Nicotinamide Phosphoribosyltransferase Attenuates Methotrexate Response in Juvenile Idiopathic Arthritis and In Vitro.

Variability in response to methotrexate (MTX) in the treatment of juvenile idiopathic arthritis (JIA) remains unpredictable and poorly understood. Based on previous studies implicating an interaction between nicotinamide phosphoribosyltransferase (NAMPT) expression and MTX therapy in inflammatory arthritis, we hypothesized that increased NAMPT expression would be associated with reduced therapeutic response to MTX in patients with JIA. A significant association was found between increased plasma concentrations of NAMPT and reduced therapeutic response in patients with JIA treated with MTX. Inhibition of NAMPT in cell culture by either siRNA-based gene silencing or pharmacological inhibition with FK-866 was found to result in a fourfold increase in the pharmacological activity of MTX. Collectively, these findings provide evidence that NAMPT inhibits the pharmacological activity of MTX and may represent a predictive biomarker of response, as well as a therapeutic target, in the treatment of JIA with MTX.

Asthma-related SNP in FCER2 is associated with increased expression of IL-4R on human B cells.

A SNP (rs2228137) (R62W) in FCER2 has been linked with severe exacerbations in asthmatics. Transfectants expressing the SNP exhibited increased IL-4Rα expression after stimulation through CD23 compared with wild-type. Our data suggest that the SNP may favour increased IgE production through increased responsiveness to IL-4 in patients possessing this genotype.

Training status influences T-cell responses in women following acute resistance exercise.

The purpose of this investigation was to examine white blood cell counts (WBC), immunoglobulin (IgA, IgG, IgM) levels, and T-cell proliferation following acute resistance training in 9 untrained (UT) and 6 trained (TR) women. Resistance training on 7 Universal machines at the subject's 10 repetition maximum (IORM) was performed at 89 +/- 5% for UT and 88 +/- 3% for TR. Blood was analyzed for WBCs and Ig levels pre-exercise, immediately postexercise, and 1.5, 3, and 24 hours postexercise. T-cell proliferation was determined pre-exercise and 3 hours postexercise through response to phytohemagglutanin (PHA). WBCs were significantly elevated in the UT subjects 1.5 and 3 hours postexercise compared with pre- and immediately postexercise; no differences (p < 0.05) were observed in TR subjects. No significant differences were found for Ig levels either between or within groups, although there was a trend for decreased IgG following exercise. T-cell proliferation was significantly decreased in the UT at 3 hours postexercise (0.27 +/- 0.06 units) compared with pre-exercise (0.41 +/- 0.06 units), whereas the proliferative response in TR was not significantly different from pre-exercise (0.48 +/- 0.04 units) to 3 hours postexercise (0.34 +/- 0.06 units). These data indicate that UT subjects experience an increase in WBC counts and a decrease in T-cell proliferative ability after acute resistance training, whereas TR subjects experience no significant change in these parameters.

MIP-1alpha induces activation of phosphatidylinositol-3 kinase that associates with Pyk-2 and is necessary for B-cell migration.

The chemokine macrophage inflammatory protein-1 alpha [MIP-1alpha] causes migration of B cells and also induces changes in antibody secretion. However, the signal transduction pathways leading to these phenotypic changes remain undefined. We have identified a signal transduction pathway initiated by MIP-1alpha in B cells. Here we report that stimulation of tonsil B cells with MIP-1alpha induces phosphatidylinositol 3-kinase [PI3-K] activation. Kinase activity was transient with peak induction occurring within 2.5 to 5 min after stimulation and was dose-dependent. In addition stimulation with MIP-1alpha induces tyrosine phosphorylation of the proline-rich tyrosine kinase Pyk-2. Immunoprecipitation analysis showed a constitutive association between Pyk-2 and PI3-K and pretreatment of MIP-1alpha-stimulated B cells with wortmannin, a specific inhibitor of PI3-K, resulted in a loss of PI3-K activity. The PI3-K inhibitor wortmannin prevented B cells from migrating in response to MIP-1alpha. Hence, PI3-K and Pyk-2 seem to be components of a signal transduction pathway induced by stimulation of B cells with MIP-1alpha, and this pathway may play a role in B-cell migration.

