376
ACUTE AND CHRONIC SUPPRESSION OF LEUKOTRIENE B, SYNTHESIS EX VIVO IN NEUTROPHILS FROM PATIENTS WITH RHEUMATOID ARTHRITIS BEGINNING TREATMENT WITH METHOTREXATE RICHARD I. SPERLING, ANTHONY I. BENINCASO, RONALD J. ANDERSON, JONATHAN S. COBLYN, K. FRANK AUSTEN, and MICHAEL E. WEINBLATT
Objective. To compare the cumulative effects of oral methotrexate (MTX) therapy (after 68 weeks) with the acute effects (24 hours after a dose) on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from patients with active rheumatoid arthritis (RA) who were beginning therapy with MTX. Methods. Neutrophils and monocytes were isolated from whole blood from 7 patients with RA, immediately before and 24 hours after their first weekly dose of 7.5 mg of MTX, and again after their dose at 6-8 weeks. Results. Total immunoreactive leukotriene B, (LTB,) formation in neutrophils activated ex vivo with calcium iono-
From the Department of Medicine, Harvard Medical School, and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts. Supported by NIH grants AR-35907, AI-2253 1, AI-23401, RR-05950, and AR-38638, a postdoctoral research grant and an Arthritis Biomedical Research Center grant from the Arthritis Foundation, and by a research grant from Lederle Laboratories. Richard I. Sperling, AM, MD: Assistant Professor of Medicine, Harvard Medical School, and 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; Ronald J. Anderson, MD: Associate 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; 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 July 5, 1991; accepted in revised form November 13, 1991. Arthritis and Rheumatism, Vol. 35, No. 4 (April 1992)
phore A23187 was significantly suppressed (by 33%) before the 68-week dose, compared with the level before the first dose (mean f SEM 8.29 f 1.24 ng/106 cells at predose 68 weeks versus 12.29 f 2.13 ng/106 cells at predose 1; P = 0.03). Reductions were also observed after the first dose (27%; P = 0.07) and after the 6-8-week dose (43%; P = 0.05) compared with the respective predose levels. MTX treatment produced significant reductions in the total generation of 5-LO pathway 6-trans-LTB4 products (5-hydroxyeicosatetraenoicacid LTB, -oxidation products of LTB,) by calcium ionophore-activated neutrophils, as quantitated by integrated optical density after resolution on reverse-phase high-performance liquid chromatography. Decreases were observed after the first dose dose (26%;P = 0.025), immediately before the --week (23%; P = 0.05), and after the 6-8-week dose (47%; P = 0.0033) compared with levels before the first dose, and after the 68-week dose compared with the level before it (32%; P = 0.04). The generation of LTB, by calcium ionophorwwtivated monocytes was not significantly affected by MTX therapy. Conclusion. The significant decreases in the formation of *oxidation products of LTB, and in the total generation of neutrophil 5-LO pathway products in the absence of a significant change in the release of 'H-arachidonic acid or the generation of platelet-activating factor suggest that the activity of the 5-LO enzyme in neutrophils is inhibited. We conclude that weekly oral MTX therapy in patients with active RA inhibits neutrophil 5-LO pathway product generation in a pattern consistent with inhibition of the activity of the 5-LO enzyme; an effect is observed after the first dose. The inhibition of 5-LO is cell-selective and cumulative, with a superimposed incremental inhibition observed after the weekly MTX dose.
