Immunostimulatory effect of laminarin on RAW 264.7 mouse macrophages.

This study investigated the immunostimulatory effects of laminarin with respect to inflammatory mediators such as hydrogen peroxide, calcium, nitric oxide, various cytokines, transcription factors, and immune response gene in RAW 264.7 mouse macrophages. Laminarin did not reduce the cell proliferation of RAW 264.7 mouse macrophages at concentrations up to 500 µg/mL. Laminarin significantly increased the release of hydrogen peroxide, calcium, nitric oxide, monocyte chemotactic protein-1, vascular endothelial growth factor, leukemia inhibitory factor, and granulocyte-colony stimulating factor with enhancing expression of Signal Transducer and Activator of Transcription 1 (STAT1), STAT3, c-Jun, c-Fos, and cyclooxygenase-2 mRNA in RAW 264.7 cells. The results suggest that laminarin has immunostimulatory properties, strengthening immune reactions via the transcription factor pathway in macrophages.

Ran overexpression leads to diminished T cell responses and selectively modulates nuclear levels of c-Jun and c-Fos.

Ras-related nuclear protein (Ran) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found in various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 h after their activation. The function of Ran in the immune system was investigated using Ran transgenic (Tg) mice. In Ran Tg T cells, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor activation in vitro when compared with wild type T cells. Tg mice also manifested defective delayed type hypersensitivity in vivo. Upon PMA and ionomycin stimulation, Tg T cells were defective in nuclear accumulation of AP-1 factors (c-Jun and c-Fos) but not NF-kappaB family members. Our experiments showed that Ran had important regulatory function in T cell activation. One of the possible mechanisms is that intracellular Ran protein levels control the nuclear retention for selective transcription factors such as c-Jun and c-Fos of AP-1, which is known to be critical in T cell activation and proliferation and lymphokine secretion.

Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

Characterization of a novel interaction between transcription factor TFII-I and the inducible tyrosine kinase in T cells.

TCR signaling leads to the activation of kinases such as inducible tyrosine kinase (Itk), a key regulatory protein in T-lymphocyte activation and function. The homolog of Itk in B cells is Bruton's tyrosine kinase, previously shown to bind and phosphorylate the transcription factor TFII-I. TFII-I plays major roles in transcription and signaling. Our purpose herein was twofold: first, to identify some of the molecular determinants involved in TFII-I activation downstream of receptor crosslinking in T cells and second, to uncover the existence of Itk-TFII-I signaling in T lymphocytes. We report for the first time that TFII-I is tyrosine phosphorylated upon TCR, TCR/CD43, and TCR/CD28 co-receptor engagement in human and/or murine T cells. We show that Itk physically interacts with TFII-I and potentiates TFII-I-driven c-fos transcription. We demonstrate that TFII-I is phosphorylated upon co-expression of WT, but not kinase-dead, or kinase-dead/R29C mutant Itk, suggesting these residues are important for TFII-I phosphorylation, presumably via an Itk-dependent mechanism. Structural analysis of TFII-I-Itk interactions revealed that the first 90 residues of TFII-I are dispensable for Itk binding. Mutations within Itk's kinase, pleckstrin-homology, and proline-rich regions did not abolish TFII-I-Itk binding. Our results provide an initial step in understanding the biological role of Itk-TFII-I signaling in T-cell function.

Individual contribution of metabotropic glutamate receptor (mGlu) 2 and 3 to c-Fos expression pattern evoked by mGlu2/3 antagonism.

UNLABELLED: OBJECTIVES AND MATERIALS AND METHODS: The aims of the present study were (1) to determine the neuronal activation pattern elicited by the group II mGlu antagonist LY341495 and (2) to evaluate the contribution of each group II mGlu subtype by using wild-type (WT) and knockout (KO) mice lacking either mGlu2 or mGlu3. c-Fos expression was used as a marker of neuronal activation. RESULTS AND DISCUSSION: In WT mice, LY341495 induced widespread c-Fos expression in 68 out of 92 brain areas, including limbic areas such as the amygdala, septum, prefrontal cortex, and hippocampus. LY341495-induced c-Fos response was markedly decreased in the medial part of the central amygdala (CeM) and lateral septum (LS) in mGlu3-KO mice, as well as in the lateral parabrachial nucleus (LPB) in both KO strains. In the majority of investigated areas, LY341495-induced c-Fos expression was similar in KO and WT mice. Analysis of the cellular and subcellular distribution of mGlu2 and mGlu3 revealed a prevailing presence of mGlu3-immunoreactivity in the CeM in glial processes and in postsynapstic neuronal elements, whereas only rare presynaptic axon terminals were found immunoreactive for mGlu2. CONCLUSION: In conclusion, our data indicate that group II mGlu blockade increases neuronal activation in a variety of brain areas, including many stress- and anxiety-related areas. The activation of two key brain areas, the CeM and LS, is mediated via mGlu3, while activation in the LPB involves both subtypes. Moreover, in the majority of investigated areas, LY341495-mediated neuronal activation appears to require a complex cross talk between group II mGlu subtypes or the action of LY341495 on additional receptors.

AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation.

Human T cell leukemia virus type 1 (HTLV-1) has previously been shown to infect antigen-presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV-1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV-1 viral gene expression in established monocytic cell lines was examined. U-937 promonocytic cells transiently transfected with a HTLV-1 long-terminal repeat (LTR) luciferase construct were treated with phorbol 12-myristate 13-acetate (PMA) to induce cellular differentiation. PMA-induced cellular differentiation resulted in activation of basal and Tax-mediated transactivation of the HTLV-1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA-induced cellular differentiation induced DNA-binding activity of cellular transcription factors to Tax-responsive element 1 (TRE-1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP-1) family of basic region/leucine zipper proteins (Fra-1, Fra-2, JunB, and JunD) were induced to bind to TRE-1 repeat II during cellular differentiation. Inhibition of AP-1 DNA-binding activity by overexpression of a dominant-negative c-Fos mutant (A-Fos) in transient expression analyses resulted in severely decreased levels of HTLV-1 LTR activation in PMA-induced U-937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV-1 viral gene expression may become up-regulated by AP-1 during differentiation into macrophages or dendritic cells.

Serotonin, estrus, and social context influence c-Fos immunoreactivity in the inferior colliculus.

A fundamental task of sensory systems is to extract relevant social information from a range of environmental stimuli in the face of changing behavioral contexts and reproductive states. Neuromodulatory pathways that interact with such contextual variables are 1 mechanism for achieving this. In the mouse inferior colliculus (IC), a midbrain auditory region, the neuromodulator serotonin increases in females interacting with courting males, but events downstream of serotonin release have not been investigated. Here, we manipulated serotonin levels in female mice with the serotonin releaser fenfluramine or the serotonin depleter para-chlorophenylalaninemethyl ester (pCPA). Females were then exposed to an empty cage, a male partner, or a playback of courtship vocalizations, and the numbers of neurons in the IC with positive immunoreactivity for the immediate early gene product c-Fos were measured. The effects of drug treatments depended on social context and estrous state. Fenfluramine had greater effects in the nonsocial than in the partner social treatments. Females in proestrus or estrus and given fenfluramine had higher densities of c-Fos immunoreactive neurons, while females in diestrus had fewer immunoreactive neurons. The drug pCPA had the expected opposite effect of fenfluramine, causing a decreased response in pro/estrus females and an increased response in diestrus females. These findings show that the effects of serotonin on c-Fos activity in the IC of females is dependent on both external context and reproductive state, and suggest that these effects occur downstream of serotonin release. (PsycINFO Database Record

The thalamus as a site of action of antipsychotic drugs.

OBJECTIVE: Because recent data suggest that there are abnormalities of the thalamus in schizophrenia, the authors tested the effects of antipsychotic drugs on thalamic sites. METHOD: Nine rats were given acute doses of the typical neuroleptic haloperidol (N = 3), the atypical neuroleptic clozapine (N = 3), or a drug-free vehicle (N = 3). Cellular activation was assessed by using immunohistochemistry to determine expression of the immediate response gene c-fos. RESULTS: Both antipsychotic drugs induced increased Fos immunoreactivity, suggesting increased activity in cells of the midline nuclei of the thalamus. CONCLUSIONS: The shared clinical effects of antipsychotic drugs may be mediated in part through actions on the thalamus.

Sublytic terminal complement attack induces c-fos transcriptional activation in myotubes.

Sublytic C5b-9 alters the molecular phenotype of myotubes by inhibiting muscle-specific gene expression. Here, we showed that C5b-9 induced c-fos mRNA and transcription. Using c-fos promoter-CAT constructs and electrophoretic mobility shift assay (EMSA), the minimal c-fos promoter activity was shown to increase within 30-min exposure to serum C5b-9, which also induced the binding of serum response factor (SRF), along with ternary complex factor (TCF) Elk1 and Sap1a to the serum response element. C5b-9 activated ERK1, which in turn activated Elk1 in myotubes. We propose that c-fos gene transcription associated with myotube dedifferentiation is induced by C5b-9 through ERK1-mediated assembly of serum response factor-ternary complex.

