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INHIBITION O F LEUKOTRIENE B, SYNTHESIS IN NEUTROPHILS FROM PATIENTS WITH RHEUMATOID ARTHRITIS BY A SINGLE ORAL DOSE O F METHOTREXATE RICHARD I. SPERLING, JONATHAN S. COBLYN, JOHN K. LARKIN, ANTHONY I. BENINCASO, K. FRANK AUSTEN, and MICHAEL E. WEINBLATT We studied the effects of a single, oral dose of methotrexate (MTX) on arachidonic acid metabolism in neutrophils from 6 patients with rheumatoid arthritis, which were obtained 1 day before and 1 day after their usual weekly MTX dose. The 6 patients had received a mean weekly MTX dose of 9.6 mg (range 5-15) for a mean of 61.7 months (range 58-64), and none received concomitant corticosteroids. Total generation of leukotriene B, (LTB,) in neutrophils stimulated ex vivo with 10 pI4 calcium ionophore A23187 for 20 minutes was significantly suppressed, by a mean of 53%, after the MTX dose compared with the predose levels (mean f SEM 13.0 f 1.4 ng/106 cells versus 6.0 f 0.9 ng/106 From the Department of Rheumatology and Immunology, Brigham and Women’s Hospital, and the Department of Medicine, Haward Medical School, Boston, Massachusetts. Supported by NIH grants AR-35907, A1-22531, and AR38638, a postdoctoral research grant and a Biomedical Research Center grant from the Arthritis Foundation, and supported in part by a research grant from Lederle Laboratories. Richard 1. Sperling, AM, MD: Assistant Professor of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women’s Hospital; Jonathan S. Coblyn, MD: Assistant Professor of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women’s Hospital; John K. Larkin. BS: Research Technician, Department of Rheumatology and Immunology, Brigham and Women’s Hospital; Anthony I. Benincaso, BS: Research Technician, Department of Rheumatology and Immunology, Brigham and Women’s Hospital; K. Frank Austen, MD: Theodore Bevier Bayles Professor of Medicine, Harvard Medical School. and Chairman, Department of Rheumatology and Immunology, Brigham and Women’s Hospital; Michael E. Weinblatt. MD: Associate Professor of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women’s Hospital. Address reprint requests to Richard I. Sperling, MD, Harvard Medical School, Seeley G. Mudd Building, 250 Longwood Avenue, Room 618, Boston, MA 02115. Submitted for publication December 6, 1989; accepted in revised form March 6, 1990. Arthritis and Rheumatism, Vol. 33, No. 8 (August 1990)
cells; P = 0.0019), reflecting a comparable suppression of both released and cell-retained LTB,. A 49% decrease in *oxidation products of LTB, demonstrates that decreased LTB, synthesis, rather than increased degradation, is responsible for the decrease in LTB, generation. The absence of a significant change in either ’H-labeled arachidonic acid release or plateletactivating factor generation indicates that the observed decrease in LTB, synthesis was apparently not caused by diminished phospholipase A, activity. A 28% decrease in the total formation of the 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid and the 6trans-LTB, diastereoisomers, and a 48% suppression of production of LTB, plus its *oxidation metabolites after the MTX dose suggest inhibition of 5-lipoxygenase activity and possible suppression of leukotriene A, epoxide hydrolase activity.
Although low-dose methotrexate (MTX) has been demonstrated to be an efficacious therapy for patients with active rheumatoid arthritis (RA) ( 1 4 , its mechanism of action in RA remains controversial (2,3,5-7). The immunosuppressive effects of high-dose MTX became apparent (8) after MTX and its related antifolate congeners became widely used antineoplastic chemotherapeutic agents in the decade following their clinical introduction in 1948 (9). Some investigators have suggested that MTX exerts its therapeutic effects in RA through an antiinflammatory mechanism (2,3,5,7), because immunosuppressive effects of lowdose MTX therapy in patients with RA have not been observed consistently and because evidence of a therapeutic response may be observed as early as 3-6 weeks after initiation of therapy (2,4). MTX has been reported to inhibit the chemo-
SPERLING ET AL
1150
tactic response of neutrophils in vivo in patients with psoriasis (10,l I), in vitro in neutrophils from healthy human volunteers (12). and in vivo in mice (13). Recently, O'Callahan et a1 (14) have demonstrated that the chemotactic activity of neutrophils obtained from patients with RA after treatment with an initial oral dose of MTX was decreased in comparison with the pretreatment level. In this study, we investigated the antiinflammatory effects of a single, oral dose of MTX by studying the chemotaxis and the calcium ionophore-stimulated generation of lipid mediators in neutrophils from patients with RA who received long-term, weekly, oral MTX therapy.