+
+
+
Low-dose, weekly therapy with methotrexate
(MTX)is efficacious in patients with active rheumatoid arthritis (RA) (1-4); however, its mechanism of action in RA remains to be defined (2,3,5-7). In the decade
LTB, SYNTHESIS AND MTX THERAPY following the clinical introduction of MTX and its related antifolate congeners as antineoplastic chemotherapeutic agents in 1948 (8), the immunosuppressive effects of high-dose MTX became apparent (9). Some investigators have inferred that low-dose, pulse MTX exerts its therapeutic effect in RA through an unknown antiinflammatory mechanism (2,3,5,7), since a therapeutic response occurs as early as 3-6 weeks (2,4) after the initiation of therapy, in the absence of consistent immunosuppressive effects seen ex vivo (2,3,5). MTX has been reported to inhibit the chemotactic response of neutrophils in vivo in patients with psoriasis (lO,ll), in vivo in mice (12), in vitro in healthy human volunteer donors (13), and ex vivo in patients with RA after treatment with an initial oral dose of MTX (14). In a previous study, we evaluated the effects of a single oral dose of MTX on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from 6 patients with RA, which had been obtained 1 day before and 1 day after their usual weekly MTX dose (15). The patients had received a mean of 61 months of weekly MTX therapy before enrolling in the study. One day after the weekly MTX dose, the total generation of leukotriene B, (LTB,) by neutrophils stimulated ex vivo for 20 minutes with 10 @ calcium ionophore A23 187 was suppressed significantly, by a mean of 53%, compared with the predose level (15). Both the released and the cell-retained LTB,, as well as the *oxidation products of LTB, were comparably suppressed, indicating that decreased intracellular LTB, biosynthesis, rather than impaired release or augmented degradation, was responsible for the MTX-related decrease in LTB, generation. In the present study, we investigated the curnulative effects of MTX therapy on the generation of neutrophil 5-lipoxygenase pathway products after 6 8 weeks of treatment, a time point at which most patients begin to show a clinical response to MTX, as well as the acute effects 24 hours after a single dose. We compared the effects on neutrophils with the effects on peripheral blood monocytes. The findings are presented below.
PATIENTS AND METHODS Patients. Informed consent was obtained from 7 patients with RA who were to begin weekly oral MTX therapy. Their mean & SD age was 58.3 ? 10.1 years (range 42-72), and all met the American College of Rheumatology
377 (formerly, the American Rheumatism Association) 1987 revised criteria for RA (16). The mean SD duration of disease was 1 1 . 1 f 8.3 years (range 2-25). Six of the 7 patients were rheumatoid factor positive. Five of the 7 patients were women. The patients received 7.5 mg of MTX orally each week. Five patients received concomitant therapy with nonsteroidal antiinflammatory drugs; none had received corticosteroids within a month before the study or during the study. Each patient was interviewed and examined in the early morning by the same physician-investigator, at study entry and at the followup visit 6-8 weeks later. The following clinical variables were assessed at both evaluations: the number of swollen joints (among 66 diarthrodial joints), the number of joints with tenderness on pressure or with pain on passive motion (or both), the duration of morning stiffness (in minutes), and the physician's and patient's global assessment of disease activity (graded on a scale of 0-4). Serum MTX levels were measured in samples obtained immediately before and the day after the MTX doses. Cell isolation and activation. Blood samples were obtained immediately before and the day after the MTX dose. Blood was drawn into tubes containing sodium citrate, and leukocytes were isolated by dextran sedimentation and discontinuous Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradient centrifugation (17). The pelleted neutrophils were purified of contaminating erythrocytes by hypotonic lysis. To quantitate the generation of LTB, and platelet-activating factor (PAF), neutrophils were suspended at a cell density of 1 x lo7 cells/ml in Tyrode's-HEPES-gelatin buffer (a modified Tyrode's buffer without bicarbonate and with 2.5 mM K+,0.5 mM MgZ+,1.5 mM Ca2', 30 mM HEPES, and 1 mg/ml gelatin, pH 7.4). Portions of the cells (400 pl each) were preincubated in a 37°C shaking water-bath for 5 minutes, and duplicate samples were activated by the addition of 400 pl of prewarmed Tyrode's-HEPES-gelatin buffer containing 20 pM calcium ionophore A23187 (final concentration 10 pM). Duplicate control samples were treated in the same way, except that calcium ionophore was omitted from the added Tyrode's-HEPES-gelatin buffer. The reactions were stopped after 15 minutes by cooling the samples to 0°C. The cells were sedimented at 10,OOOg for 1 minute at O"C, the supernatants were harvested, and the cell pellets were extracted with 800 pl of methanol (18). Both fractions were stored under a nitrogen atmosphere at -20°C. Monocytes from the mononuclear cells at the interfaces of the discontinuous gradients were purified further by adhesion to plastic (18). Monocyte monolayers were preincubated in Tyrode's-HEPES-gelatin buffer at 37°C for 15 minutes. The supernatants were discarded, and duplicate monolayers were incubated for 15 minutes either with 1 ml of 10 p M calcium ionophore A23187 in Tyrode's-HEPESgelatin buffer or with the buffer alone. At the end of the incubation period, the fluid over the monocyte monolayers was decanted and retained, and the monocyte monolayers were extracted with 1 ml of methanol. Both the supernatants and the extracts were stored under a nitrogen atmosphere at -20°C.
