Spin90 deficiency increases CXCL13-mediated B cell migration.

SPIN90 regulates actin dynamics, which is important for cell migration control. CXCL13-mediated B cell migration is essential for B cell immune responses. In this study, we investigated the role of SPIN90 in CXCL13-mediated B cell migration using Spin90 gene-deficient mice. Our chemokinesis analysis and transwell cell migration assay showed that SPIN90 is involved in CXCL13-mediated B cell migration. Moreover, the level of CXCR5, which is CXCL13 receptor, was increased in SPIN90-deficient B cells compared with wild-type B cells. Overall, our data suggest that SPIN90 plays an important role in B cell immune responses through the regulation of CXCL13-mediated B cell migration.

An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes.

Several methods have been developed for the immortalization of B lymphocytes by Epstein-Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester-induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders.

Synergistic cooperation between thapsigargin and phorbol ester for induction of nitric oxide synthesis in murine peritoneal macrophages.

The biochemical transductional events involved in NO synthesis are not fully understood. These studies, therefore, were undertaken to elucidate the role of intracellular calcium and protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Thapsigargin (TG), Ca(2+)-ATPase inhibitor of endoplasmic reticulum, had modest activity on NO synthesis by itself, whereas phorbol ester, PKC activator, alone had no effect. When TG was used in combination with phorbol ester, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of phorbol ester was shown in the first 6 h after TG treatment. In addition, the ability of TG with phorbol ester on NO synthesis could be mimicked by another chemically unrelated inhibitor of Ca(2+)-ATPase, 2,5-di-(t-butyl)-1, 4-benzohydroquinone, and Ca2+ ionophore, A23187. This increase of NO synthesis was reflected as increased amount of NO synthase (NOS) mRNA, as determined by Northern blotting. Intracellular Ca2+ transient by TG was not affected in the presence or absence of extracellular Ca2+, indicating that TG must be effective on cytosolic Ca2+ pool. In addition, chelation of intracellular Ca2+ by acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracellular Ca2+ chelating agent, blocked TG- or TG + PMA-induced NO production. PKC inhibitors such as staurosporine or polymyxin B reduced only the synergistic cooperative effect of TG with phorbol ester without affecting TG-induced NO production. In addition, when the cells were pretreated with phorbol ester before TG treatment, there was no synergy between TG and phorbol ester, indicating that PKC is not directly involved in the expression of NOS but involved in "triggering" signal. Secretion of NO corresponded with tumor cell killing, but TG plus phorbol ester-activated macrophages failed to kill tumor cell targets in the presence of Ng-monomethyl-L-arginine. Collectively, these data illustrate that mobilization of intracellular Ca2+ provides a "priming" signal for induction of NOS gene expression by itself and it also requires PKC as a "triggering" signal for macrophage tumoricidal activity.

Synergistic cooperation between water-soluble chitosan oligomers and interferon-gamma for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages.

The effects of water-soluble chitosan oligomers (WSCO) on the synthesis of nitric oxide (NO) by murine peritoneal macrophages and on macrophage-mediated cytotoxicity towards murine fibrosarcoma Meth A cells were investigated. WSCO alone had no effect on NO synthesis and killing of tumor cells. However, treatment of macrophages with a combination of WSCO and interferon-gamma (IFN-gamma) synergically increased NO synthesis and enhanced killing of tumor cells. The synergism between IFN-gamma and WSCO in NO synthesis and tumoricidal activity was mainly dependent on increased secretion of tumor necrosis factor-alpha by WSCO.

Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer.

Water-soluble chitosan oligomer (WSCO) has been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we have shown that WSCO is able to inhibit proliferation of human leukemia HL-60 cells and induce these cells to differentiate. Treatment with WSCO for 4 days resulted in a concentration-dependent reduction in HL-60 cell growth as measured by cell counting and MTT assay. This effect was accompanied by a marked increase in the proportion of G(0)/G(1) cells as measured by flow cytometry. WSCO also induced differentiation of the cells as measured by phorbol ester-dependent reduction of NBT, morphological changes as examined by Wright-Giemsa staining and expression of CD11b but not of CD14 as analysed by flow cytometry, indicating differentiation of HL-60 cells toward granulocyte-like cells. A combination of low dose of WSCO with all-trans retinoic acid, a differentiating agent toward granulocyte-like cells, exhibited a synergistic effect on the differentiation. In addition, treatment of HL-60 cells with WSCO for 6 or 8 days resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis and quantitatively by Annexin V technique using flow cytometry. Collectively, there is a potential for WSCO in the treatment of myeloid leukemia.

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.

Effect of qigong training on proportions of T lymphocyte subsets in human peripheral blood.