Minimal influence of carbohydrate ingestion on the immune response following acute resistance exercise.

The effect of carbohydrate supplementation (CHO) on the lymphocyte response to acute resistance exercise was examined in 10 resistance-trained males. Subjects completed a randomized double-blind protocol with sessions separated by 14 days. The exercise session consisted of a high intensity, short rest interval squat workout. Subjects consumed 1.0 g á kg body mass(-1) CHO or an equal volume of placebo (PLC) 10 min prior to and 10 min following exercise. Blood was collected at rest (REST), immediately post exercise (POST), and at 1.5 hours and 4.0 hours of recovery, and analyzed for plasma glucose, serum cortisol, leukocyte subsets, and phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. A significant Treatment 3 Time effect was observed for lymphocyte proliferation between CHO and PLC, but post hoc analyses revealed no between-treatment differences at any post-exercise time point. Lymphocyte proliferation was significantly depressed below REST at POST (-39.2% for PLC, -25.7% for CHO). Significant fluctuations in leukocyte subset trafficking were observed for both treatments at POST, 1.5 hours, and 4.0 hours. Plasma glucose was significantly increased POST in CHO compared to PLC. Cortisol was significantly increased from REST to POST in both treatments. These data support a minimal effect of carbohydrate ingestion on the lymphocyte response to high-intensity resistance exercise.

Linear and cyclic LFA-1 and ICAM-1 peptides inhibit T cell adhesion and function.

Short peptides derived from functional proteins have been used in several instances to inhibit activity of the parent proteins. In some cases, stability and efficacy were found to be increased by cyclization of these peptides. Inhibition of interaction of the two cell adhesion counter receptors leukocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1 is being studied as a method for modulating autoimmune diseases such as rheumatoid arthritis and for facilitating organ transplantation. Here, several 10-amino acid peptides derived from the contact domains of LFA-1 and ICAM-1 were evaluated for their ability to interfere with intercellular adhesion by T cells and to inhibit a more biologic, mixed lymphocyte reaction. Both linear and cyclic forms of the peptides were effective at inhibiting intercellular adhesion. Cyclic forms were effective at inhibiting T cell activation and proliferation in the mixed lymphocyte reaction.

Induction of PYK-2 phosphorylation during LFA-1/ICAM-1-dependent homotypic adhesion of fresh human B-cells.

Stimulation with the combination of PDB plus ionomycin induced LFA-1/ICAM-1-dependent homotypic adhesion of tonsil B cells. Adhesion of tonsil B cells in our system induced tyrosine phosphorylation of Pyk-2. Disruption of homotypic adhesion and concomitant inhibition of induction of protein tyrosine phosphorylation was achieved by physical separation of the cells and by treatment with methyl-2.5-dihydroxycinnamate (MDHC), an inhibitor of protein tyrosine phosphorylation. These results suggest that protein tyrosine phosphorylation that is associated with homotypic adhesion is mediated by LFA-1/ICAM-1 interactions.

MIP-1alpha induces binding of nuclear factors to the kappaB DNA element in human B cells.

The chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) stimulates migration of B cells through an unknown mechanism. Furthermore, little is known about signal transduction mechanisms through which MIP-1alpha might signal phenotypic changes in B cells. We are investigating the role of MIP-1alpha in B cell signaling. Here we report that stimulation of the Ramos B cell line or tonsil B or peripheral blood-B (PBL-B) cells with MIP-1alpha caused the transcription factor NF-kappaB to bind to DNA. NF-kappaB induction was dose dependent, and was transient, with peak induction occurring at 30 min. MIP-1alpha treatment stimulated the degradation of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. The biological significance of NF-kappaB activation by MIP-1alpha is currently unknown, but it is known that NF-kappaB modulates expression of genes involved in many inflammatory and immune responses. Here we show that NF-kappaB activation is a target of signals sent into B cells by MIP-1alpha.