*
SPERLING ET AL
378
Quantitation of LTB, and PAF by radioimmunoassay. Portions of the methanolic cell pellet extracts were dried under reduced pressure and redissolved in 100 pl of IsogelTris buffer (10 mM Tris HCI, pH 7.4, in O.15M NaCI, with 1 mg/ml of gelatin) before radioimmunoassay. Portions of the cell supernatants (diluted in Isogel-Tris buffer) and the redissolved cell pellet extracts from control and calcium ionophore-activated samples were assayed for LTB, by radioimmunoassay, using a commercial kit (Du Pont-New England Nuclear, Boston, MA) with less than 3% crossreactivity for other arachidonic acid-derived 5-LO products (l7,19). Supernatants and redissolved cell pellet extracts from control and calcium ionophoreactivated samples were assayed for PAF with a commercial radioimmunoassay kit (Du Pont-New England Nuclear). Quantities of immunoreactive LTB, and PAF were determined on the linear portion of the displacement curve (17,19,20). Quantitation of 5-lipoxygenase products by reversephase high-performance liquid chromatography (RP-HPLC). Duplicate portions of neutrophils suspended in Tyrode'sHEPES buffer with 1 mglml bovine serum albumin added (Tyrode's-HEPES-albumin buffer) were preincubated in parallel for 5 minutes at 37°C and activated by the addition of an equal volume of 20 pM calcium ionophore A23187 in Tyrode's-HEPES-albumin buffer; the samples were processed as described above. Portions (600 PI) of the cell supernatant and the corresponding methanolic cell pellet extract were combined, and -100,OOO counts per minute 'H-labeled LTB, (as an internal standard) and 100 ng of prostaglandin B, (PGB,; as a retention-time standard) were added. The samples were chilled at 0°C for 10 minutes and centrifuged at 1 ,OOOg for 10 minutes to remove any precipitate. The samples were injected into a 3-pm Beckman Ultrasphere ODS column (4.6 X 75-mm; Beckman, Palo Alto, CA) which had been preequilibrated with 89.6% solvent A (0.1% aqueous ammonium acetate (pH 5.6): methanol:acetonitrile, 70:25:5 volumeholume) and 10.4% solvent B (methanol:acetonitrile, 9 5 5 v/v). The column was eluted at a flow rate of 1 muminute with a 30-minute linear gradient from 10.4% to 100% solvent B. The RP-HPLC system consisted of a 2-pump Rainin Rabbit HPLC, a Macintosh controller running the Dynamax program (Rainin, Woburn, MA), and a Gilson 116 online ultraviolet detector. 5-LO products were quantitated by integrated optical density. With this system, mean SEM retention times were as follows: 20-carboxy-LTB4 8.47 0.15 minutes, 20-hydroxy-LTB4 14.26 ? 0.08 minutes, PGB, 23.50 0.04 minutes, 5S, 12R-6-trans-LTB4 (6-trans-LTB4) 27.09 5 0.46 minutes, LTB, 29.41 ? 0.18 minutes, and 5-hydroxyeicosatetraenoicacid (5-HETE) 48.06 2 0.20 minutes. The recoveries of 'H-labeled LTB, and 'H-labeled 5-HETE from the HPLC column were 93.7 ? 3.4% and 90.2 2 1.8%, respectively (mean 2 SD); the overall recovery of 'H-labeled LTB, was 72.2 -+ 3.3%. Synthetic LTB, and 6trans-LTB4 diastereoisomers were prepared as described elsewhere (21); synthetic 5-HETE and 20-hydroxy- and 20-carboxy-LTB4 were obtained from Cayman Chemical (Ann Arbor, MI). Tritium-labeled arachidonic acid (AA) released from prelabeled neutrophils was measured as described previ-
*
*
*
ously (22). Briefly, neutrophils were incubated with 'HHlabeled AA for 1 hour at 37"C, washed 3 times with Hanks' balanced salt solution without divalent cations and containing bovine serum albumin, prewarmed for 5 minutes, and activated with the calcium ionophore, A23 187, as described above. Chemotaxis assay. The chemotactic responses of neutrophils to concentrations of synthetic LTB, and N-formyl-methionyl-leucyl-phenylalanine(FMLP) were assessed in Boyden microchambers by determining the distance migrated by the leading edge and the number of cells that migrated the same distance as the leading edge in control samples (17,23). The experiments were performed in duplicate; the interassay and intraassay coefficients of variation were