The effect of Qigong training on proportions of T lymphocyte subsets was investigated in human peripheral blood. We observed that the ratio of CD4+/CD8+ T lymphocytes was increased as much as 50% in a trainee group who practiced Qigong training more than 5 months compared to a normal healthy group who did not practice. The absolute number of CD4+ T lymphocytes was also elevated in trainee group with 100 cells/mm3 more than in normal healthy group. The positive correlation between the ratio of CD4+/CD8+ T lymphocytes and the ratio of CD4+45RA-/CD4+CD45RA+ T lymphocytes was shown in the trainee group. In contrast, there was a negative correlation between the ratio of CD4+/CD8+ T lymphocytes and the ratio of CD8+CD57+/CD8+CD57- T lymphocytes in the trainee group. The data indicate that Qigong training affects the profile of lymphocyte subsets in human peripheral blood, especially the proportion of CD4+ T lymphocytes.

Delayed cutaneous hypersensitivity reactions in Qigong (chun do sun bup) trainees by multitest cell mediated immunity.

To determine the difference of cellular immunity between a Qigong trainee group and a normal healthy group, skin tests for delayed cutaneous hypersensitivity (DCH) were carried out with ubiquitous seven antigens. The maximal antigen response time was faster in Qigong trainee group (24 hr) and the response antigen number was also higher in the Qigong trainee group (6 antigens) than in normal healthy person (48 hr and 4 antigens). Qigong trainee also had a larger induration diameter (5.14 mm) than normal healthy person (3.79 mm) at 24 hr. Our results represent the difference in cell mediated immunity (CMI) between Qigong trainees and normal healthy subjects.

DA-9601 inhibits activation of the human mast cell line HMC-1 through inhibition of NF-kappaB.

Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 with immune regulatory properties. The formulated ethanol extract of Artemisia asiatica Nakai (DA-9601) has been reported to have antioxidative and anti-inflammatory activities. In this report, we investigated the effect of DA-9601 on the expression of pro-inflammatory cytokines by the activated human mast cell line HMC-1 and studied its possible mechanisms of action. DA-9601 dose-dependently decreased the gene expression and production of TNF-alpha, IL-1beta, and IL-6 on phorbol 12-myristate 13-acetate (PMA)- and calcium ionophore A23187-stimulated HMC-1 cells. In addition, DA-9601 attenuated PMA- and A23187-induced activation of NF-kappaB as indicated by inhibition of degradation of IkappaBalpha, nuclear translocation of NF-kappaB, NF-kappaB/DNA binding, and NF-kappaB-dependent gene reporter assay. Our in vitro studies provide evidence that DA-9601 might contribute to the treatment of mast cell-derived allergic inflammatory diseases.

Dual effect of nitric oxide in immortalized and malignant human oral keratinocytes: induction of apoptosis and differentiation.

BACKGROUND: Nitric oxide (NO) is known to act cytostatically on several tumor cell when functioning as an effector molecule of activated macrophages, but the differential effects of NO on immortalized and malignant oral keratinocytes have not been examined. METHODS: We investigated the influence of NO on the proliferation, cell cycle, apoptosis, and differentiation of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, sulforhodamine B (SRB) assay, flow cytometry, nuclear DNA staining, and Western blotting. RESULTS: The MTT and SRB assays indicated inhibited growth of IHOK and HN4 cells that were treated with sodium nitroprusside (SNP) at concentrations higher than 1 mM but not at lower SNP concentrations. The higher concentrations of SNP up-regulated the apoptosis-related protein expression, which is consistent with the analyses of sub-G(1) phase arrest, annexin V-FITC (fluorescein isothiocynate) staining, nuclear staining, and DNA fragmentation. On the other hand, the lower concentrations of SNP enhanced the expression of keratinocyte differentiation markers in IHOK and HN4 cells. CONCLUSIONS: These data suggest that high concentrations of NO can inhibit the growth of IHOK and HN4 cells through the induction of apoptosis, while low concentrations of NO can induce cytodifferentiation. The dual effects of NO, namely, the induction of apoptosis or cytodifferentiation, have important implications for the possible anti-oral cancer treatment.

Dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion.

Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single "fully competent" LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an alpha-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/DeltaD1-2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/DeltaD1-2 and with an approximately 2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/DeltaD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/DeltaD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, "fully competent" LFA-1-binding surface.

Nitric oxide induces ADP-ribosylation of actin in murine macrophages: association with the inhibition of pseudopodia formation, phagocytic activity, and adherence on a laminin substratum.

