Two Inhibitors of DNA‐Synthesis Lead to Inhibition of Cytokine Production via a Different Mechanism

Methotrexate (MTX) and mycophenolic acid (MPA) are used in the clinic for their immunosuppressive properties. MTX is widely used for the treatment of rheumatoid arthritis (RA). MPA is used to prevent graft rejection and is now experimentally used in systemic lupus erythematosis and RA. It is known that both drugs interfere with DNA synthesis. However, the precise mechanism of action is still debated. We have analysed the effect of the drugs on cytokine production in whole blood during short cultures. The production of T‐cell cytokines was inhibited by both drugs. MTX inhibits cytokine production because MTX induces apoptosis in activated T‐cells. MPA inhibits cytokine production by preventing T‐cells to progress to the S‐phase of the cell cycle. Cytokine production by monocytes was slightly decreased by the drugs. The reason for this inhibition is not clear. These results indicate that T‐cells are the main target cells of the immunosuppressive drugs MPA and MTX.     

Mycophenolic acid and methotrexate inhibit lymphocyte cytokine production via different mechanisms

Mycophenolic acid (MPA) and methotrexate (MTX) are immunosuppressive drugs used for the treatment of various immunological disorders. MPA is an inhibitor of inosine monophosphate dehydrogenase and MTX is a folate antagonist that inhibits tetrahydrofolate reductase. Production of T cell cytokines in whole blood cultures, as well as in PBMC cultures, is inhibited by a low concentration of both drugs. Inhibition of cytokine production after monocyte stimulation was less evident. The mechanism by which inhibition is achieved is different for both drugs. Inhibition of T cell cytokine production by MPA was more profound and started earlier compared to the inhibition by MTX. MTX induced apoptosis in T cells that became activated, whereas MPA prevented activation of T cells by arresting the cell cycle in the G0/G1 phase. Addition of guanosine and adenosine can overcome this cell cycle arrest, even after several days. Furthermore MPA inhibited the expression of activation markers HLA-DR and CD71 on T cells. The observation that MTX cannot prevent T cell activation but induces apoptosis in activated T cells, and that MPA reversibly prevents activation of T cells could explain the immunosuppressive effects of both these drugs.     

Effect of methotrexate and anti-TNF on Epstein-Barr virus T-cell response and viral load in patients with rheumatoid arthritis or spondylarthropathies

Introduction

There is a suspicion of increased risk of Epstein-Barr virus (EBV)-associated lymphoproliferations in patients with inflammatory arthritides receiving immunosuppressive drugs. We investigated the EBV load and EBV-specific T-cell response in patients treated with methotrexate (MTX) or anti-TNF therapy.

Methods

Data for patients with rheumatoid arthritis (RA) (n = 58) or spondylarthropathy (SpA) (n = 28) were analyzed at baseline in comparison with controls (n = 22) and after 3 months of MTX or anti-TNF therapy for EBV load and EBV-specific IFNγ-producing T cells in response to EBV latent-cycle and lytic-cycle peptides.

Results

The EBV load and the number of IFNγ-producing T-cells after peptide stimulation were not significantly different between groups at baseline (P = 0.61 and P = 0.89, respectively). The EBV load was not significantly modified by treatment, for RA with MTX (P = 0.74) or anti-TNF therapy (P = 0.94) or for SpA with anti-TNF therapy (P = 1.00). The number of EBV-specific T cells was not significantly modified by treatment, for RA with MTX (P = 0.58) or anti-TNF drugs (P = 0.19) or for SpA with anti-TNF therapy (P = 0.39). For all patients, the EBV load and EBV-specific T cells were significantly correlated (P = 0.017; R = 0.21). For most patients, short-term exposure (3 months) to MTX or anti-TNF did not alter the EBV load or EBV-specific T-cell response but two patients had discordant evolution.

