American Journal of Pathology, Vol. 140, No. 5, May 1992 Copright © American Association of Pathologizsts
Rapid Communication Interleukin-1 Receptor Antagonist Protein Inhibits Interleukin-8 Expression in Lipopolysaccharide-stimulated Human Whole Blood L. E. DeForge,* D. E. Tracey,t J. S. Kenney, and D. G. Remick* From the Department of Pathology,* The University of Michigan Medical School, Ann Arbor, Michigan, Hypersensitivity Diseases Research,J The Upjohn Company, Kalamazoo, Michigan, and the Department of Cellular Immunologvy, Syntex Research, Inc., Palo Alto, California
Interleukin-8 (IL-8) is a neutrophil and lymphocyte chemoattractant and activator that may play an important role in mediating events at sites of imflammation. IL-8 is produced by many cell types in response to a variety of inducers, including interleukin-i (IL-I). Studies were conducted to address the question of whether an inhibitor of IL-I action, IL-I receptor antagonist protein (IRAP), would suppress IL -8 production. L ipopolysaccharide (LPS) stimulated human whole blood was used as an ex vivo model of local cytokineproduction. Preliminary time course studies showed that plasma IL- 1a levels were fully induced by 6 hours (approximately 15 ng/ml) and persisted at this level over 24 hours. IL-8 production; in contrast, reached a plateau between 6 to 12 hours (5 ng/ml) and then increased rapidly to 17 nglml at 24 hours. Addition of IRAP was found to dose-dependently inhibitIL-8 expression at the levels of both protein and mRNA A 50% reduction in IL-8 protein release occurred at an IRAP dose of 8 Rg/ml (5 x 10- 7 mol/l) at 24 hours. A 2 hour delay in the addition of IRAP relative to LPS still permitted optimal reduction in IL-8 release, whereas delays of 4-8 hours reduced or eliminated the inhibitory effect IRAP was found to have no effect on the LPSstimulated production of IL-loa orIL-1p. In addition; experiments performed with isolated peripheral blood cells demonstrated that, whereas monocytes were the major producers of IL-8, IRAP was equally
effective in reducing IL-8 production in neutrophil and mononuclear cell suspensions. These studies further document the role of IL-I in inducing the production of IL-8 and indicate that the ability of IRAP to suppress IL-8 expression may be an important mechanism of the anti-inflammatory properties of this molecule. (AmJ Pathol 1992, 140:1045-1054)
Lipopolysaccharide (LPS) and other microbial products are known to be potent inducers of cytokine production. Interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a) are two such cytokines that serve as central, early mediators of the immuneAlnflammatory response. These molecules are capable of eliciting a diverse range of effects, including fever, hypotension, neutrophilia, endothelial cell expression of leukocyte adhesion molecules, and the production of a cascade of other mediators such as prostaglandin E2, prostacyclin, platelet-activating factor, and other cytokines.1 2 A more recently characterized property of IL-1 and TNF is their ability to stimulate the production of interleukin-8 (IL-8), a neutrophil chemoattractant/activator and a T-lymphocyte chemoattractant.34 The ability of these cytokines to induce IL-8 synthesis was first described for peripheral blood monocytes5 and has been extended to include endothelial cells, epithelial cells, fibroblasts, and keratinocytes.3'4 Although the role of IL-8 in the inflammatory response is as yet unclear, circulating levels of IL-8 have been detected in baboons injected with a lethal dose of Escherichia coli (E. co/i), LPS, or IL-1 x,6 in human volunteers injected with LPS,7 Supported by grants GM 44918 and DK 42455 and a grant-in-aid from the American Heart Association of Michigan. Accepted for publication February 20, 1992. Address reprint requests to Dr. Daniel G. Remick, Department of Pathclogy, M2210 Medical Science Building I, The University of Michigan Medical School, Ann Arbor, Ml 48109-0602.
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and in patients with septic shock.8 Local production of IL-8 has been documented in synovial fluid,9 psoriatic plaques,10 burn-damaged skin,11 and in the sputum of trauma patients with nosocomial pneumonia.12 These studies suggest that IL-8 may potentially be involved in mediating events in these diverse inflammatory states. The IL-1 receptor antagonist protein (IRAP, also termed IL-ira) is a naturally occurring molecule that binds specifically to IL-1 receptors with no agonist effects.13 First characterized in the supernatants of human monocytes cultured on adherent IgG,14 this molecule has been cloned and expressed.15'16 Its availability has made possible the re-examination of the specific role of IL-1 in a variety of inflammatory processes. Notably, several studies using in vivo models of septic shock and local inflammation have shown that IRAP treatment markedly reduced the infiltration of inflammatory cells to tissue sites. 17-21 This effect may be accounted for by the known ability of IRAP to inhibit both neutrophil release from bone marrow-' and the expression of leukocyte adhesion proteins by endothelial cells.16, Alternatively, reduced neutrophil infiltration could potentially arise from an IRAPmediated reduction in IL-8 expression. The potential of IRAP to decrease IL-8 gene expression was evaluated using LPS-stimulated human whole blood as an ex vivo model of local cytokine production. Whole blood represents a readily available, intact human tissue that has been successfully used in several different studies.23'0 Moreover, this model obviates potential artifacts arising from isolation procedures and cellular adherence and also maintains the cells within a physiologic environment where cellular interactions are preserved. These studies demonstrated that IRAP dose-dependently reduced the expression of both IL-8 protein and mRNA by approximately 50% with no effect on the LPS-induced production of IL-1a or IL-1 3.