A recently recognized property of nitric oxide (NO), which would be expected to alter cell function, is the capacity to induce the ADP-ribosylation of various proteins. In these studies we demonstrate that actin present in murine macrophages is a substrate for NO-dependent ADP-ribosylation and that this modification is associated with the modification of cellular functions in murine peritoneal macrophages. A 42-kDa substrate for NO-dependent ADP-ribosylation was identified as actin by binding to DNAse-I and immunoprecipitation with anti-actin antibodies. The amount of actin ADP-ribosylation was correlated with the concentration of sodium nitroprusside (SNP), a NO generating agent, used in each experiment and with the amount of NO produced by activated macrophages. However, a specific inhibitor for NO synthase, N(G)-monomethyl-L-arginine (N(G)MMA), inhibited the ADP-ribosylation of actin by blocking the NO production in the interferon (IFN)-gamma plus lipopolysaccharide (LPS)-stimulated cells. Because the integrity of cytoskeletal protein is involved in shape change, adhesion, and phagocytosis of cells, we elucidated the role of NO-dependent ADP-ribosylation of actin in murine macrophages. A morphology kinetics assay comparing pseudopodial extension values over a 72-hr period showed that IFN-gamma plus LPS-treated macrophages underwent a wave of morphological changes, returning to a round shape after 32 hr. However, incubation of the cells with IFN-gamma plus LPS in the presence of N(G)MMA resulted in spindle-shaped pseudopodia formation and an altered composition of F-actin in macrophages. Adding either SNP or botulinum C2 toxin also inhibited IFN-gamma plus LPS-induced pseudopodia formation even in the presence of N(G)MMA. Flow cytometry revealed that NO inhibits the phagocytosis of fluorescent particles in a reversible manner. Preincubation of the cells with SNP (2 mM) also diminished LPS- or phorbol 12-myristate 13-acetate-induced macrophage adhesion on a laminin substratum. Collectively, in addition to its better-characterized role as a cytolytic mediator, the data illustrate that NO shows negative regulatory roles in cytoskeletal assembly, pseudopodia formation, phagocytosis, and adherence of murine macrophages in association with the ADP-ribosylation of actin.

Involvement of protein kinase C during taxol-induced activation of murine peritoneal macrophages.

Taxol has been known to block cell division by stabilizing microtubules with promising anticancer activity. However, taxol has distinct cell cycle-independent effects. Recently, this novel drug has been shown to provide a second signal for murine macrophage activation to tumoricidal activity via L-arginine-dependent nitric oxide (NO) synthesis. To investigate the mechanism of taxol-induced NO synthesis, we evaluated the ability of protein kinase C (PKC) inhibitors such as staurosporine (STSN) or polymyxin B to block taxol-induced effects. Taxol alone had only a small effect, whereas taxol in combination with rIFN-gamma markedly increased NO synthesis in a dose-dependent manner. STSN and polymyxin B decreased NO synthesis, which had been induced by rIFN-gamma plus taxol. Furthermore, prolonged incubation of the cells with phorbol ester, which down-regulates PKC activity, abolished synergistic cooperative effect of taxol with rIFN-gamma on NO synthesis. Synergy between IFN-gamma and taxol was mainly dependent on taxol-induced TNF-alpha secretion because not only the increase of inducible NO synthase (iNOS) gene expression by rIFN-gamma plus taxol was associated with the increased expression of TNF-alpha gene but also taxol-induced NO production was decreased by the treatment of anti-murine TNF-alpha neutralizing Abs. STSN and polymyxin B potently inhibited taxol-induced TNF-alpha secretion and TNF-alpha gene expression as well as iNOS gene expression by rIFN-gamma plus taxol. However, rIFN-gamma plus TNF-alpha-induced NO synthesis was not blocked by STSN or polymyxin B. This result indicates that TNF-alpha-induced signaling for induction of NO synthesis is not dependent on PKC activation, and further suggests that the point at which TNF-alpha acts on the NO synthesis from rIFN-gamma-primed macrophages lies next to the point of PKC activation. In conclusion, the present results strongly suggest that the capacity of taxol to increase NO synthesis from rIFN-gamma-primed macrophages is the result of taxol-induced TNF-alpha secretion via the signal transduction pathway of PKC activation.

Fibronectin activates murine peritoneal macrophages for tumor cell destruction in the presence of IFN-gamma.