ACUTE AND CHRONIC SUPPRESSION OF LEUKOTRIENE B, SYNTHESIS EX VIVO IN NEUTROPHILS FROM PATIENTS WITH RHEUMATOID ARTHRITIS BEGINNING TREATMENT WITH METHOTREXATE RICHARD I. SPERLING, ANTHONY I. BENINCASO, RONALD J. ANDERSON, JONATHAN S. COBLYN, K. FRANK AUSTEN, and MICHAEL E. WEINBLATT
Objective. To compare the cumulative effects of oral methotrexate (MTX) therapy (after 68 weeks) with the acute effects (24 hours after a dose) on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from patients with active rheumatoid arthritis (RA) who were beginning therapy with MTX. Methods. Neutrophils and monocytes were isolated from whole blood from 7 patients with RA, immediately before and 24 hours after their first weekly dose of 7.5 mg of MTX, and again after their dose at 6-8 weeks. Results. Total immunoreactive leukotriene B, (LTB,) formation in neutrophils activated ex vivo with calcium iono-
From the Department of Medicine, Harvard Medical School, and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts. Supported by NIH grants AR-35907, AI-2253 1, AI-23401, RR-05950, and AR-38638, a postdoctoral research grant and an Arthritis Biomedical Research Center grant from the Arthritis Foundation, and by a research grant from Lederle Laboratories. Richard I. Sperling, AM, MD: Assistant Professor of Medicine, Harvard Medical School, and 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; Ronald J. Anderson, MD: Associate 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; 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 July 5, 1991; accepted in revised form November 13, 1991. Arthritis and Rheumatism, Vol. 35, No. 4 (April 1992)
phore A23187 was significantly suppressed (by 33%) before the 68-week dose, compared with the level before the first dose (mean f SEM 8.29 f 1.24 ng/106 cells at predose 68 weeks versus 12.29 f 2.13 ng/106 cells at predose 1; P = 0.03). Reductions were also observed after the first dose (27%; P = 0.07) and after the 6-8-week dose (43%; P = 0.05) compared with the respective predose levels. MTX treatment produced significant reductions in the total generation of 5-LO pathway 6-trans-LTB4 products (5-hydroxyeicosatetraenoicacid LTB, -oxidation products of LTB,) by calcium ionophore-activated neutrophils, as quantitated by integrated optical density after resolution on reverse-phase high-performance liquid chromatography. Decreases were observed after the first dose dose (26%;P = 0.025), immediately before the --week (23%; P = 0.05), and after the 6-8-week dose (47%; P = 0.0033) compared with levels before the first dose, and after the 68-week dose compared with the level before it (32%; P = 0.04). The generation of LTB, by calcium ionophorwwtivated monocytes was not significantly affected by MTX therapy. Conclusion. The significant decreases in the formation of *oxidation products of LTB, and in the total generation of neutrophil 5-LO pathway products in the absence of a significant change in the release of 'H-arachidonic acid or the generation of platelet-activating factor suggest that the activity of the 5-LO enzyme in neutrophils is inhibited. We conclude that weekly oral MTX therapy in patients with active RA inhibits neutrophil 5-LO pathway product generation in a pattern consistent with inhibition of the activity of the 5-LO enzyme; an effect is observed after the first dose. The inhibition of 5-LO is cell-selective and cumulative, with a superimposed incremental inhibition observed after the weekly MTX dose.