Induction of c-FOS immunoreactivity in the hippocampus following potassium stimulation.

In microdialysis procedures high potassium ion concentrations are generally used to induce neurotransmitter release. However, the widespread effects, if any, of such a treatment have not been described. In order to establish a possible link between c-fos expression and stimulating conditions for neurotransmitter release in microdialysis procedures we administered KCl (100 mM) into the hippocampus. Proto-oncogene c-FOS-like immunoreactivity is upregulated in granule cells of the dentate gyrus, pyramidal cells of the hippocampus, cingulate, piriform and frontoparietal cortices at 2 h, but not 24 h after K+ administration. Neither implantation of the probes nor perfusion with artificial cerebrospinal fluid resulted in similar patterns of c-FOS immunoreactivity. In addition, we investigated whether the impairment of the cholinergic septohippocampal pathway would modify the K(+)-induced expression of the immediate early gene c-fos in the hippocampus. The expression of c-fos induced by KCl was not altered in the animals with fimbria-fornix lesion despite the marked decrease in acetylcholine release in the hippocampus. Glutamate concentrations measured in the same superfusates showed that a significantly greater glutamate release occurs in denervated hippocampi. Furthermore, abolishment of seizure-like activity (induced by KCl) in anesthetized animals did not alter expression of c-FOS immunoreactivity in the K(+)-stimulated hippocampi. The results from these studies confirm that most of the releasable acetylcholine of the hippocampus is linked to the fimbrial input and may suggest that c-FOS upregulation in this model does not respond to any cholinergic input from the medial septum via the fimbria-fornix.

Deregulated c-fos modulates IgG2b production of B cells mediated by lipopolysaccharide.

We have examined effects of the deregulated c-fos protein on IgG2b production of B cells cultured with lipopolysaccharide (LPS) using splenic B cells from a transgenic line carrying the mouse c-fos gene under the control of the interferon alpha/beta (IFN) inducible Mx promoter (Mx-c-fosD). High c-fos expression was induced in the Mx-c-fosD B cells during the first two days of culture. DNA synthesis and IgG2b production were augmented in the culture. When IFN was added together with LPS, the high c-fos expression was prolonged until day 3 of culture. IgG2b production was remarkably suppressed. However, the production was not suppressed by upregulation of c-fos via exogenous IFN on day 4 of culture. These results suggest a regulatory effect of the c-fos protein on the differentiation of B cells to IgG2b producing cells at a distinct period.

Synergy between signal transduction pathways is obligatory for expression of c-fos in B and T cell lines: implication for c-fos control via surface immunoglobulin and T cell antigen receptors.

Expression of the protooncogene c-fos is controlled by three main regulatory pathways involving kinase C, cAMP, and calcium. Kinase C mediates its effects via phosphorylation of serum response factor (SRF) which interacts with the serum response element (SRE); cAMP and calcium mediate their effects via phosphorylation of CREB (cAMP regulatory element binding protein) presumably by activation of a protein kinase A or calmodulin-regulated kinase. We have examined the function of these elements in Burkitt's lymphoma cells (Ramos and Daudi) as well as a T lymphocytic cell line (Jurkat). We have found that stimulation of any one of these pathways alone has little or no effect on c-fos induction. However, kinase C activation (PMA stimulation) combined with either cAMP (forskolin plus MIX) or calcium stimulation (ionophore) leads to greatly enhanced c-fos induction. By contrast, cAMP in the presence of calcium shows no synergy in c-fos induction. Okadaic acid augments PMA- as well as calcium-mediated activation of c-fos, and has little or no effect when combined with cAMP. The main difference between Ramos (B cells) and Jurkat (T cells) in the regulation of c-fos is that cAMP plus calcium is strongly synergistic in Jurkat and is without effect in Ramos. Analysis of AP-1 activity using gel mobility shift assays confirms that the requirements for synergy in c-fos mRNA induction are paralleled by requirements for synergy in induction of AP-1 activity. Signaling in B cells due to anti-Ig stimulation involves both kinase C activation and release of intracellular calcium, and results in c-fos mRNA induction. Our results indicate that synergy between the kinase C activation and calcium is needed for efficient c-fos induction since neither of these two alone induces c-fos well. That synergy of signaling pathways is relevant for the anti-Ig induction of c-fos is supported by the fact that cAMP-inducing agents and okadaic acid further enhance anti-Ig induction of c-fos. These results suggest that cell-specific patterns of synergy are an essential feature for c-fos induction and may be relevant for c-fos control through B and T cell antigen receptors.

Specific expression of spinal Fos after PAR-2 stimulation in mast cell-depleted rats.