Conclusions

These data are reassuring and suggest there is no short-term defect in EBV-immune surveillance in patients receiving MTX or anti-TNF drugs. However, in these patients, long term follow-up of EBV-specific T-cell response is necessary and the role of non-EBV-related mechanisms of lymphomagenesis is not excluded.     

甲氨蝶呤对大鼠尿液蛋白质组的影响

甲氨蝶呤是常见的免疫抑制剂,曾被大剂量用于治疗癌症,近年来其被小剂量用于治疗类风湿性关节炎。文中尝试研究甲氨蝶呤对大鼠尿蛋白质组的影响。大鼠口服甲氨蝶呤构造用药模型,再收集大鼠10 h内的尿液,并且采用液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)分析大鼠的尿蛋白。总共鉴定到31个差异蛋白,其中7个蛋白与甲氨蝶呤药物作用和类风湿性关节炎症状有关。部分大鼠的生物学过程反映了甲氨蝶呤对机体谷胱甘肽代谢以及对JAK/STAT信号通路的影响。结果显示,尿蛋白具有反映甲氨蝶呤对大鼠机体影响的能力。对单个大鼠个体差异蛋白的分析体现出不同个体对该药物的反应有较大差异。     

4-Hydroxy-oxyphenbutazone is a potent inhibitor of cytokine production

4-Hydroxy-oxyphenbutazone (4OH-OPB), is currently in phase II trials for its immunosuppressive effect in patients with rheumatoid arthritis. 4OH-OPB and other compounds related to phenylbutazone were tested for their effect on in vitro cytokine production by monocytes and lymphocytes present in peripheral mononuclear cells (PBMC) or whole blood (WB) cultures, and compared against phenylbutazone and oxyphenbutazone, two known anti-inflammatory drugs. In PBMC cultures, 4OH-OPB was by far the most potent inhibitor, and both monokines and Th1 and Th2 lymphokines were efficiently inhibited at low concentrations. In WB cultures, 4OH-OPB was less effective than in PBMC cultures, but was still the best inhibitor of lymphokine production and, furthermore, was the only inhibitor of monokine production. The increase in 4OH-OPB concentration needed to induce the same inhibition of cytokine production in WB as in PBMC culture could be mimicked by the addition of erythrocytes to the PBMC cultures. Experiments with radioactively-labeled 4OH-OPB suggest that 4OH-OPB is taken up very rapidly into erythrocytes and is secreted by the erythrocytes with much slower kinetics via a multidrug-resistance-associated protein. The secreted compound is most likely structurally different from 4OH-OPB, as in PBMC and WB cultures, the inhibition of cytokine production seems to be caused by a different mechanism. In PBMC cultures, the inhibition of cytokine production is accompanied by a loss of cell viability, while this is not the case when 4OH-OPB inhibits cytokine production in WB. Our data suggest that 4OH-OPB may be useful as an immunosuppressive drug for patients with inflammatory diseases.     

Free radicals and redox signalling in T-cells during chronic inflammation and ageing

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Immunomodulatory effects of BXL-01-0029, a less hypercalcemic vitamin D analogue, in human cardiomyocytes and T cells

Current immunosuppressive protocols have reduced rejection occurrence in heart transplantation; nevertheless, management of heart transplant recipients is accompanied by major adverse effects, due to drug doses close to toxic range. In allograft rejection, characterized by T-helper 1 (Th1) cell-mediated response, the CXCL10-CXCR3 axis plays a pivotal role in triggering a self-promoting inflammatory loop. Indeed, CXCL10 intragraft production, required for initiation and development of graft failure, supports organ infiltration by Th1 cells. Thus, targeting the CXCL10-CXCR3 axis while avoiding generalized immunosuppression, may be of therapeutic significance. Based on preclinical evidence for immunoregulatory properties of vitamin D receptor agonists, we propose that a less hypercalcemic vitamin D analogue, BXL-01-0029, might have the potential to contribute to rejection management. We investigated the effect of BXL-01-0029 on CXCL10 secretion induced by proinflammatory stimuli, both in human isolated cardiomyocytes (Hfcm) and purified CD4+ T cells. Mycophenolic acid (MPA), the active agent of mycophenolate mofetil, was used for comparison. BXL-01-0029 inhibited IFNγ and TNFα-induced CXCL10 secretion by Hfcm more potently than MPA, impairing cytokine synergy and pathways. BXL-01-0029 reduced also CXCL10 protein secretion and gene expression by CD4+ T cells. Furthermore, BXL-01-0029 did not exert any toxic effect onto both cell types, suggesting its possible use as a dose-reducing agent for conventional immunosuppressive drugs in clinical transplantation.     

