[60]
TRANSCELLULARBIOSYNTHESIS
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ethidium permeability can be monitored in monolayers grown on coverslips as described by Gomperts. 2s Contraction of Mesangial Cells. Low-density monolayers of mesangial cells on three-dimensional, type I collagen gels are incubated with contractile agonists and the decrease in cross-sectional area of individual cells is measured using computer-aided microscopy or photomicroscopy. 29"3° Type I collagen gels are prepared by mixing 8.0 ml of 2.9 mg/ml collagen in 0.012 N HCI, 1.0 mi of 1 N NaOH, and 1.0 ml of 10x minimal essential medium, all from sterile solutions at 4 °, and adjusting to pH 7.4. Aliquots (1 ml) are plated into two-chamber Lab-Tek slides and gelation initiated by warming to 37° for 1 hr. Contraction is tested 24 hr after subculture onto the collagen gel by incubating cells plus or minus agonists in HBSS with 10 mM HEPES (pH 7.4), and 2 mg/ml fatty acid-free bovine serum albumin. The change in cross-sectional for individual cells area over time is measured using computer-aided microscopy as previously described, z9'3° A cell is scored positive for contraction with a decrease in cross-sectional area ->7%. Acknowledgments This work was supported by National Institutes of Health Grants HL-22563 and HL-37117. z8 B. D. Gomperts, Nature (London) 306, 64 (1983). z9 M. S. Simonson and M. J. Dunn, Kidney Int. 30, 524 (1986). 30 M. S. Simonson, BioTechniques 3, 484 (1985).
[ 6 0 ] L e u k o t r i e n e B4 B i o s y n t h e s i s b y Erythrocyte-Neutrophil Interactions
By DAVID A. JONES and FRANK A. FITZPATRICK Enzymatic cooperation between different cell types has been observed for biosynthesis of several eicosanoids. 1-12 This process, termed transcellular biosynthesis, represents a novel mechanism for the production of i S. Bunting, R. Gryglewski, S. Moncada, and J. R. Vane, Prostaglandins 12, 897 (1976). 2 A. Marcus, B. Weksler, E. Jaffe, and J. Broekman, J. Clin. Invest. 66, 979 (1980). 3 A. Marcus, M. J. Broekman, B. Weksler, E. Jaffe, L. Sailer, H. Ullman, and K. TackGoldman, Philos. Trans. R. Soc. London B294, 343 (1981).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
554
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[60]
prostaglandins and leukotrienes. Transcellular biosynthesis is important from several perspectives. For instance, the quantitative and qualitative features of eicosanoid production via cell-cell cooperation differ from the sum of the separate cellular biosynthetic capabilities. Second, cell combinations encountered in diseases often differ from those encountered in normal circumstances. Thus, better characterization of transcellular eicosanoid biosynthesis should improve our comprehension of the role of eicosanoids in the cellular biology of inflammation and thrombosis. Experiments on transcellular biosynthesis should conform to certain criteria: (i) are the cells under investigation likely to occur together in vivo; (ii) are the ratios of the respective cells likely to occur together in vivo; (iii) are the cells sufficiently pure to provide unambiguous conclusions regarding the origin of the eicosanoid under study; and (iv) do transformations of synthetic substrate and cellularly "donated" substrate correspond. We outline below the experimental procedures to assess these criteria for investigations on erythrocyte-neutrophil interactions. The original investigations demonstrated that combinations of human neutrophils plus erythrocytes, with appropriate stimulation, produce more leukotriene B4 (LTB4) than neutrophil suspensions alone. ~2 This was significant because erythrocytes do not contain detectable phospholipase A2 or 5-1ipoxygenase activity and have therefore been regarded physiologically inert in terms of leukotriene formation. This opinion is no longer warranted.
Experimental Procedure Reagents Sodium citrate (Sigma, St. Louis, MO): 3.8% (w/v) in sterile saline NaCI (Sigma): 0.9% (w/v) in sterile, distilled water Cellulose mixture: microcrystalline cellulose (Sigma cat. No. C-8002), a-cellulose (Sigma cat. No. S-5504) (1 : 1, w/w) suspended in 0.9% NaC1 4 j. Maclouf and R. C. Murphy, J. Biol. Chem. 263, 174 (1988). 5 C. Dahinden, T. Clancy, M. Gross, J. Chiller, and T, Hugh, Proc. Natl. Acad. Sci. U.S.A. 82, 6632 (1985). 6 A. Marcus, L. Sailer, H. Ullman, M. Broekman, N. Islam, T. Oglesby, and R. Gorman, Proc. Natl. Acad. Sci. U.S.A. 81, 907 (1984). 7 S. Feinmark and P. Cannon, J. Biol. Chem. 261, 16466 (1986). s S. Feinmark and P. Cannon, Biochim. Biophys. Acta 922, 125 (1987). 9 j. A. Claesson and J. Haeggstrom, Adv. Prostaglandin. Thromboxane Leukotriene 17, 115 (1987). to p. Needleman, A. Wyche, and A. Raz, J. Clin. Invest. 63, 345 (1979). 1~ G. Hornstra, E. Haddeman, and J. Don, Nature (London) 279, 66 (1979). 12 j. E. McGee and F. A. Fitzpatrick, Proc. Natl. Acad. Sci. U.S.A. 83, 1349 (1986).
