside (3-30 microM) significantly increased the generation of osteoclasts. These results indicated that NO appears to be an important regulatory molecule in the processes of bone formation and resorption. Hence, NO may be involved in the pathogenesis of bone loss in diseases associated with cytokine activation, such as periodontal disease and rheumatoid arthritis.