The induction of apoptosis by methotrexate in activated lymphocytes as indicated by fluorescence hyperpolarization: a possible model for predicting methotrexate therapy for rheumatoid arthritis patients

The objectives of this study were to test the in vitro response of healthy non-activated, activated, and rheumatoid arthritis (RA) lymphocytes to methotrexate (MTX), and design an in vitro model for predicting the efficiency of MTX treatment for RA patients. Considering the RA profile of clonal-expanded CD4(+) T cells, phytohemagglutinin-activated mononuclear cells taken from healthy donors were incubated with different concentrations of MTX. The MTX-immunosuppressive effect was tested by fluorescence intensity measurements, including PI assay and annexin V assay. For simple detection, we used the Individual Cell Scanner (IC-S), which enables the measurement of early events in individual cells. Healthy mononuclear cells (MNC), and MNC derived from RA patients, were tested by the IC-S while utilizing fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) as an established marker of activation or suppression. In healthy activated MNC, we found that MTX, through its early incubation period, interferes with the activation signal obtained by PHA and exerts an apoptotic signal, which is noted by increases in the FP. Comparing our model to six long-standing RA patients and five newly-diagnosed patients revealed significant differences in the FP measurements, including fluorescence depolarization as an early established measurement of lymphocyte activation, and hyperpolarization as a measurement of an early immunosuppressive effect. We conclude that MTX, an effective therapy for RA patients, could easily be tested by fluorescence polarization measurements of FDA before (or during) clinical use in order to predict its efficiency on a specific RA patient. Moreover, the FP measurements can be used for the diagnosis, and making timing and dosage decisions.     

7'-(3',4'-dihydroxyphenyl)-N-[(4-methoxyphenyl)ethyl]propenamide (Z23), an effective compound from the Chinese herb medicine Fissistigma oldhamii (Hemsl.) Merr, suppresses T cell-mediated immunity in vitro and in vivo

Fissistigma oldhamii (Hemsl.) Merr [F. oldhamii], a traditional Chinese herb medicine, is widely used for treating rheumatoid arthritis (RA) in China. Following bioactivity-guided isolation, a representative immunosuppressive compound with low cytotoxicity, 7'-(3',4'-dihydroxyphenyl)-N-[(4-methoxyphenyl)ethyl]propenamide (Z23), was been identified in this herb medicine. We investigated the immunosuppressive effects of Z23 on T cells in vitro and in vivo. The results showed that Z23 in a dose-dependent manner significantly inhibited the proliferation of splenocytes induced by concanavalin A (ConA) and by the mixed lymphocyte culture reaction (MLR), with half inhibitive concentration (IC(50)) values of 6.22 microM and 0.78 microM, respectively. Z23 also dose-dependently inhibited the proliferation and type 1 cytokine (IFN-gamma and IL-2) production of primary T cells stimulated by anti-CD3/CD28 mAbs, but did not affect IL-12 production by mouse peritoneal macrophages (pMphi) stimulated with LPS plus IFN-gamma in vitro. Administration of Z23 (6.25 mg/kg, 12.5 mg/kg, 25 mg/kg, i.p.) dose-dependently suppressed 2,4-dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) reactions. Furthermore, administration of Z23 (25 mg/kg, i.p.) significantly reduced the incidence and severity of type II bovine collagen (CII)-induced arthritis (CIA), which was associated with the inhibition of CII-specific T cell proliferation and type 1 cytokine (IFN-gamma and IL-2) production. In this study, we report that a representative immunosuppressive compound from F. oldhamii, Z23, effectively inhibits murine immune responses in vitro and in vivo, and that the immunosuppressive effects of Z23 might be attributed to suppression of T cell activation and function and Th1 type cytokine production.     

