In vitro effects of methotrexate on peripheral blood monocytes: modulation by folinic acid and S-adenosylmethionine.

The mechanism of action of low dose methotrexate in rheumatoid arthritis has not been established. It has been shown to have an anti-inflammatory effect and to inhibit neutrophil chemotaxis, but the effect on monocytes has not been widely studied. Normal donor peripheral blood monocytes were incubated with methotrexate in vitro and their superoxide production, chemotaxis, and phagocytosis subsequently assessed. Additionally, the influence of different culture media, and of folinic acid, and the methyl donor S-adenosylmethionine, and spermidine on the methotrexate mediated effects were evaluated. It was found that methotrexate in low concentrations inhibited in vitro monocyte chemotaxis and superoxide production but only after prolonged incubation. This inhibition was augmented by incubation in medium containing a low methionine concentration and was abolished by folinic acid and S-adenosylmethionine, suggesting that methotrexate may interfere with specific methylation reactions.     

Pharmacokinetics of salazosulfapyridine in a hemodialysis patient]

The patient was a 62-year-old female. Total gastrectomy was performed due to gastric ulcer in 1969. She was diagnosed as rheumatoid arthritis (RA) in 1985 and was developed to amyloidosis in 1991. She was started on hemodialysis (HD) for chronic renal failure in 1996. In 1998, her arthralgia was aggravated, and 100 mg/day of bucillamine was administered on the day of HD. Her arthralgia persisted, and switching to salazosulfapyridine (SASP) was considered. As there were no standards and no reports for the use of SASP in HD patients, we examined the pharmacokinetics of SASP and its metabolites, and compared our patient with the results of phase one study in normal subjects in Japan. In this case, the blood concentration of SASP was similar to that in healthy controls after single administration of 500 mg of SASP on the day of non-HD, while the concentration of sulfapyridine (SP) was higher than that in healthy donors. However, the blood concentrations of SASP, SP, and N4-acetyl-SP (AcSP) at 24 hours after administration were similar to those obtained in healthy men. SASP was not dialyzed, while about half of SP and AcSP, were dialyzed. In a five-day consecutive administration study also, the blood concentrations of these compounds on Day 5 were similar to those of phase one study, suggesting no accumulation. No adverse drug reaction was observed. As this case had the past history of total gastrectomy and amyloidosis, it is possible that this result is influenced by the factors. Therefore it is necessary to examine pharmacokinetics of SASP and its metabolites beforehand when administering this agent to other HD/RA patients.     

Effects of dialysis on the pharmacokinetics of salazosulfapyridine

There was no standard or report for the treatment of rheumatoid arthritis (RA) patients on hemodialysis with Salazosulfapyridine (SASP). We examined the pharmacokinetics of SASP and its metabolites in RA patient on hemodialysis. Hemodialysis was started 2 h after administration of SASP at a dose of 250 or 500 mg. Blood samples were took 8 times during the observation period. The concentration of SASP and its metabolites (SP, Ac-SP) in blood sample were measured. There was no difference for the concentration of SASP before and after hemodialysis. Results showed SASP was nondialyzable, but SP and AC-SP were dialyzable. At a dose of 500 mg, AUC0-∞ of SASP and SP were higher than healthy volunteer. Therapy with SASP for hemodialysis RA should be started at a lower dose for adverse event risk.     

Human Neutrophil Functions Are Inhibited In Vitro by Clinically Relevant Ethanol Concentrations

Neutrophils [polymorphonuclear neutrophils (PMNs)] play a pivotal role in host defense in man. These defenses may be compromised, however, in alcohol users and abusers. We therefore evaluated the effect of ethanol levels (12.5 to 500 mg/dl), on key functions of human PMNs—chemotaxis and production of reactive oxygen species—and on changes in cytosolic-free calcium ([Ca²⁺]_i), a pivotal intracellular mechanism of PMN activation. Ethanol significantly inhibited chemotaxis as evaluated by formyl-methionyl-leucyl-phenylalanine (fMLP)-induced upregulation of surface adhesion molecules (CD11b), fMLP-induced PMN elongation was only inhibited by a very high ethanol concentration of 500 mg/dl. Production of reactive oxygen species by normal PMNs was assessed by either chemiluminescence (CL) for hypochlorous acid or ferricytochrome c reduction (FCR) for superoxide anions. For PMN stimulated by fMLP, ethanol inhibited CL but not FCR. For PMNs activated by phorbol myristate acetate, ethanol inhibited both CL and FCR. Ethanol did not alter baseline [Ca²⁺]_i, as assessed by videomicroscopy using the Ca²⁺-sensing fluorescent dye Fura-2-AM, but did significantly potentiate the increase in peak [Ca²⁺]_i, levels that occurs in response to stimulation by fMLP. Calcium channel blockers attenuated ethanol's inhibition of CL. Thus, acute in vitro ethanol, at clinically relevant concentrations, can inhibit several critical aspects of PMN functions. But, in PMNs, unlike neural cells, these inhibitory effects do not seem to be mediated by decreases in Ca²⁺ influx or in [Ca²⁺]_i.     