Protease-activated receptor-2 (PAR-2) in the sensory neurons may be involved in nociceptive processing. We attempted to detect and characterize specific expression of spinal Fos, a marker of nociception, in mast cell-depleted rats. Intraplantar (i.pl.) administration of not only the PAR-2 agonist SLIGRL-NH2, but also the control peptide LSIGRL-NH2, induced Fos expression in naive rats, whereas only the former specifically produced Fos expression in mast cell-depleted rats. This Fos expression was blocked by intrathecal DAMGO, a mu-opioid agonist, and, in part, by i.pl. calphostin C, a protein kinase C (PKC) inhibitor. Thus, specific expression of spinal Fos following peripheral PAR-2 activation is detectable in mast cell-depleted rats, suggesting activation of spinal nociceptive neurons, which is partially mediated by activation of PKC.

Immune stimulation induces Fos expression in brainstem amygdala afferents.

Adaptation to infectious diseases or models of infectious diseases such as immune stimulation with exogenous administration of bacterial lipopolysaccharide (LPS) or cytokines involve complex autonomic, endocrine, and behavioral responses mediated by the central nervous system. The purpose of this study is to determine the neural pathways from the brainstem activating the central nucleus of the amygdala after LPS injections in rats. Fos immunohistochemistry was performed as a marker of neuronal activation in rats prepared with injections of the retrograde tracing agents Fluorogold or cholera toxin B subunit directed at the central nucleus of the amygdala, and subsequently treated with intravenous LPS. The dose of LPS was titrated to achieve behavioral suppression ("sickness behavior") and fever, while avoiding hypotension and shock. Low-dose LPS induced Fos in central amygdala afferent neurons in the periaqueductal gray, lateral parabrachial nucleus, and solitary nucleus, as indicated by neurons containing both Fos and retrograde tracing agent. The lateral parabrachial nucleus had approximately 10-fold higher numbers of double-labeled cells than the solitary nucleus and periaqueductal gray; 95% of the double-labeled neurons in the lateral parabrachial nucleus were located in the outer zone of the external lateral subnucleus. These results suggest that a prominent source of limbic activation from the brainstem after LPS involves a restricted subdivision of the lateral parabrachial nucleus.

Changes in c-fos expression in the rat heart during morphine withdrawal. Involvement of alpha2-adrenoceptors.

We previously demonstrated an increase in Fos expression in the heart during morphine withdrawal. In the present study we examined the role of beta- and alpha-adrenoceptors in naloxone-precipitated increases in Fos expression in the heart. Dependence on morphine was induced by 7-day chronic subcutaneous implantation of six morphine pellets (75 mg). Morphine withdrawal was precipitated by administration of naloxone (5 mg/kg subcutaneously) on day 8. Using immunohistochemical staining of Fos, the present results indicate that morphine withdrawal induced marked Fos immunoreactivity (Fos-IR) within the cardiomyocyte nuclei. Moreover, Western blot analysis revealed a peak expression of c-fos in the right and left ventricles after naloxone-precipitated withdrawal in parallel with an increase in noradrenaline (NA) turnover. In the second study, the effects of the administration of adrenoceptor antagonists on withdrawal-induced Fos expression in the heart were studied. Pretreatment with the beta antagonist, propranolol (3 mg/kg intraperitoneally) or alpha1-adrenoceptor antagonist, prazosin (1 mg/kg intraperitoneally) did not block the marked Fos-IR or the hyperactivity of catecholaminergic neurons observed in the heart during withdrawal. However, pre-treatment with alpha2-adrenoceptor antagonist, yohimbine (1 mg/kg intraperitoneally), 20 min before naloxone administration to morphine-dependent rats antagonized Fos expression and the enhancement of NA turnover in the heart. Collectively, these results suggest that noradrenergic neurons in the heart are active during morphine withdrawal, and that activation of transcriptional responses mediated by Fos are dependent upon cardiac alpha2-adrenoceptor.

Circadian regulation of Fos-like expression in the brain of the blow fly Calliphora vicina.

Expression of Fos-like immunoreactivity (FOS-lir) was examined in the brains of the blow fly Calliphora vicina for evidence of circadian regulation by photic stimuli. Fos-lir in various parts of the brain was investigated as a function of light and time of day. Immunohistochemistry demonstrated that photic stimuli have an inductive effect on c-fos expression in the various parts of the brain, but only in the neurons of the pars intercerebralis did the clear photic induction of c-fos expression occur at times when light was capable of phase-shifting circadian locomotor activity rhythms. This suggests that the c-fos gene may play a role in the photic pathway for circadian entrainment and that these neurons may be involved in the transduction of photic signals. Whether changes in c-fos expression are essential components of this pathway remains to be determined.