[60]
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Hanks' balanced salt solution (Gibco Labs, Gaithersburg, MD) A23187 divalent calcium ionophore (Calbiochem, San Diego, CA) Ethyl acetate (Burdick & Jackson, Muskegon, MI) Mobile phase: methanol/water/glacial acetic acid (70:30:0.1) Prostaglandin B ~ (Sigma) L T A 4 methyl ester (The Upjohn Co., Kalamazoo, MI) Human serum albumin (Sigma) LiOH (Sigma) Tetrahydrofuran (Burdick & Jackson)
Isolation o f Human Erythrocytes
This isolation procedure is a modification of that presented by Beutler et al. 13 The coagulation step is omitted to avoid activation of different cell types. Human venous blood (from drug-free volunteers) is collected using sterile sodium citrate as an anticoagulant (9: 1, v/v). The whole blood is then centrifuged at 200 g for 30 rain at 25 ° and the platelet-rich plasma pipetted off, removing as much of the buffy coat as possible. The sedimented cells are resuspended to the original volume in 0.9% NaCI and again centrifuged at 200 g for 20 min at 25 °. The supernatant is discarded and the wash repeated. Suspended erythrocytes are further purified by filtering through a microcrystalline cellulose-a-cellulose column to remove residual neutrophils and platelets. Columns can be prepared in 5 c m 3 syringes, sedimenting the cellulose mixture to a depth of 1.5 cm. Suspensions are filtered at a flow rate of 2 ml/min using 0.9% NaCI as an elution buffer. The erythrocytes are resuspended to the original whole blood volume in saline. Finally, erythrocytes, neutrophils, and platelet concentrations are determined by differential cell counting with a Coulter Diff. Typically, at each stage of sample purification, 20/zl of the cell suspension are diluted with 20.0 ml of Isoton II counting buffer prior to particle counting. Values for six representative preparations (Table I) illustrate that purified erythrocytes typically contain --
TRANSCELLULARBIOSYNTHESIS
553
ethidium permeability can be monitored in monolayers grown on coverslips as described by Gomperts. 2s Contraction of Mesangial Cells. Low-density monolayers of mesangial cells on three-dimensional, type I collagen gels are incubated with contractile agonists and the decrease in cross-sectional area of individual cells is measured using computer-aided microscopy or photomicroscopy. 29"3° Type I collagen gels are prepared by mixing 8.0 ml of 2.9 mg/ml collagen in 0.012 N HCI, 1.0 mi of 1 N NaOH, and 1.0 ml of 10x minimal essential medium, all from sterile solutions at 4 °, and adjusting to pH 7.4. Aliquots (1 ml) are plated into two-chamber Lab-Tek slides and gelation initiated by warming to 37° for 1 hr. Contraction is tested 24 hr after subculture onto the collagen gel by incubating cells plus or minus agonists in HBSS with 10 mM HEPES (pH 7.4), and 2 mg/ml fatty acid-free bovine serum albumin. The change in cross-sectional for individual cells area over time is measured using computer-aided microscopy as previously described, z9'3° A cell is scored positive for contraction with a decrease in cross-sectional area ->7%. Acknowledgments This work was supported by National Institutes of Health Grants HL-22563 and HL-37117. z8 B. D. Gomperts, Nature (London) 306, 64 (1983). z9 M. S. Simonson and M. J. Dunn, Kidney Int. 30, 524 (1986). 30 M. S. Simonson, BioTechniques 3, 484 (1985).