Defective monocyte cytotoxicity in rheumatoid arthritis

The cytotoxic activities of human blood mononuclear cells against certain established cell lines were evaluated prospectively in 17 patients with rheumatoid arthritis before and after treatment with low (150 mg per week) and moderate doses (300 mg per week) of levamisole. Spontaneous or “natural” killer activity (NK) and antibody-dependent cellular cytotoxicity (ADCC) of plastic adherent cells (“monocytes”) and lymphocytes were studied. We report a selective cytotoxic defect in monocyte NK and a correlation of this defect with severely active disease. The patients with the most severe defect responded to low-dose levamisole, but others with normal values did not respond as well to treatment. This cytotoxic defect may be an important pathogenetic factor in rheumatoid arthritis and this new assay may be helpful in selecting candidates who are most likely to respond to levamisole.     

Insulin differentially regulates monocyte and polymorphonuclear neutrophil functions in healthy young and elderly humans

CONTEXT: Insulin can regulate immune cell function. Aging is associated with various degrees of insulin resistance together with reduced immune cell activity. OBJECTIVE: We investigated the hypothesis that blood monocytes and polymorphonuclear neutrophils (PMNs) are less responsive to the action of insulin in elderly subjects. DESIGN-INTERVENTION: We evaluated the effect of hyperinsulinemia (0.7 mU/kg(-1) fat-free mass per minute(-1)) on monocyte and PMN activity using a 4-h euglycemic clamp technique. PARTICIPANTS: Eight young (24 +/- 6 yr old) and nine elderly (69 +/- 4 yr old) healthy volunteers participated in the study. MAIN OUTCOME MEASURES: Monocyte and PMN receptor expression and density were measured using flow cytometric detection. PMN chemotaxis toward formyl-Met-Leu-Phe (fMLP) was evaluated using a two-compartment chamber. PMN and monocyte phagocytosis was determined by measuring the engulfment of opsonized particles. Microbicidal functions were determined based on the production of reactive oxygen species (ROS) and bactericidal protein by stimulated cells. RESULTS: The density of PMN and monocyte insulin receptors was not affected by age or insulin clamp treatment regardless of the age. Insulin was able to regulate the expression of receptors involved in PMN action in the young-adult group only. PMN chemotaxis was up-regulated by insulin in both groups. In contrast, although insulin stimulated phagocytosis and bactericidal activity in young-adult subjects, the ability of PMN to adapt to physiological hyperinsulinemia was blunted in the older group. The effect of insulin on monocyte bactericidal properties seemed to be limited, although a suppressive action on fMLP-induced ROS production was detected in young adults. CONCLUSIONS: We confirmed the presence of the insulin receptor on monocyte and PMN membranes. We revealed that insulin has a limited action on monocyte function. Insulin has a priming effect on the main PMN functions. Immune cell function adapted poorly to insulin infusion in the elderly subjects.     

Treatment with sulfasalazine or sulfapyridine, but not 5-aminosalicyclic acid, inhibits basic fibroblast growth factor-induced endothelial cell chemotaxis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression. METHODS: SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites. RESULTS: HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78. CONCLUSION: These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.     