Disease-modifying antirheumatic drugs are associated with a reduced risk for cardiovascular disease in patients with rheumatoid arthritis: a case control study

Rheumatoid arthritis (RA) is characterized by inflammation and an increased risk for cardiovascular disease (CVD). This study investigates possible associations between CVD and the use of conventional disease-modifying antirheumatic drugs (DMARDs) in RA. Using a case control design, 613 RA patients (5,649 patient-years) were studied, 72 with CVD and 541 without CVD. Data on RA, CVD and drug treatment were evaluated from time of RA diagnosis up to the first cardiovascular event or the end of the follow-up period. The dataset was categorized according to DMARD use: sulfasalazine (SSZ), hydroxychloroquine (HCQ) or methotrexate (MTX). Odds ratios (ORs) for CVD, corrected for age, gender, smoking and RA duration, were calculated per DMARD group. Patients who never used SSZ, HCQ or MTX were used as a reference group. MTX treatment was associated with a significant CVD risk reduction, with ORs (95% CI): 'MTX only', 0.16 (0.04 to 0.66); 'MTX and SSZ ever', 0.20 (0.08 to 0.51); and 'MTX, SSZ and HCQ ever', 0.20 (0.08 to 0.54). The risk reductions remained significant after additional correction for the presence of rheumatoid factor and erosions. After correction for hypertension, diabetes and hypercholesterolemia, 'MTX or SSZ ever' and 'MTX, SSZ and HCQ ever' showed significant CVD risk reduction. Rheumatoid factor positivity and erosions both increased CVD risk, with ORs of 2.04 (1.02 to 4.07) and 2.36 (0.92 to 6.08), respectively. MTX and, to a lesser extent, SSZ were associated with significantly lower CVD risk compared to RA patients who never used SSZ, HCQ or MTX. We hypothesize that DMARD use, in particular MTX use, results in powerful suppression of inflammation, thereby reducing the development of atherosclerosis and subsequently clinically overt CVD.     

Fluorescent cell barcoding as a tool to assess the age-related development of intracellular cytokine production in small amounts of blood from infants

Fluorescent Cell Barcoding (FCB) is a flow cytometric technique which has been used for assessing signaling proteins. This FCB technique has the potential to be applied in other multiparameter analyses. Since data on antigen (Ag)-specific T-cell immune responses, like intracellular cytokine production, are still lacking in infants because limited blood volumes can be obtained for analysis, the FCB technique could be very useful for this purpose. The objectives of this study were to modify the FCB method to be able to measure multiple Ag-specific cytokine reponses in T-cells upon simultaneous stimulation by various antigens and mitogens in small amounts of blood and to investigate the cytokine pattern of T-cell subsets in healthy infants aged six and twelve months. Blood samples, collected from 20 healthy infants aged six and twelve months, were stimulated in vitro with the antigens: phorbol-myristate-acetate (PMA), purified-protein-derivative (PPD), Tetanus-toxoid (TT), Staphylococcal-enterotoxin-B (SEB), and phytohemagglutinin (PHA). Each stimulus was barcoded by labelling with different intensities of fluorescent cell barcoding (FCB) markers. Intracellular production of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha was measured simultaneously in just one blood sample of 600 μl whole blood. Significant age-related differences in cytokine production were shown for PMA, PHA, and TT in CD4(+) T-cells, and for PMA, PHA, SEB, and TT in CD8(+) T-cells. The intracellular cytokine production by CD4(+) and CD8(+) T-cells was higher at twelve months compared to six months of age for all antigens, except for PMA, which was lower at the age of twelve months. Based on the consistency in both T-cell subsets, we conclude that the new FCB method is a promising tool to investigate the age-related development of intracellular cytokine production in infants.     

