DOWNREGULATION OF TUMOR NECROSIS FACTOR RECEPTORS ON MACROPHAGES AND ENDOTHELIAL CELLS BY MICROTUBULE DEPOLYMERIZING AGENTS BY
AIHAO H . DING, FRANCOISE PORTEU, ELIZABETH SANCHEZ, AND CARL F. NATHAN
From the Beatrice and Samuel A . Seaver Laboratory, Division of Hematology-Oncology, Department of Medicine, Cornell University Medical College, New York, New York 10021
TNFa is a potent mediator ofinflammation (1, 2). Its actions are believed to result from binding to a single class of receptors (TNF ctR) on the surface of a wide variety of cells (3-6). In principle, agents that affect the number or affinity of TNFc1R, such as IFN- ,y (7-9), LPS (10), or activators of protein kinase C or A (11-14),
may modulate some of TNF-a's biologic effects . Among antineoplastic and nonsteroidal antiinflammatory drugs are compounds, such as colchicine, nocodazole, vincristine, vinblastine, and podophyllotoxin, which bind tubulin and inhibit its polymerization. Disassembly of cytoplasmic microtubules (MT)1 by these agents causes mitotic arrest in dividing cells, suggesting a mechanism of action for their antineoplastic effects . It is less clear how colchicine exerts antiinflammatory actions in conditions as diverse as gout, familial Mediterranean fever, cirrhosis, pericarditis, and Behcet's disease . Possible mechanisms include inhibition of the ability of polymorphonuclear leukocytes to migrate (15), degranulate (16), or translocate 5-lipoxygenase (17). We have observed that exposure of murine and human macrophages and human umbilical vein endothelial cells to micromolar concentrations of five different MT depolymerizing agents resulted in a rapid decrease in surface TNF ciR . MT appeared to be required either for the synthesis of TNFaR or for their translocation to the plasma membrane . This effect may contribute to the antiinflammatory actions of MT depolymerizing agents .
Materials and Methods
Materials . Human rTNFce (4.1 x 107 U/mg protein), murine rINFy (5 .2 x 107 U/mg protein) and antiserum 15E against murine TNFa (6 .4 x 105 neutralizing units/ml) were kindly provided by Genentech, Inc . (South San Francisco, CA) . Na 1211 (carrier-free) was from the Radiochemical Center, Amersham, UK . Thioglycollate broth (Brewer) was obtained from Difco Laboratories (Detroit, MI) . The following were from Sigma Chemical Co. (St . Louis, MO) : colchicine, fl-lumicolchicine, y-lumicolchicine, nocodazole, vinblastine, vincristine, podophyllotoxin, cytochalasin B, dihydrocytochalasin B, cytochalasin E, PGE,, dibutyryl-cyclic adenosine monophosphate, indomethacin, ibuprofen, acetylsalicylic acid, and cycloheximide.
Address correspondence to Dr. A. H. Ding, Cornell University Medical College, 1300 York Avenue, Box 57, New York, NY 10021 . ' Abbreviation used in this paper. MT, microtubule. MED. C The Rockefeller University Press " 0022-1007/90/03/0715/13 $2 .00 Volume 171 March 1990 715-727
J . Exp.