Diclofenac inhibits monocyte superoxide production ex vivo in rheumatoid arthritis

Summary Effects of the nonsteroidal anti-inflammatory drug, diclofenac, on stimulated monocyte superoxide production were assessed directly in vitro and following treatment of patients with rheumatoid arthritis ex vivo. Diclofenac inhibited superoxide generation provoked by serum treated zymosan (STZ) and fluoride anion (F) but not by phorbol myristate acetate (PMA) in vitro. Following patient therapy, inhibition of superoxide production occurred when STZ and PMA, but not F were used as stimuli. No changes were seen in control subjects. The contrasting profiles of inhibition seen in vitro and ex vivo suggest an indirect effect on superoxide production during clinical use of the agent. These data are consistent with the hypothesis that anti-inflammatory drugs may act in rheumatoid arthritis by inhibiting phagocyte super-oxide anion production.     

Effect of sulphasalazine and its metabolites on the generation of reactive oxygen species.

The relative in vitro anti-oxidant efficacy of sulphasalazine (salicylazosulphapyridine, SASP) and its metabolites (5-aminosalicylic acid, 5-ASA; sulphapyridine, SP) was examined by studying their effects on the generation of reactive oxygen species (ROS) using zymosan-stimulated polymorphonuclear leucocytes (PMNs) and a cell free, xanthine-xanthine oxidase system. Salicylazosulphapyridine, 5-ASA, and SP showed anti-oxidant effects to the various degrees. In particular, production of OH, which is one of the most potent reactive oxygen species, was remarkably suppressed by 5-ASA dose relatedly. These findings suggest that SASP and its metabolites play an important role in the inhibition of respiratory bursts. As the potent products of the respiratory burst by polymorphonuclear leucocytes are thought to be important inflammatory mediators, suppression of toxic reactive oxygen species generation by these agents may partly explain the therapeutic efficacy of SASP in ulcerative colitis, which is characterised by an acute mucosal inflammation dominated by polymorphonuclear leucocytes accumulation.     

Scavenger effect of sulfasalazine, 5-aminosalicylic acid,and olsalazine on superoxide radical generation

Thein vitro antioxidant capacity of sulfasalazine (SASP), its metabolites (SP, 5-SSA), and olsalazine (OAZ), was studied by evaluating their effects on superoxide (O₂ ^– ) production. Assay systems were the xanthine-xanthine oxidase (X/XOD) reaction and phorbol myristate acetate (PMA)-activated polymorphonuclear leukocytes (PMNs), using the cytochromec (cyt-c) reduction assay and a luminol-dependent chemiluminescence method. 5-ASA, SASP, and OAZ showed a dose-dependent scavenger effect in both O₂ ^– generating systems, 5-ASA being the most powerful (>50% of inhibition in the PMNs system and >70% in the X/XOD system at 10 M concentration). SP had an inhibitory effect only in the PMNs system but did not modify the activity of xanthine oxidase, thus excluding a scavenger action. These data suggest that the scavenger effect of 5-ASA, SASP, and OAZ may be an important mechanism of action.     

Defective Fc-, CR1- and CR3-mediated monocyte phagocytosis and chemotaxis in common variable immunodeficiency and X-linked agammaglobulinemia patients.

Blood monocyte phagocytic functions were evaluated by chemotaxis, phagocytosis, and superoxide anion production in nine patients with common variable immunodeficiency (CVI), eight patients with X-linked agammaglobulinemia (XLA), and in 17 normal subjects. Further laboratory diagnosis included the determination of the Bruton's tyrosine kinase (Btk) protein expression in monocytes using flow cytometry. The analysis of monocyte phagocytic function demonstrated that CR3-, CR1-, and Fc-mediated phagocytosis (p = 0.0001) were significantly decreased in CVI and XLA patients, and chemotaxis of monocytes (p = 0.0082) was reduced in XLA patients. Superoxide anion production, however, did not differ between the CVI, XLA, and the control groups. The cytoplasmic expression of Btk protein in monocytes was normal in CVI patients and decreased or not detected in XLA patients. It is proposed that impaired chemotaxis and phagocytosis by monocytes may be a characteristic of the innate immune system in CVI and XLA patients, providing a new direction for the physiopathology of these immunodeficiencies.     

Effects of gold sodium thiomalate on functional correlates of human monocyte maturation

The mechanism of action of gold salts in the treatment of rheumatoid arthritis is unknown. Effects of gold on monocyte-macrophage function could be due to inhibition of maturation and differentiation. We found that 3 markers of monocyte differentiation, loss of peroxidase activity, spontaneous synthesis of C2, and spontaneous cytotoxicity for chicken erythrocytes, were all inhibited by gold treatment. This was not a general toxic effect since phorbol myristate acetate could still induce gold-treated monocytes to lyse chicken erythrocytes. Also, phorbol myristate acetate-stimulated superoxide production, a monocyte function not requiring further differentiation, was not inhibited by incubation with gold. Lymphokine-stimulated cytotoxicity for nucleated target cells, another function of monocytes, was inhibited only partially for certain target cells and not at all for others. These data suggest that gold has the capacity to selectively inhibit some monocyte functions which are associated with macrophage differentiation.     