Engineering Human T Cells for Resistance to Methotrexate and Mycophenolate Mofetil as an In Vivo Cell Selection Strategy

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR^FS; L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2^IY; T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt⁺DHFR^FS+IMPDH2^IY+ T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR^FS/MTX and IMPDH2^IY/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.     

Kinetically controlled drug resistance: how Penicillium brevicompactum survives mycophenolic acid

The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 10(3)-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate.     

Association between SLCO1A2 genetic variation and methotrexate toxicity in human rheumatoid arthritis treatment

Methotrexate (MTX), one of the important disease‐modifying anti‐rheumatic drugs, is the first‐line drug for rheumatoid arthritis (RA) treatment. However, its adverse drug effects (ADEs) often lead to the abortion of MTX therapy. Human organic anion‐transporting polypeptide 1A2 (OATP1A2, also referred as OATP‐A or OATP1) encoded by SLCO1A2 gene is an important isoform of the solute carrier transporter (SLC) family. It is known to participate in the cellular uptake of MTX. In our previous study, we identified four OATP1A2 natural variants (E184K, D185N, T259P, and D288N) with impaired MTX uptake activity. This study aimed to evaluate the association of the SLCO1A2 genetic variations encoding these OATP1A2 variants and MTX‐related toxicity in RA patients. A total of 60 RA patients were genotyped for these four polymorphisms (G550A, G553A, A775C, and G862A). The association between SLCO1A2 genetic variations and MTX toxicity was analyzed by binary logistic regression analysis. Single nucleotide polymorphisms (SNPs) analysis revealed that A775C and G862A SNPs were not detected in RA patients enrolled in this study, and the presence of 550AA genotype was associated with a high risk of MTX ADEs. Haplotype analysis revealed that H3 (H3 = AG) showed a high risk of MTX ADEs. Furthermore, there was a significant association of 550AA genotype and impaired MTX disposition, which might be the cause of the increased incidence of MTX ADEs in RA patients. Therefore, genetic variations in SLCO1A2 gene are risk factors for MTX toxicity and its information contributes to the prediction of MTX‐related toxicity in RA treatment.     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.     

Suppression of B cell function by methotrexate and trimetrexate. Evidence for inhibition of purine biosynthesis as a major mechanism of action

Methotrexate (MTX) is a widely used drug in the treatment of a variety of human neoplasms. Trimetrexate (TMQ) is a lipid-soluble quinazoline derivate of MTX that, unlike MTX, is not dependent upon membrane folate transport for cellular entry. A number of studies have demonstrated that MTX and, more recently, TMQ possess potent immunosuppressive properties. To examine the cellular events associated with the immunomodulatory effects of anti-folates on humoral immunity, a murine B cell maturation model was used. In vitro, MTX and TMQ reduced the number of antibody-forming cells to SRBC, as well as IgM production. B cells stimulated with anti-Ig demonstrated a dose-related suppression in [3H]UdR incorporation after addition of either drug, suggestive of a decrease in de novo DNA synthesis. B cell activation events preceding S phase were also suppressed by both anti-folates, as evidenced by inhibition of RNA synthesis. However, neither drug affected surface expression of Ia Ag nor inositol phosphate accumulation. Addition of TdR caused a slight non-significant increase in the antibody-forming cell response in the presence of 10(-7) M MTX. However, addition of hypoxanthine or adenine, but not guanine, resulted in complete restoration. Timed addition revealed that the ability of MTX to suppress antibody responses was diminished if added after 48 h of culture, similar to the reversal of this suppression mediated by hypoxanthine. Cell cycle analysis of LPS-stimulated B lymphocytes demonstrated that both drugs modulated events preceding, as well as during, the S phase. The present studies suggest that although drug-induced impairments in dTMP biosynthesis may be responsible for deficient lymphoid proliferation, anti-folate-induced impairment in purine biosynthesis is a major mechanism in anti-folate-induced suppression of humoral immunity.     