+
+
+
Low-dose, weekly therapy with methotrexate
(MTX)is efficacious in patients with active rheumatoid arthritis (RA) (1-4); however, its mechanism of action in RA remains to be defined (2,3,5-7). In the decade
LTB, SYNTHESIS AND MTX THERAPY following the clinical introduction of MTX and its related antifolate congeners as antineoplastic chemotherapeutic agents in 1948 (8), the immunosuppressive effects of high-dose MTX became apparent (9). Some investigators have inferred that low-dose, pulse MTX exerts its therapeutic effect in RA through an unknown antiinflammatory mechanism (2,3,5,7), since a therapeutic response occurs as early as 3-6 weeks (2,4) after the initiation of therapy, in the absence of consistent immunosuppressive effects seen ex vivo (2,3,5). MTX has been reported to inhibit the chemotactic response of neutrophils in vivo in patients with psoriasis (lO,ll), in vivo in mice (12), in vitro in healthy human volunteer donors (13), and ex vivo in patients with RA after treatment with an initial oral dose of MTX (14). In a previous study, we evaluated the effects of a single oral dose of MTX on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from 6 patients with RA, which had been obtained 1 day before and 1 day after their usual weekly MTX dose (15). The patients had received a mean of 61 months of weekly MTX therapy before enrolling in the study. One day after the weekly MTX dose, the total generation of leukotriene B, (LTB,) by neutrophils stimulated ex vivo for 20 minutes with 10 @ calcium ionophore A23 187 was suppressed significantly, by a mean of 53%, compared with the predose level (15). Both the released and the cell-retained LTB,, as well as the *oxidation products of LTB, were comparably suppressed, indicating that decreased intracellular LTB, biosynthesis, rather than impaired release or augmented degradation, was responsible for the MTX-related decrease in LTB, generation. In the present study, we investigated the curnulative effects of MTX therapy on the generation of neutrophil 5-lipoxygenase pathway products after 6 8 weeks of treatment, a time point at which most patients begin to show a clinical response to MTX, as well as the acute effects 24 hours after a single dose. We compared the effects on neutrophils with the effects on peripheral blood monocytes. The findings are presented below.
PATIENTS AND METHODS Patients. Informed consent was obtained from 7 patients with RA who were to begin weekly oral MTX therapy. Their mean & SD age was 58.3 ? 10.1 years (range 42-72), and all met the American College of Rheumatology
377 (formerly, the American Rheumatism Association) 1987 revised criteria for RA (16). The mean SD duration of disease was 1 1 . 1 f 8.3 years (range 2-25). Six of the 7 patients were rheumatoid factor positive. Five of the 7 patients were women. The patients received 7.5 mg of MTX orally each week. Five patients received concomitant therapy with nonsteroidal antiinflammatory drugs; none had received corticosteroids within a month before the study or during the study. Each patient was interviewed and examined in the early morning by the same physician-investigator, at study entry and at the followup visit 6-8 weeks later. The following clinical variables were assessed at both evaluations: the number of swollen joints (among 66 diarthrodial joints), the number of joints with tenderness on pressure or with pain on passive motion (or both), the duration of morning stiffness (in minutes), and the physician's and patient's global assessment of disease activity (graded on a scale of 0-4). Serum MTX levels were measured in samples obtained immediately before and the day after the MTX doses. Cell isolation and activation. Blood samples were obtained immediately before and the day after the MTX dose. Blood was drawn into tubes containing sodium citrate, and leukocytes were isolated by dextran sedimentation and discontinuous Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradient centrifugation (17). The pelleted neutrophils were purified of contaminating erythrocytes by hypotonic lysis. To quantitate the generation of LTB, and platelet-activating factor (PAF), neutrophils were suspended at a cell density of 1 x lo7 cells/ml in Tyrode's-HEPES-gelatin buffer (a modified Tyrode's buffer without bicarbonate and with 2.5 mM K+,0.5 mM MgZ+,1.5 mM Ca2', 30 mM HEPES, and 1 mg/ml gelatin, pH 7.4). Portions of the cells (400 pl each) were preincubated in a 37°C shaking water-bath for 5 minutes, and duplicate samples were activated by the addition of 400 pl of prewarmed Tyrode's-HEPES-gelatin buffer containing 20 pM calcium ionophore A23187 (final concentration 10 pM). Duplicate control samples were treated in the same way, except that calcium ionophore was omitted from the added Tyrode's-HEPES-gelatin buffer. The reactions were stopped after 15 minutes by cooling the samples to 0°C. The cells were sedimented at 10,OOOg for 1 minute at O"C, the supernatants were harvested, and the cell pellets were extracted with 800 pl of methanol (18). Both fractions were stored under a nitrogen atmosphere at -20°C. Monocytes from the mononuclear cells at the interfaces of the discontinuous gradients were purified further by adhesion to plastic (18). Monocyte monolayers were preincubated in Tyrode's-HEPES-gelatin buffer at 37°C for 15 minutes. The supernatants were discarded, and duplicate monolayers were incubated for 15 minutes either with 1 ml of 10 p M calcium ionophore A23187 in Tyrode's-HEPESgelatin buffer or with the buffer alone. At the end of the incubation period, the fluid over the monocyte monolayers was decanted and retained, and the monocyte monolayers were extracted with 1 ml of methanol. Both the supernatants and the extracts were stored under a nitrogen atmosphere at -20°C.