[ 6 0 ] L e u k o t r i e n e B4 B i o s y n t h e s i s b y Erythrocyte-Neutrophil Interactions
By DAVID A. JONES and FRANK A. FITZPATRICK Enzymatic cooperation between different cell types has been observed for biosynthesis of several eicosanoids. 1-12 This process, termed transcellular biosynthesis, represents a novel mechanism for the production of i S. Bunting, R. Gryglewski, S. Moncada, and J. R. Vane, Prostaglandins 12, 897 (1976). 2 A. Marcus, B. Weksler, E. Jaffe, and J. Broekman, J. Clin. Invest. 66, 979 (1980). 3 A. Marcus, M. J. Broekman, B. Weksler, E. Jaffe, L. Sailer, H. Ullman, and K. TackGoldman, Philos. Trans. R. Soc. London B294, 343 (1981).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
554
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[60]
prostaglandins and leukotrienes. Transcellular biosynthesis is important from several perspectives. For instance, the quantitative and qualitative features of eicosanoid production via cell-cell cooperation differ from the sum of the separate cellular biosynthetic capabilities. Second, cell combinations encountered in diseases often differ from those encountered in normal circumstances. Thus, better characterization of transcellular eicosanoid biosynthesis should improve our comprehension of the role of eicosanoids in the cellular biology of inflammation and thrombosis. Experiments on transcellular biosynthesis should conform to certain criteria: (i) are the cells under investigation likely to occur together in vivo; (ii) are the ratios of the respective cells likely to occur together in vivo; (iii) are the cells sufficiently pure to provide unambiguous conclusions regarding the origin of the eicosanoid under study; and (iv) do transformations of synthetic substrate and cellularly "donated" substrate correspond. We outline below the experimental procedures to assess these criteria for investigations on erythrocyte-neutrophil interactions. The original investigations demonstrated that combinations of human neutrophils plus erythrocytes, with appropriate stimulation, produce more leukotriene B4 (LTB4) than neutrophil suspensions alone. ~2 This was significant because erythrocytes do not contain detectable phospholipase A2 or 5-1ipoxygenase activity and have therefore been regarded physiologically inert in terms of leukotriene formation. This opinion is no longer warranted.
Experimental Procedure Reagents Sodium citrate (Sigma, St. Louis, MO): 3.8% (w/v) in sterile saline NaCI (Sigma): 0.9% (w/v) in sterile, distilled water Cellulose mixture: microcrystalline cellulose (Sigma cat. No. C-8002), a-cellulose (Sigma cat. No. S-5504) (1 : 1, w/w) suspended in 0.9% NaC1 4 j. Maclouf and R. C. Murphy, J. Biol. Chem. 263, 174 (1988). 5 C. Dahinden, T. Clancy, M. Gross, J. Chiller, and T, Hugh, Proc. Natl. Acad. Sci. U.S.A. 82, 6632 (1985). 6 A. Marcus, L. Sailer, H. Ullman, M. Broekman, N. Islam, T. Oglesby, and R. Gorman, Proc. Natl. Acad. Sci. U.S.A. 81, 907 (1984). 7 S. Feinmark and P. Cannon, J. Biol. Chem. 261, 16466 (1986). s S. Feinmark and P. Cannon, Biochim. Biophys. Acta 922, 125 (1987). 9 j. A. Claesson and J. Haeggstrom, Adv. Prostaglandin. Thromboxane Leukotriene 17, 115 (1987). to p. Needleman, A. Wyche, and A. Raz, J. Clin. Invest. 63, 345 (1979). 1~ G. Hornstra, E. Haddeman, and J. Don, Nature (London) 279, 66 (1979). 12 j. E. McGee and F. A. Fitzpatrick, Proc. Natl. Acad. Sci. U.S.A. 83, 1349 (1986).
[60]
TRANSCELLULAR BIOSYNTHESIS
555
Hanks' balanced salt solution (Gibco Labs, Gaithersburg, MD) A23187 divalent calcium ionophore (Calbiochem, San Diego, CA) Ethyl acetate (Burdick & Jackson, Muskegon, MI) Mobile phase: methanol/water/glacial acetic acid (70:30:0.1) Prostaglandin B ~ (Sigma) L T A 4 methyl ester (The Upjohn Co., Kalamazoo, MI) Human serum albumin (Sigma) LiOH (Sigma) Tetrahydrofuran (Burdick & Jackson)
Isolation o f Human Erythrocytes
This isolation procedure is a modification of that presented by Beutler et al. 13 The coagulation step is omitted to avoid activation of different cell types. Human venous blood (from drug-free volunteers) is collected using sterile sodium citrate as an anticoagulant (9: 1, v/v). The whole blood is then centrifuged at 200 g for 30 rain at 25 ° and the platelet-rich plasma pipetted off, removing as much of the buffy coat as possible. The sedimented cells are resuspended to the original volume in 0.9% NaCI and again centrifuged at 200 g for 20 min at 25 °. The supernatant is discarded and the wash repeated. Suspended erythrocytes are further purified by filtering through a microcrystalline cellulose-a-cellulose column to remove residual neutrophils and platelets. Columns can be prepared in 5 c m 3 syringes, sedimenting the cellulose mixture to a depth of 1.5 cm. Suspensions are filtered at a flow rate of 2 ml/min using 0.9% NaCI as an elution buffer. The erythrocytes are resuspended to the original whole blood volume in saline. Finally, erythrocytes, neutrophils, and platelet concentrations are determined by differential cell counting with a Coulter Diff. Typically, at each stage of sample purification, 20/zl of the cell suspension are diluted with 20.0 ml of Isoton II counting buffer prior to particle counting. Values for six representative preparations (Table I) illustrate that purified erythrocytes typically contain --