PATIENTS AND METHODS Patients. Informed consent was obtained from 6 patients who had participated in a previous controlled trial of MTX in RA (2) and are now enrolled in a long-term prospective study (15). The selected patients, with a mean 2 SEM age of 63.5 2 2.6 years (range 51-68), met the American Rheumatism Association 1987 revised criteria for RA (16). The patients had received long-term, weekly, oral MTX therapy for a mean 2 SEM period of 61.7 2 2.7 months (range 58-64), with a mean f SEM dose of 9.6 4 1.8 mg/week (range 5-15). Three patients received concomitant therapy with nonsteroidal antiinflammatory drugs; none had received corticosteroids within a month before the study or during the study. Cell activation and quantitation of LTB.,and plateletactivating factor (PAF) generation by radioimmunoassays. Neutrophils were isolated by dextran sedimentation, discontinuous Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradient centrifugation,and hypotonic lysis (17)from whole blood obtained the day before and the day after the usual weekly MTX dose. For quantitation of LTB, and PAF generation, neutrophils were suspended at a cell density of 1 X 10' cells/ml in Tyrode's-HEPES-gelatin buffer (a modified Tyrode's buffer without bicarbonate containing 2.5 mM K+,0.5 mM Mg2+, 1.5 mM Ca*+, 30 mM HEPES, and 1 mg/ml gelatin, pH 7.4). Portions of cells (400 pl each) were incubated at 37°C for 5 minutes, with agitation, and duplicate samples were activated by the addition of 400 pl of warmed Tyrode's-HEPES-gelatin buffer containing 20 pM calcium ionophore A23187, resulting in a final concentration of 10 pR4. Duplicate control samples were treated in the same manner, except that calcium ionophore was omitted from the added Tyrode's-HEPES-gelatin buffer. The reactions were stopped after 20 minutes by cooling the samples to 0°C. The cells were sedirnented by centrifugation at 10,OOOg for 1 minute, the supernatants were removed, and the cell pellets were extracted with 800 pl of methanol (18). Serum MTX levels were measured the day before and the day after the usual MTX dose; all levels were below the limits of detection (
INHIBITION O F LEUKOTRIENE B, SYNTHESIS IN NEUTROPHILS FROM PATIENTS WITH RHEUMATOID ARTHRITIS BY A SINGLE ORAL DOSE O F METHOTREXATE RICHARD I. SPERLING, JONATHAN S. COBLYN, JOHN K. LARKIN, ANTHONY I. BENINCASO, K. FRANK AUSTEN, and MICHAEL E. WEINBLATT We studied the effects of a single, oral dose of methotrexate (MTX) on arachidonic acid metabolism in neutrophils from 6 patients with rheumatoid arthritis, which were obtained 1 day before and 1 day after their usual weekly MTX dose. The 6 patients had received a mean weekly MTX dose of 9.6 mg (range 5-15) for a mean of 61.7 months (range 58-64), and none received concomitant corticosteroids. Total generation of leukotriene B, (LTB,) in neutrophils stimulated ex vivo with 10 pI4 calcium ionophore A23187 for 20 minutes was significantly suppressed, by a mean of 53%, after the MTX dose compared with the predose levels (mean f SEM 13.0 f 1.4 ng/106 cells versus 6.0 f 0.9 ng/106 From the Department of Rheumatology and Immunology, Brigham and Women’s Hospital, and the Department of Medicine, Haward Medical School, Boston, Massachusetts. Supported by NIH grants AR-35907, A1-22531, and AR38638, a postdoctoral research grant and a Biomedical Research Center grant from the Arthritis Foundation, and supported in part by a research grant from Lederle Laboratories. Richard 1. Sperling, AM, MD: Assistant Professor of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women’s Hospital; Jonathan S. Coblyn, MD: Assistant Professor of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women’s Hospital; John K. Larkin. BS: Research Technician, Department of Rheumatology and Immunology, Brigham and Women’s Hospital; Anthony I. Benincaso, BS: Research Technician, Department of Rheumatology and Immunology, Brigham and Women’s Hospital; K. Frank Austen, MD: Theodore Bevier Bayles Professor of Medicine, Harvard Medical School. and Chairman, Department of Rheumatology and Immunology, Brigham and Women’s Hospital; Michael E. Weinblatt. MD: Associate Professor of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women’s Hospital. Address reprint requests to Richard I. Sperling, MD, Harvard Medical School, Seeley G. Mudd Building, 250 Longwood Avenue, Room 618, Boston, MA 02115. Submitted for publication December 6, 1989; accepted in revised form March 6, 1990. Arthritis and Rheumatism, Vol. 33, No. 8 (August 1990)
cells; P = 0.0019), reflecting a comparable suppression of both released and cell-retained LTB,. A 49% decrease in *oxidation products of LTB, demonstrates that decreased LTB, synthesis, rather than increased degradation, is responsible for the decrease in LTB, generation. The absence of a significant change in either ’H-labeled arachidonic acid release or plateletactivating factor generation indicates that the observed decrease in LTB, synthesis was apparently not caused by diminished phospholipase A, activity. A 28% decrease in the total formation of the 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid and the 6trans-LTB, diastereoisomers, and a 48% suppression of production of LTB, plus its *oxidation metabolites after the MTX dose suggest inhibition of 5-lipoxygenase activity and possible suppression of leukotriene A, epoxide hydrolase activity.