715
716
MICROTUBULE REGULATION OF TNF RECEPTORS
Cell Culture. Murine macrophages were washed from the peritoneal cavities offemale CDl mice (Charles River Breeding Laboratories, Wilmington, MA) . Where indicated, mice were injected intraperitoneally with 2 ml of thioglycollate broth (Brewer, 4%) 4 d earlier as described (18). Adherent monolayers were obtained by plating 8 x 105 cells per well in 24-well plastic trays or 2 x 105 cells per well in 96-well plastic trays in RPMI 1640 (KC Biological, Inc., Lenexa, KS) containing 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT) . Monolayers were maintained at 37°C in 5% C02/95 17o air for 1-4 d before the experiments . Human macrophages were allowed to mature from peripheral blood monocytes in Teflon beakers as described (19). Second-passage human umbilical vein endothelial cells were the gift ofDr. EricJaffe (Cornell University Medical College, New York, NY) and were prepared as described (20). Human polymorphonuclear leukocytes (PMN) were isolated from heparinized venous blood on Neutrophil Isolation Medium (Los Alamos Diagnostics, Los Alamos, NM) as described (21). Iodination ofrTNF-a and rIFN-y. Human rTNFa and murine rIFN- y were iodinated according to the method o£ Aggarwal et al. (22), and stored at VC for no longer than 6 wk. In brief, 100 p.l of 0.1 M phosphate buffer (pH 7 .4) was added to a glass tube coated with 5 lAg of Iodogen (Pierce Chemical Co., Rockford, IL) and incubated with 1 mCi of Na' 25I for 10 min at 4oC . The mixture was then transferred to a tube containing 10 Rg of rTNFa or rIFN-y for 10 min at 4°C . The reaction was stopped by addition of one drop of 1 M KI. Iodinated material was separated from free iodide by filtration through a PD-10 column (Pharmacia Fine Chemicals, Piscataway, NJ) that had been equilibrated with 0.1 M phosphate (for rTNF-a) or 20 mM Tris buffer (for rIFN-y), both containing 0.1% gelatin, pH 7.4. '25 1-rTNFa had a specific activity of -300 Ci/mmol monomer, and migrated as a single band with an apparent molecular mass of -16 kD on an 1117o polyacrylamide gel under nonreducing conditions. '21I-rIFN-y had a specific activity of -100 Ci/mmol monomer. Both cytokines retained their ability to activate resident mouse peritoneal macrophages for enhanced H202 release (18). Receptor Binding Assay. Cell monolayers in 24-well trays were overlaid with 0.3 ml per well ofbinding buffer (HBSS containing 10% FCS) and incubated with indicated concentrations of 1251-rTNFot in the presence or absence of a 100-fold excess of unlabeled rTNFa . After 3-4 h at 4oC, binding buffer was removed and the monolayers rinsed four times with ice cold 0.9% NaCl. Cells were solubilized in 0.3 ml of 0.3 N NaOH and radioactivity determined in a gamma counter (Packard Instrument Co., Downers Grove, IL) . Binding to human PMN in suspension was carried out as described (10). Nonspecific binding was
AIHAO H . DING, FRANCOISE PORTEU, ELIZABETH SANCHEZ, AND CARL F. NATHAN
From the Beatrice and Samuel A . Seaver Laboratory, Division of Hematology-Oncology, Department of Medicine, Cornell University Medical College, New York, New York 10021
TNFa is a potent mediator ofinflammation (1, 2). Its actions are believed to result from binding to a single class of receptors (TNF ctR) on the surface of a wide variety of cells (3-6). In principle, agents that affect the number or affinity of TNFc1R, such as IFN- ,y (7-9), LPS (10), or activators of protein kinase C or A (11-14),
may modulate some of TNF-a's biologic effects . Among antineoplastic and nonsteroidal antiinflammatory drugs are compounds, such as colchicine, nocodazole, vincristine, vinblastine, and podophyllotoxin, which bind tubulin and inhibit its polymerization. Disassembly of cytoplasmic microtubules (MT)1 by these agents causes mitotic arrest in dividing cells, suggesting a mechanism of action for their antineoplastic effects . It is less clear how colchicine exerts antiinflammatory actions in conditions as diverse as gout, familial Mediterranean fever, cirrhosis, pericarditis, and Behcet's disease . Possible mechanisms include inhibition of the ability of polymorphonuclear leukocytes to migrate (15), degranulate (16), or translocate 5-lipoxygenase (17). We have observed that exposure of murine and human macrophages and human umbilical vein endothelial cells to micromolar concentrations of five different MT depolymerizing agents resulted in a rapid decrease in surface TNF ciR . MT appeared to be required either for the synthesis of TNFaR or for their translocation to the plasma membrane . This effect may contribute to the antiinflammatory actions of MT depolymerizing agents .