Peptides derived from ICAM-1 and LFA-1 modulate T cell adhesion and immune function in a mixed lymphocyte culture.

BACKGROUND: The counter receptors intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are lymphocyte cell surface adhesion proteins the interaction of which can provide signals for T cell activation. This binding event is important in T cell function, migration, and general immune system regulation. The ability to inhibit this interaction with monoclonal antibodies has proved to be therapeutically useful for several allograft rejection and autoimmune disease models. METHODS: Short peptides representing counter-receptor contact domains of LFA-1 and ICAM-1 were examined for their ability to inhibit T cell adhesion and T cell function. RESULTS: Peptides encompassing amino acids Q1-C21 and D26-K50 of ICAM-1, I237-I261 and G441-G466 of the LFA-1 alpha-subunit, and D134-Q159 of the LFA-1 beta-subunit inhibited LFA-1/ICAM-1-dependent adhesion in a phorbol-12,13-dibutyrate-induced model of tonsil T cell homotypic adhesion. This inhibition was specific to the peptide sequence and occurred without stimulation of T cell proliferation. The peptides also were effective in preventing T cell function using a one-way mixed lymphocyte reaction model for bone marrow transplantation. CONCLUSIONS: Our data suggest that these peptides or their derivatives may be useful as therapeutic modulators of LFA-1/ICAM-1 interaction during organ transplants.

Triptolide is more effective in preventing T cell proliferation and interferon-gamma production than is FK506.

The Chinese traditional herb Tripterygium wilfordii Hook. F (TWHF) has been reported to be effective in the treatment of a variety of autoimmune disorders. The major active component of this herb is triptolide and most of the efficacy of this herb immunosuppression is attributed to triptolide. FK506 is also a potent immunosuppressive agent and is currently being used clinically. The present studies compare the effectiveness of triptolide and FK506 to suppress certain human T cell functions. Specifically human T cell proliferation, IL-2 and IFNgamma were compared. The results show that, overall, triptolide is more effective at inhibiting T cell proliferation and IFNgamma production than FK506 and the two compounds inhibit IL-2 production in an equivalent manner.

Sequences at the 3' side of the c-fos SRE mediate gene expression via an Sob1-dependent, TCF-independent pathway.

We previously described a 110-kDa tyrosine phosphoprotein, Sob 1, that regulates formation of the DNA binding complex Band A at the c-fos serum response element (SRE) during T cell activation. Using competition and mutant oligonucleotide analysis, we have determined that both the core CArG box of the c-fos SRE and the 3' sequences flanking the CArG box are necessary for stable Band A complex formation. Moreover, using transient transfection and reporter assays, we show that mutations affecting Band A complex formation in vitro also impaired serum induction of c-fos gene expression in vivo. Since mutation at this site has no effect on SRF binding, our results suggest that in combination with SRE/SRF, Sob 1-regulated factor(s) bind at the 3' side of SRE to form Band A, and this confers maximal serum induction of c-fos gene expression via the SRE.

A Ca2+ binding cyclic peptide derived from the alpha-subunit of LFA-1: inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion.

The objective of this work is to study the conformation of cyclic peptide (1), cyclo (1, 12) Pen1-Gly2-Val3-Asp4-Val5-Asp6-Gln7-+ ++Asp8-Gly9-Glu10-Thr11-Cys12, in the presence and absence of calcium. Cyclic peptide 1 is derived from the divalent cation binding sequence of the alpha-subunit of LFA-1. This peptide has been shown to inhibit ICAM-1-LFA-1 mediated T-cell adhesion. In order to understand the structural requirements for this biologically active peptide, its solution structure was studied by nuclear magnetic resonance (NMR), circular dichroism (CD) and molecular dynamics simulations. This cyclic peptide exhibits two types of possible conformations in solution. Structure I is a loop-turn-loop type of structure, which is suitable to bind cations such as EF hand proteins. Structure II is a more extended structure with beta-hairpin bend at Asp4-Val5-Asp6-Gln7. There is evidence that alterations in the conformation of LFA-1 upon binding to divalent cations cause LFA-1 to bind to ICAM-1. To understand this mechanism, the cation-binding properties of the peptide were studied by CD and NMR. CD studies indicated that the peptide binds to calcium and forms a 1 : 1 (peptide: calcium) complex at low calcium concentrations and multiple types of complexes at higher cation concentrations. NMR studies indicated that the conformation of the peptide is not significantly altered upon binding to calcium. The peptide can inhibit T-cell adhesion by directly binding to ICAM-1 or by disrupting the interaction of the alpha and beta-subunits of LFA-1 protein. This study will help us to understand the mechanism(s) of action of this peptide and will improve our ability to design a better inhibitor of T-cell adhesion.