Suppression of inflammatory gene expression in T cells by Porphyromonas gingivalis is mediated by targeting MAPK signaling.

There is increasing awareness of the effects of Porphyromonas gingivalis on host immune responses. Degradation of cytokines and chemokines by cysteine proteinases has previously been reported. However, the precise mechanisms by which P. gingivalis is able to alter intracellular signaling, and thus proliferation and inflammation, have not been described. We have previously reported suppression of activator protein-1 (AP-1) and degradation of IL-2 by proteinases from P. gingivalis. In the present study, we have analyzed the effects of P. gingivalis on Jurkat T-cell signal transduction and subsequent IL-2 and CXCL8 expression. We found that CXCL8, but not IL-2, gene expression levels were significantly suppressed by viable P. gingivalis. Analysis of intracellular signaling revealed an inhibitory effect of P. gingivalis on c-Jun and c-Fos, but not NFκB (p50 and p65), NFAT or STAT5 expression. This inhibitory effect was not due to suppression of mitogen-activated protein kinase (MAPK) (p38, erk and JNK) gene expression, but was rather due to prevention of protein kinase C (PKC) and p38 phosphorylation, as demonstrated by western blot analysis. Furthermore, SOCS1 and SOCS3 expression levels decreased following treatment of Jurkat T cells with viable P. gingivalis. The results indicate that P. gingivalis is able to suppress inflammatory gene expression by targeting the activity of MAPK pathways in T cells, which was confirmed by using specific inhibitors of NF-κB, PKC, ERK, p38 and JNK.

MUC1 is a novel costimulatory molecule of human T cells and functions in an AP-1-dependent manner.

MUC1 mucin, primarily known as an epithelial antigen, has been demonstrated to be expressed on activated human T cells. In the present study, we first examined the expression of MUC1 on different subsets of T cells (naive, effector, effector/memory). MUC1 appears to be strongly upregulated on activated CD4(+) T cells in comparison with CD8(+) T cells. The cytoplasmic tail of MUC1 contains both immune tyrosine-based activation and inhibitory motifs; therefore, we investigated whether MUC1 can also act as a costimulatory molecule on human T cells. Nonpurified T-cell cultures from human peripheral blood exhibited enhanced proliferation and an increase in cytokine production when CD3 and MUC1 were cross-linked and coligated. The intracellular mechanism of MUC1-mediated costimulation was determined to be mediated by the calcium-dependent NF-AT pathway. We further demonstrated that the cytoplasmic tail of MUC1 binds to the AP-1 transcription factors c-Fos and c-Jun, with c-Fos binding constitutively and c-Jun binding only after MUC1 stimulation. Their nuclear migration is then facilitated in a CD3-dependent manner. Our findings clearly demonstrate that MUC1 is a novel T-cell costimulatory molecule involved in immune regulation. These studies delineate important mechanisms of T-cell activation and regulation.

Anti-IgM and anti-IgG modulate c-fos RNA levels differently in human B lymphocytes.

One component of the B cell activation cascade is the induction of the protooncogene c-fos. Data presented here demonstrate that stimulation of mIgM-bearing cells with either anti-IgM or the combination of PDB plus ionomycin generated comparable levels of c-fos RNA. Furthermore, a synergistic response was observed when the cells were treated with selected concentrations of anti-IgM plus either PDB or ionomycin. In contrast, stimulation of mIgG-bearing B cells with anti-IgG did not induce the c-fos RNA levels that were seen when these cells were treated with the combination of PDB plus ionomycin. Treatment of mIgG-bearing cells with only the combination of anti-IgG plus ionomycin yielded a synergistic response and anti-IgG plus PDB did not. Thus, induction of c-fos RNA appears to be different in mIgM- and mIgG-bearing B cells after stimulation through mIg.

Cooling decreases fos-immunoreactivity in the rat after formalin injection.

The effects of cooling on inflammatory response after injection of formalin in rats were examined by counting Fos-labeled cells in the dorsal horn of the spinal cord and evaluating swelling. A small amount of dilute formalin was injected into the right hindpaw of rats. The rats were divided into Cooling (-) and Cooling (+) groups and Fos-labeled cells in the spinal cord and paw volumes were compared between the two groups. In the Cooling (+) group, Fos-labeled cells were reduced significantly and the peak time of expression was delayed compared with the Cooling (-) group. Paw volumes were significantly smaller in the Cooling (+) group than in the Cooling (-) group. These results suggest that cooling therapy is effective by delaying the progress of inflammatory reactions and provides extra time for administration of analgesics or implementation of other clinical treatments.