Materials and Methods
Materials . Human rTNFce (4.1 x 107 U/mg protein), murine rINFy (5 .2 x 107 U/mg protein) and antiserum 15E against murine TNFa (6 .4 x 105 neutralizing units/ml) were kindly provided by Genentech, Inc . (South San Francisco, CA) . Na 1211 (carrier-free) was from the Radiochemical Center, Amersham, UK . Thioglycollate broth (Brewer) was obtained from Difco Laboratories (Detroit, MI) . The following were from Sigma Chemical Co. (St . Louis, MO) : colchicine, fl-lumicolchicine, y-lumicolchicine, nocodazole, vinblastine, vincristine, podophyllotoxin, cytochalasin B, dihydrocytochalasin B, cytochalasin E, PGE,, dibutyryl-cyclic adenosine monophosphate, indomethacin, ibuprofen, acetylsalicylic acid, and cycloheximide.
Address correspondence to Dr. A. H. Ding, Cornell University Medical College, 1300 York Avenue, Box 57, New York, NY 10021 . ' Abbreviation used in this paper. MT, microtubule. MED. C The Rockefeller University Press " 0022-1007/90/03/0715/13 $2 .00 Volume 171 March 1990 715-727
J . Exp.
715
716
MICROTUBULE REGULATION OF TNF RECEPTORS
Cell Culture. Murine macrophages were washed from the peritoneal cavities offemale CDl mice (Charles River Breeding Laboratories, Wilmington, MA) . Where indicated, mice were injected intraperitoneally with 2 ml of thioglycollate broth (Brewer, 4%) 4 d earlier as described (18). Adherent monolayers were obtained by plating 8 x 105 cells per well in 24-well plastic trays or 2 x 105 cells per well in 96-well plastic trays in RPMI 1640 (KC Biological, Inc., Lenexa, KS) containing 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT) . Monolayers were maintained at 37°C in 5% C02/95 17o air for 1-4 d before the experiments . Human macrophages were allowed to mature from peripheral blood monocytes in Teflon beakers as described (19). Second-passage human umbilical vein endothelial cells were the gift ofDr. EricJaffe (Cornell University Medical College, New York, NY) and were prepared as described (20). Human polymorphonuclear leukocytes (PMN) were isolated from heparinized venous blood on Neutrophil Isolation Medium (Los Alamos Diagnostics, Los Alamos, NM) as described (21). Iodination ofrTNF-a and rIFN-y. Human rTNFa and murine rIFN- y were iodinated according to the method o£ Aggarwal et al. (22), and stored at VC for no longer than 6 wk. In brief, 100 p.l of 0.1 M phosphate buffer (pH 7 .4) was added to a glass tube coated with 5 lAg of Iodogen (Pierce Chemical Co., Rockford, IL) and incubated with 1 mCi of Na' 25I for 10 min at 4oC . The mixture was then transferred to a tube containing 10 Rg of rTNFa or rIFN-y for 10 min at 4°C . The reaction was stopped by addition of one drop of 1 M KI. Iodinated material was separated from free iodide by filtration through a PD-10 column (Pharmacia Fine Chemicals, Piscataway, NJ) that had been equilibrated with 0.1 M phosphate (for rTNF-a) or 20 mM Tris buffer (for rIFN-y), both containing 0.1% gelatin, pH 7.4. '25 1-rTNFa had a specific activity of -300 Ci/mmol monomer, and migrated as a single band with an apparent molecular mass of -16 kD on an 1117o polyacrylamide gel under nonreducing conditions. '21I-rIFN-y had a specific activity of -100 Ci/mmol monomer. Both cytokines retained their ability to activate resident mouse peritoneal macrophages for enhanced H202 release (18). Receptor Binding Assay. Cell monolayers in 24-well trays were overlaid with 0.3 ml per well ofbinding buffer (HBSS containing 10% FCS) and incubated with indicated concentrations of 1251-rTNFot in the presence or absence of a 100-fold excess of unlabeled rTNFa . After 3-4 h at 4oC, binding buffer was removed and the monolayers rinsed four times with ice cold 0.9% NaCl. Cells were solubilized in 0.3 ml of 0.3 N NaOH and radioactivity determined in a gamma counter (Packard Instrument Co., Downers Grove, IL) . Binding to human PMN in suspension was carried out as described (10). Nonspecific binding was