Differential display screening for specific gene expression induced by dietary nonsteroidal estrogen.

The dietary phytoestrogen, daidzein, produced a biphasic response in cell proliferation of cultured, estrogen-responsive human breast carcinoma MCF-7 cells. Cell growth was stimulated at a daidzein concentration of 0.25 microg/ml whereas the addition of daidzein at concentrations >25 microg/ml significantly inhibited cell growth in a dose-dependent fashion, resulting in an IC50 value of 50 microg/ml. Upon exposure to 50 microg/ml of daidzein, cell morphology was severely altered, cell volume decreased, and condensation of the chromosomes was clearly noticeable. To identify genes whose expression were inhibited by daidzein, a differential display reverse transcriptase polymerase chain reaction assay (DD-RT-PCR) was performed and the cDNA fragments of several daidzein-regulated genes were visualized. The sequence of one of the cloned cDNA fragments that showed differential mRNA expression level in response to daidzein at a concentration of 50 microg/ml had a high homology with a cDNA expressed in fetal human brain, EST 06411.

Inhibition of homotypic adhesion of T-cells: secondary structure of an ICAM-1-derived cyclic peptide.

The objective of this study was to elucidate the solution conformation of cyclo-(1,12) Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Ar g9-Gly10-Gly11-Cys12 (1) derived from the intercellular adhesion molecule-1 (ICAM-1). Cyclic peptide 1 inhibits homotypic adhesion of T-cells (Molt-3) mediated by ICAM-1 and the leukocyte function-associated antigen-1 (LFA-1) on the surface of T-cells. Cyclic peptide 1 is more potent than is the linear peptide Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Ar g9-Gly10-Gly11-Cys12 (2) in inhibiting homotypic adhesion. The difference in biological activity of peptides 1 and 2 may be due to the more stable conformation of cyclic peptide 1 compared to linear peptide 2 or because cyclization prevents the peptide from adopting non-productive conformation. Therefore, conformational studies of cyclic peptide 1 will give a better understanding of its biological active conformation. The conformational studies of cyclic peptide 1 were done by NMR, CD and molecular dynamics simulations. NMR studies indicated that the major conformation of cyclic peptide 1 contained trans-configuration at both X-Pro peptide bonds. Type I beta-turns at Lys4-Val5-Ile6-Leu7 and Leu7-Pro8-Arg9-Gly10 were found in cyclic peptide 1. The C- and N-terminal regions of this peptide were stabilized by antiparallel beta-sheet-like structure with the presence of intramolecular hydrogen bonds. The overall structure of this peptide exposed the hydrophobic side chains on one face of the molecule and the hydrophilic side chains on the other.

Cross-linking of ICAM-1 on T cells induces transient tyrosine phosphorylation and inactivation of cdc2 kinase.

Intercellular adhesion molecules (ICAM)-1 and -3 coexist on T lymphocytes and are counter-receptors for the integrin LFA-1. Signaling through ICAM-3 stimulates a number of T cell functions and involves phosphorylation of Fyn, Lck, CD45, and other proteins. In contrast, this type of specific signaling event has not been described for signaling through ICAM-1. Here, tyrosine phosphorylation of cellular proteins was examined after cross-linking of ICAM-1. Tyrosine phosphorylation of the 34-kDa cdc2 protein kinase was induced transiently after stimulation of the leukemic T cell line, Molt-3, or peripheral blood T cells. Stimulation through ICAM-1 had no effect on constitutive presence of cdc2 or phosphorylation of cdc2 on threonine. cdc2 kinase activity was constitutive in peripheral blood T cells, and transient inhibition of kinase activity after ICAM-1 stimulation correlated kinetically with phosphorylation of cdc2 on tyrosine.