The effect of sulphasalazine on neutrophil superoxide generation in rheumatoid arthritis

The production of superoxide by the peripheral blood neutrophils of 19 patients with active rheumatoid arthritis was measured during treatment with sulphasalazine (SASP). The response to drug treatment was determined by change in plasma viscosity, CRP, early morning stiffness and articular index over a 10-point scale. Of the 19 patients studied, eight were considered to have responded well to SASP and seven to have responded poorly or not at all. Over the treatment period, plateau levels of superoxide production fell in seven of the eight responders (P = 0.028) compared with a non-significant fall in 3/7 of the non- responder groups. The initial rate of superoxide production also fell in the responder group, but this was not statistically significant. Initial values in both the responder and non-responder groups were comparable with those seen for normal controls. Analysis of drug levels showed all patients to be compliant with drug treatment; however, drug levels and neutrophil activity were not correlated. Studies of the effect of SASP and sulphapyridine on superoxide production in vitro showed no difference between good and poor responders. These results suggest that there is no inherent difference between good and poor responders regarding the susceptibility of their neutrophils to SASP. SASP's action on neutrophils, therefore, appears not to be its main mechanism of disease-modifying activity in RA.      

Blood polymorphonuclear behavior in patients with ankylosing spondylitis

The oxidative metabolism and chemotaxis of polymorphonuclear leukocytes (PMNs) collected from patients with ankylosing spondylitis and healthy subjects were studied in parallel. The responses to opsonized zymosan were significantly lowered considering oxygen consumption and release of superoxide anions, whereas no modification of these parameters to phorbol myristate acetate and calcium ionophore (A 23187) stimulations were observed. A seric factor was not involved but the characterization of a specific intrinsic abnormality of the PMNs needs further investigations. PMN chemotaxis, assessed by two methods performed in parallel, remained unchanged.     

Monocytes in rheumatoid arthritis. Regulatory, effector, and phenotypic properties

We reported that rheumatoid arthritis (RA) blood mononuclear cells (MNC) secreted less Ig and IgM rheumatoid factor (RF) than synovial cells. Since antibody elaboration is partly monocyte dependent, we compared regulatory, effector, and phenotypic properties of monocytes from 31 patients with RA with those of 21 normal subjects. RA IgG and IgM elaboration was less than normal. Monocyte, T or B cell numbers were comparable in RA and normal MNC and monocyte enriched/depleted preparations. RA and normal Ig production were monocyte dependent and this differed for IgG and IgM for RA and normals. IgM RF elaboration by stimulated RA MNC was also monocyte dependent and addition of normal monocytes/monocyte culture supernatants to RA monocyte depleted cultures and vice versa had inconsistent effects. RA MNC (Ficoll-Hypaque) phagocytosis was less than normal; killing (acridine orange fluorescent microscopy) was not. RA synovial monocyte phagocytosis - but not killing was also reduced. MNC from RA patients and normals contained similar numbers of monocytes; RA monocyte enriched populations (Percoll) showed less phagocytosis than normal; with similar killing. RA phagocytosis was reduced at 1,2,3,4 and 5 h and differed from normal at 3 and 5 h. Different sera - FCS, autologous, normal, RA - exhibited inconsistent effects on phagocytosis. RA and normal neutrophil phagocytosis and killing were comparable. Lastly, RA monocyte enriched preparations contained populations of OKM1, OKM3, OKM5, OKM6, Leu M1, Leu M2, Leu M3, and HLA-DR-positive cells (FACS analysis) comparable with normals. Regulatory and effector but not phenotypic properties of RA blood monocytes differed from normal and may contribute to inappropriate autoantibody production and chronic inflammation.     