Although low-dose methotrexate (MTX) has been demonstrated to be an efficacious therapy for patients with active rheumatoid arthritis (RA) ( 1 4 , its mechanism of action in RA remains controversial (2,3,5-7). The immunosuppressive effects of high-dose MTX became apparent (8) after MTX and its related antifolate congeners became widely used antineoplastic chemotherapeutic agents in the decade following their clinical introduction in 1948 (9). Some investigators have suggested that MTX exerts its therapeutic effects in RA through an antiinflammatory mechanism (2,3,5,7), because immunosuppressive effects of lowdose MTX therapy in patients with RA have not been observed consistently and because evidence of a therapeutic response may be observed as early as 3-6 weeks after initiation of therapy (2,4). MTX has been reported to inhibit the chemo-
SPERLING ET AL
1150
tactic response of neutrophils in vivo in patients with psoriasis (10,l I), in vitro in neutrophils from healthy human volunteers (12). and in vivo in mice (13). Recently, O'Callahan et a1 (14) have demonstrated that the chemotactic activity of neutrophils obtained from patients with RA after treatment with an initial oral dose of MTX was decreased in comparison with the pretreatment level. In this study, we investigated the antiinflammatory effects of a single, oral dose of MTX by studying the chemotaxis and the calcium ionophore-stimulated generation of lipid mediators in neutrophils from patients with RA who received long-term, weekly, oral MTX therapy.
PATIENTS AND METHODS Patients. Informed consent was obtained from 6 patients who had participated in a previous controlled trial of MTX in RA (2) and are now enrolled in a long-term prospective study (15). The selected patients, with a mean 2 SEM age of 63.5 2 2.6 years (range 51-68), met the American Rheumatism Association 1987 revised criteria for RA (16). The patients had received long-term, weekly, oral MTX therapy for a mean 2 SEM period of 61.7 2 2.7 months (range 58-64), with a mean f SEM dose of 9.6 4 1.8 mg/week (range 5-15). Three patients received concomitant therapy with nonsteroidal antiinflammatory drugs; none had received corticosteroids within a month before the study or during the study. Cell activation and quantitation of LTB.,and plateletactivating factor (PAF) generation by radioimmunoassays. Neutrophils were isolated by dextran sedimentation, discontinuous Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradient centrifugation,and hypotonic lysis (17)from whole blood obtained the day before and the day after the usual weekly MTX dose. For quantitation of LTB, and PAF generation, neutrophils were suspended at a cell density of 1 X 10' cells/ml in Tyrode's-HEPES-gelatin buffer (a modified Tyrode's buffer without bicarbonate containing 2.5 mM K+,0.5 mM Mg2+, 1.5 mM Ca*+, 30 mM HEPES, and 1 mg/ml gelatin, pH 7.4). Portions of cells (400 pl each) were incubated at 37°C for 5 minutes, with agitation, and duplicate samples were activated by the addition of 400 pl of warmed Tyrode's-HEPES-gelatin buffer containing 20 pM calcium ionophore A23187, resulting in a final concentration of 10 pR4. Duplicate control samples were treated in the same manner, except that calcium ionophore was omitted from the added Tyrode's-HEPES-gelatin buffer. The reactions were stopped after 20 minutes by cooling the samples to 0°C. The cells were sedirnented by centrifugation at 10,OOOg for 1 minute, the supernatants were removed, and the cell pellets were extracted with 800 pl of methanol (18). Serum MTX levels were measured the day before and the day after the usual MTX dose; all levels were below the limits of detection (