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.

Regulation of the B cell immune response: induction of competence and progression in a surface IgM-bearing subset.

We have targeted the specific surface IgM-bearing subset of human B lymphocytes within a mixed population and investigated cell cycle activation in these cells stimulated with phorbol ester and antibody directed against surface immunoglobulin. Stimulation with phorbol 12,13-dibutyrate (PDB) in combination with anti-IgM led to induction of the state of competence followed by progression to proliferation of the surface IgM-bearing cells. In contrast, sequential stimulation with PDB and anti-IgM in either order did not induce either competence or progression. Brief exposure to both stimuli for 30 min did not induce significant proliferation, but did induce the state of competence such that the cells progressed to DNA synthesis after incubation with PDB alone. Furthermore, a competence-related gene, c-fos, was induced in cells that did not become fully competent. Thus, induction of competence to proliferate required simultaneous delivery of two signals whereas actual progression to proliferation by competent cells was accomplished with only one signal.

Increased cyclic adenosine monophosphate levels block progression but not initiation of human T cell proliferation.

The involvement of cyclic nucleotides in the regulation of cell growth is well established as are the findings that sustained high levels of intracellular cyclic adenosine monophosphate (cAMP) inhibit ligand-induced lymphocyte proliferation. However, there is considerable controversy concerning the precise role of cAMP in this regard or the target of its action. A number of studies with cAMP analogs or forskolin have implicated the early events after ligand binding as being particularly susceptible whereas others have failed to do so or offered alternate targets. We have examined the effects of cell permeant analogs of cAMP and forskolin on human T cell proliferation. After brief exposure to submitogenic concentrations of PHA or phorbol ester plus ionomycin, we have examined the effects of increased cAMP levels during initial activation (or induction of competence) and progression to DNA synthesis. We find that these drugs that inhibit T cell proliferation have little effect on induction of competence or early activation events including transmembrane Ca2+ uptake, release of Ca2+ from internal stores or induction of the genes encoding c-fos or early growth response gene. In contrast, it is the progression phase of the proliferative response which appears to be the major target for cAMP-protein kinase A-mediated inhibition of human T cell proliferation.

Expansion of B lymphocytes with an unusual immunoglobulin rearrangement associated with atypical lymphocytosis and cigarette smoking.

Persistent polyclonal lymphocytosis has been described in a group of female patients who all have the HLA-DR7 antigen in common and who are all heavy cigarette smokers. Immunoglobulin heavy chain gene rearrangement was analyzed by hybridization with specific immunoglobulin heavy chain genes to restriction enzyme-digested genomic DNA samples. The results in two of these patients showed that the lymphocytosis was associated with an expanded subpopulation of B-lineage cells represented by the presence of an unusual immunoglobulin gene rearrangement pattern. Expansion of this subpopulation of B cells appeared to be linked to cigarette smoking since the intensity of the cell population harboring the rearranged gene was much stronger in patients who were smoking heavily compared with the same patients who were temporarily not smoking.

Expression of IgE from a nonrearranged epsilon locus in cloned B-lymphoblastoid cells that also express IgM.

During development, B lymphocytes have the ability to switch from synthesis of IgM to immunoglobulins of another isotype such as IgG, IgA, or IgE. This class switching mechanism has been shown to involve DNA rearrangement and concomitant deletion of intervening CH genes. In our report, an EBV-transformed B lymphoblastoid cloned cell line is described that simultaneously expressed and secreted both IgM and IgE. DNA analysis showed the (nonproductive) rearrangement of one allele to gamma and (productive) rearrangement of the other allele to mu. Germ-line arrangement of the C epsilon gene was preserved on both alleles.