Increasing evidence indicates that interaction of cells with fibronectin (Fn) affects many aspects of cellular responses including growth, morphology, differentiation, and activation. However, it is not known whether Fn could activate macrophages for the tumor cell killing. Here we report that Fn provides a signal for murine macrophage activation to tumoricidal activity. Tumoricidal activity was determined by the release of 51Cr from prelabeled P815 mastocytoma target cells. Fn alone had no effect, whereas recombinant interferon-gamma (rIFN-gamma) weakly induced C57BL/6 murine macrophages to kill P815 mastocytoma cells. However, combination of Fn with rIFN-gamma synergized to activate macrophages to lyse tumor cells in a dose dependent manner. Secretion of nitric oxide (NO) correlated with tumor cell killing, and the activated macrophages failed to kill tumor cell targets in the presence of NG-monomethyl-L-arginine (NGMMA), a competitive inhibitor of NO synthase (NOS). Fn, unlike lipopolysaccharide (LPS), alone had no effect on NO synthesis by itself and did not induce bioactive tumor necrosis factor-alpha (TNF-alpha) secretion from murine peritoneal macrophages. The data illustrate the potential for Fn to activate macrophage-mediated antitumor mechanisms in addition to its better characterized role as a cell adhesion molecule.

Nitric oxide mediates the toxoplasmastatic activity of murine microglial cells in vitro.

Toxoplasma gondii (T. gondii), an opportunistic protozoan, is an important cause of central nervous system (CNS) infections in immunosuppressed patients. The present study focused on the interaction between T. gondii and microglial cells from the brain of neonatal Balb/c mice. Preincubation of the murine microglial cells with recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) induced significant inhibition of T. gondii replication in a dose dependent manner. This antiparasitic effect in microglial cells was correlated with the induction of the L-arginine-dependent generation of reactive nitrogen intermediates (RNIs). Tumor necrosis factor-alpha (TNF-alpha) was also involved in the toxoplasmastatic activity. Microglial cells incubated with recombinant TNF-alpha in combination with a non-activating concentration of rIFN-gamma released substantial amount of RNIs. Neutralizing antibodies against mouse TNF-alpha inhibited the release of RNI by rIFN-gamma activated macrophages. In summary, the present results show that activation of microglial cells by rIFN-gamma and LPS induce the production of nitric oxide (NO) by these cells via an L-arginine dependent pathway. NO appears to be the effector molecule mediating the toxoplasmastatic effects in these cells.

Phorbol ester synergistically increases interferon-gamma-induced nitric oxide synthesis in murine microglial cells.

The effect of phorbol ester on the synthesis of nitric oxide (NO) in murine microglial cells was examined. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, alone had no effect, whereas PMA with recombinant interferon (rIFN)-gamma synergistically increased NO synthesis in murine microglial cells. The maximal effect of PMA on NO synthesis increase always fitted with the range for full activation of PKC in these cells. The increase of NO synthesis was reflected as increased amount of immunologic NO synthase (iNOS) mRNA detected by Northern blotting. Treatment with PKC inhibitors such as staurosporine or polymyxin B decreased rIFN-gamma-plus-PMA-stimulated NO synthesis. Further, prolonged incubation of the cells with PMA, which down-regulates PKC activity, abolished the synergistic cooperative effect with IFN-gamma. NG-monomethyl-L-arginine monohydrate, an analogue of L-arginine, and arginase inhibited rIFN-gamma-plus-PMA-induced NO production in murine microglial cells. On the basis of these observations, we conclude that PKC might not be involved in the expression of iNOS, but instead, might be involved in the posttranscriptional modification of iNOS mRNA.

Nitric oxide inhibits the expression of protein kinase C delta gene in the murine peritoneal macrophages.

Since there is increasing evidence that protein kinase C (PKC) has a crucial role in the production of nitric oxide (NO) from activated macrophages, this study was undertaken to address whether NO could regulate the expression of the gene of this enzyme. Stimulation of the cells with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) after treatment with recombinant interferon-gamma (rIFN-gamma) resulted in the increased production of NO in the medium. rIFN-gamma in combination with either LPS or PMA showed marked inhibition of the expression of PKC delta gene, whereas rIFN-gamma alone showed modest inhibition. The inhibition of gene expression was correlated with the amount of NO produced by activated macrophages. The inhibitory effect of NO on the expression of PKC delta gene is mimicked by the treatment of NO generating agent, sodium nitroprusside (SNP). On the other hand, a specific inhibitor for NO synthase, NG-monomethyl-L-arginine (NGMMA), blocked the inhibition of the expression of PKC delta gene by blocking the NO production in the rIFN-gamma and LPS-stimulated cells. However, production of NO did not affect the expression of both TNF-alpha and TGF-beta gene which were induced by the stimulation of macrophages, as well as beta-actin gene, which was constitutively expressed in the macrophages. In conclusion, these findings show that NO has a regulatory role for the expression of the gene of PKC delta which is crucially involved in the process of NO synthesis.

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1).

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).