An Immunomodulating Motif of the HIV-1 Fusion Protein Is Chirality-independent: IMPLICATIONS FOR ITS MODE OF ACTION*

An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between d- and l-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a d-enantiomer of ISLAD CM (termed ISLAD d-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35–55)-specific line T-cells to the same extent as the l-motif form. Moreover, the d- and l-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD d-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35–55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the d-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies.     

The Impact of DMARD and Anti-TNF Therapy on Functional Characterization of Short-Term T-Cell Activation in Patients with Rheumatoid Arthritis – A Follow-Up Study

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by a systemic dysfunction of T-cells. In this study we tested the impact of DMARD and anti-TNF agents on short-term activation characteristics of T-cells. We enrolled 12 patients with newly diagnosed RA (naïve RA) who were treated with methothrexate (MTX) and glucocorticsteroid (GCS) and 22 patients with established RA non responding to conventional DMARD therapy who were treated with different anti-TNF agents. Nine healthy volunteers served as controls. Blood samples were taken at baseline, then at 4^th and 8^th week of therapy. The characteristics of several intracellular activation processes during short-term activation of T-cells including cytoplasmic Ca²⁺ level, mitochondrial Ca²⁺ level, reactive oxygen species (ROS) and nitric oxide (NO) generation were determined by a novel flow-cytometry technique. At baseline, the tested processes were comparable to controls in naïve RA. During GCS therapy, cytoplasmic Ca²⁺ level and ROS generation decreased. After the addition of MTX to GCS cytoplasmic Ca²⁺ level became comparable to controls, while ROS generation decreased further. In DMARD non responders, cytoplasmic Ca²⁺ level was higher than controls at baseline. The cytoplasmic Ca²⁺ level became comparable to controls and ROS generation decreased during each of the three anti-TNF-α agent therapies. Mitochondrial Ca²⁺ level and NO generation were unaltered in all of the patient groups. These results indicate that intracellular machinery is affected in T-cells of RA patients. This may alter the behavior of T-cells during activation. Different therapeutic approaches may modulate the abnormal T-cell functions.     

Platelets induce a proinflammatory phenotype in monocytes via the CD147 pathway in rheumatoid arthritis

Introduction

Activated platelets exert a proinflammatory action that can be largely ascribed to their ability to interact with monocytes. However, the mechanisms that promote dynamic changes in monocyte subsets in rheumatoid arthritis (RA) have not been clearly identified. The aim of this study was to determine whether platelet activation and the consequent formation of monocyte-platelet aggregates (MPA) might induce a proinflammatory phenotype in circulating monocytes in RA.

Methods

The surface phenotype of platelets and the frequencies of monocyte subpopulations in the peripheral blood of RA patients were determined using flow cytometry. Platelets were sorted and co-cultured with monocytes. In addition, monocyte activation was assessed by measuring the nuclear factor kappa B (NF-κB) pathway. The disease activity was evaluated using the 28-joint disease activity score.

Results

Platelet activation, circulating intermediate monocytes (Mon2) and MPA formation were significantly elevated in RA, especially in those with active disease status. Furthermore, Mon2 monocytes showed higher CD147 expression and responded to direct cell contact with activated platelets with higher cytokine production and matrix metallopeptidase 9 (MMP-9) secretion, which increased the expression of CD147. After the addition of specific antibodies for CD147, those effects were abolished. Furthermore, the NF-κB-driven inflammatory pathway may be involved in this process.

Conclusions

These findings indicate an important role of platelet activation and the consequent formation of MPA in the generation of the proinflammatory cytokine milieu and for the promotion and maintenance of the pathogenically relevant Mon2 monocyte compartment in RA, which is likely to play an important role in the pathogenesis of autoimmunity.