Preliminary study on the immunologic background of good clinical outcome in rheumatoid arthritis patients after one month therapy with leflunomide

This preliminary study focuses on early peripheral cellular immune changes after 1 month therapy with leflunomide, in 18 patients with severe rheumatoid arthritis, previously treated with methotrexate. A good clinical outcome of disease was documented and we showed that a particular target of short-time leflunomide therapy in rheumatoid arthritis was the peripheral innate immune system (NK cells and the population of granulocytes developing phagocytosis and superoxide anion production when challenged ex vivo with zymosan particles). Meanwhile, the high inter-individual variability of adaptive immunity required data analysis in subgroups of patients. We showed that the abnormal increase of peripheral leukocytes counts, or the decrease towards normal values of the CD4:CD8 lymphocytes ratio, or the inhibition of uridine uptake by ex vivo activated lymphocytes were consistent with a positive clinical evolution, proved by the reduction of tender/swollen joints, morning stiffness duration or acute phase response. We emphasized that significant benefits of short-term leflunomide therapy were associated with functional suppression of peripheral B lymphocytes. Hence, the positive evolution of rheumatoid arthritis patients seemed to be specifically linked to early drug-induced changes of trafficking or uridine metabolism of mononuclear cells.     

Pharmacology of human spontaneous monocyte-mediated cytotoxicity

The effect of various antiinflammatory agents on the spontaneous cytotoxicity of human mononuclear cells in vitro was assessed. Acetylsalicylic acid (ASA) and hydrocortisone enhanced spontaneous monocyte-mediated cytotoxicity compared to control values. This enhancement could not be mediated through inhibition of prostaglandin biosynthesis since indomethacin had no effect on cytotoxic function and since the direct addition of PGE₂ to the cell cultures did not inhibit the expression of cytotoxicity. Likewise, salicylic acid (SA), which has no effect on prostaglandin biosynthesis, also enhanced monocyte cytotoxicity. Stimulation of monocyte-mediated cytotoxicity resulting in more efficient antigen removal and thus decreasing antigen persistence may be an additional mechanism by which ASA, SA, and hydrocortisone modulate the destructive inflammatory response in rheumatoid arthritis.     

A study to determine the active moiety of sulphasalazine in rheumatoid arthritis

Thirty patients with active rheumatoid arthritis (RA) participated in an open study of 6 months' treatment with either 5-aminosalicylic acid (5-ASA) or sulphapyridine (SP), the two moieties of sulphasalazine (SASP). Patients were assessed at regular intervals using clinical and biochemical tests designed to detect specific antirheumatic activity. Patients taking SP showed significant improvement in disease activity, but those taking 5-ASA did not improve, despite the fact that high serum concentrations of 5-ASA and acetyl 5-ASA were achieved. These results suggest that SP is the active moiety of SASP. Its possible mode of action is discussed. Nausea was a frequent problem in patients taking SP. Unless this can be overcome, SP is unlikely to offer any therapeutic advantages over SASP in the treatment of RA.     

Chemoattractant-mediated stimulation of the respiratory burst in human polymorphonuclear leukocytes may require appearance of protein kinase activity in the cells' particulate fraction

Low doses of aliphatic alcohols produce divergent effects on the function of chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) since they enhance chemotaxis but inhibit stimulation of superoxide production by chemoattractants. As such, alcohols can provide useful pharmacologic tools to probe the mechanisms of stimulus- response coupling in leukocytes. A role for protein kinase C has been implicated in the activation of the respiratory burst in PMNs. Although the vast majority of this enzyme activity is located in the cytosolic fraction of unactivated PMNs, protein kinase C activity appears in the particulate fraction of the cells when they are stimulated to produce superoxide by either chemoattractants or by phorbol myristate acetate (PMA). Doses of the alcohols that selectively inhibited stimulation of superoxide production by chemoattractants also inhibited the appearance of protein kinase C activity as well as an undefined protein kinase activity in the particulate fraction of the cells. In contrast, the alcohols did not affect either the ability of PMA to stimulate the production of superoxide in PMNs nor the appearance of protein kinase activity in the cells' particulate fraction. PMA is known to bind and activate protein kinase C directly, thus bypassing receptor-mediated events. These data suggest that alcohols inhibit the stimulation of the respiratory burst by chemoattractants in PMNs by blocking the ability of receptor occupancy to induce the appearance of protein kinase activity in particulate fractions. These results moreover suggest that the appearance of protein kinase activity in the particulate fraction may be required for activation of the respiratory burst in PMNs.