*
SPERLING ET AL
378
Quantitation of LTB, and PAF by radioimmunoassay. Portions of the methanolic cell pellet extracts were dried under reduced pressure and redissolved in 100 pl of IsogelTris buffer (10 mM Tris HCI, pH 7.4, in O.15M NaCI, with 1 mg/ml of gelatin) before radioimmunoassay. Portions of the cell supernatants (diluted in Isogel-Tris buffer) and the redissolved cell pellet extracts from control and calcium ionophore-activated samples were assayed for LTB, by radioimmunoassay, using a commercial kit (Du Pont-New England Nuclear, Boston, MA) with less than 3% crossreactivity for other arachidonic acid-derived 5-LO products (l7,19). Supernatants and redissolved cell pellet extracts from control and calcium ionophoreactivated samples were assayed for PAF with a commercial radioimmunoassay kit (Du Pont-New England Nuclear). Quantities of immunoreactive LTB, and PAF were determined on the linear portion of the displacement curve (17,19,20). Quantitation of 5-lipoxygenase products by reversephase high-performance liquid chromatography (RP-HPLC). Duplicate portions of neutrophils suspended in Tyrode'sHEPES buffer with 1 mglml bovine serum albumin added (Tyrode's-HEPES-albumin buffer) were preincubated in parallel for 5 minutes at 37°C and activated by the addition of an equal volume of 20 pM calcium ionophore A23187 in Tyrode's-HEPES-albumin buffer; the samples were processed as described above. Portions (600 PI) of the cell supernatant and the corresponding methanolic cell pellet extract were combined, and -100,OOO counts per minute 'H-labeled LTB, (as an internal standard) and 100 ng of prostaglandin B, (PGB,; as a retention-time standard) were added. The samples were chilled at 0°C for 10 minutes and centrifuged at 1 ,OOOg for 10 minutes to remove any precipitate. The samples were injected into a 3-pm Beckman Ultrasphere ODS column (4.6 X 75-mm; Beckman, Palo Alto, CA) which had been preequilibrated with 89.6% solvent A (0.1% aqueous ammonium acetate (pH 5.6): methanol:acetonitrile, 70:25:5 volumeholume) and 10.4% solvent B (methanol:acetonitrile, 9 5 5 v/v). The column was eluted at a flow rate of 1 muminute with a 30-minute linear gradient from 10.4% to 100% solvent B. The RP-HPLC system consisted of a 2-pump Rainin Rabbit HPLC, a Macintosh controller running the Dynamax program (Rainin, Woburn, MA), and a Gilson 116 online ultraviolet detector. 5-LO products were quantitated by integrated optical density. With this system, mean SEM retention times were as follows: 20-carboxy-LTB4 8.47 0.15 minutes, 20-hydroxy-LTB4 14.26 ? 0.08 minutes, PGB, 23.50 0.04 minutes, 5S, 12R-6-trans-LTB4 (6-trans-LTB4) 27.09 5 0.46 minutes, LTB, 29.41 ? 0.18 minutes, and 5-hydroxyeicosatetraenoicacid (5-HETE) 48.06 2 0.20 minutes. The recoveries of 'H-labeled LTB, and 'H-labeled 5-HETE from the HPLC column were 93.7 ? 3.4% and 90.2 2 1.8%, respectively (mean 2 SD); the overall recovery of 'H-labeled LTB, was 72.2 -+ 3.3%. Synthetic LTB, and 6trans-LTB4 diastereoisomers were prepared as described elsewhere (21); synthetic 5-HETE and 20-hydroxy- and 20-carboxy-LTB4 were obtained from Cayman Chemical (Ann Arbor, MI). Tritium-labeled arachidonic acid (AA) released from prelabeled neutrophils was measured as described previ-
*
*
*
ously (22). Briefly, neutrophils were incubated with 'HHlabeled AA for 1 hour at 37"C, washed 3 times with Hanks' balanced salt solution without divalent cations and containing bovine serum albumin, prewarmed for 5 minutes, and activated with the calcium ionophore, A23 187, as described above. Chemotaxis assay. The chemotactic responses of neutrophils to concentrations of synthetic LTB, and N-formyl-methionyl-leucyl-phenylalanine(FMLP) were assessed in Boyden microchambers by determining the distance migrated by the leading edge and the number of cells that migrated the same distance as the leading edge in control samples (17,23). The experiments were performed in duplicate; the interassay and intraassay coefficients of variation were