Materials and Methods
Experimental Design Experiments were performed essentially as described.23 Briefly, blood from normal male volunteers was drawn into heparinized syringes (20 U heparin/ml blood). A total white blood cell (WBC) count was obtained using a Coulter counter (Coulter Electronics, Hialeah, FL). Aliquots of blood (1 ml) were placed in sterile 1.5 ml Eppendorf tubes (Brinkmann Instruments, Westbury, NY) and stimulated with either LPS alone or LPS combined with IRAP. In all experiments, blood was adjusted to 1 jig/ml LPS (E. coil Qi 11i:B4, Sigma Chemical Co., St. Louis, MO) using a 200 ,ug/ml stock solution prepared in RPMI-1640 media (Gibco Laboratories, Grand Island,
NY). Relative to the LPS stimulus, human recombinant IRAP (produced at the Upjohn Company, Kalamazoo, MI) was added either concomitantly or delayed by 2-8 hours. A stock solution of IRAP (5 mg/ml, 99.9% pure as assessed by Coomassie blue staining subsequent to sodium dodecylsulfate polyacrylamide gel electrophoresis) was diluted in RPMI + 10% sterile glycerol (suggested by the manufacturer as a means of preventing loss of the protein on surfaces) and added to the blood in concentrations ranging between 0.1 ng/ml to 100 jig/ml (6 x 10-12-8 X 10-6 mol/l). Controls demonstrated that the final glycerol concentration of 0.2% had no effect on the extent of cytokine release. Lastly, some samples received no additions and served as controls for spontanteous cytokine production. The closed Eppendorf tubes containing the aliquots of blood were placed on a rotator and incubated at 370C for 4-24 hours in an atmosphere containing 5% CO2. The samples were then removed from the incubator and centrifuged at 600g for 5 minutes. The plasma was removed, diluted 1:5 (a convenient starting dilution for the cytokine assays) in RPMI containing 1 % fetal calf serum (FCS), and frozen at -200C for later analysis. In some instances, the buffy coat was also removed, and the contaminating red blood cells were lysed with 0.82% NH4CI. The WBC pellet was then solubilized in 0.3 ml of an RNA extraction buffer (25 mmolA TRIS, pH 8.0, 4.2 mol/l guanidine isothiocyanate, 0.5% N-laurosarcosine, and 0.1 mol/l 2-mercaptoethanol), and the samples were stored at - 700C for later RNA extraction.
White Blood Cell Viability Aliquots of blood with and without LPS stimulation were incubated as described earlier for various time intervals up to 32 hours. The buffy coat was then isolated, subjected to NH4CI lysis twice to completely clear red blood cell contamination, and resuspended in 1 ml RPMI containing 1 % fetal calf serum (FCS), 1 00 U/mI penicillin, 100 ,ug/ml streptomycin, 1 mmol/l L-glutamine, 25 mmol/l Hepes, and 50 ,umoVI 2-mercaptoethanol. Triplicate aliquots of the cell suspension (200 RI) were placed in 96well plates, and 20 ,ul MTT-tetrazolium (5 mg/ml, Sigma) was added. This reagent is metabolized to a dark blue end product only by live cells and thereby provides a rapid, accurate means of determining cell viability in a large number of samples.31 The plates were incubated for 4 hours at 370C in a humidified atmosphere containing 5% CO2. One hundred and fifty microliters of the supernatant was then removed, 1 00 RI of 0.04 N HCI/ isopropanol was added to solubilize the blue crystals, and the plates were allowed to stand overnight protected from light. The absorbance at 550 nm was measured using a Bio-Tek microplate reader (Bio-Tek Instruments, Inc.,
IL-1 Receptor Antagonist Protein Inhibits IL-8 Gene Expression
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Winooski, VT). The triplicate values obtained at each time point for each blood donor were averaged, and the data for the five individual blood donors was then compiled.
Isolated Cell Studies Mononuclear cells were isolated from whole blood using Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and washed three times with RPMI + 1% FCS. The neutrophil/red blood cell pellets were resuspended in 20 ml Hanks' balanced salt solution (HBSS) and the red blood cells were sedimented by adding an equal volume of 3% dextran T500 (Pharmacia, Piscataway, NJ) in 0.9% NaCI. After standing at room temperature for 20 minutes, the neutrophil-rich upper layer was subjected to hypotonic lysis twice to clear residual red blood cell contamination. The mononuclear cells and neutrophils were resuspended at concentrations of 1 x 106 cells/ml and 5 x 106 cells/ml, respectively, in RPMI supplemented with 10% FCS, 100 U/ml pencillin, 100 ,ug/ml streptomycin, 1 mmol/l L-glutamine, and 25 mmol/l Hepes. Cytospin preparations were made of these final cell suspensions. These were subsequently stained with Diff-Quik (Scientific Products, McGaw Park, IL), and 300-cell differential cell counts were performed. The composition of the neutrophil suspensions (mean ± SEM from cell preparations of 6 different blood donors) was: neutrophils, 93.5 + 1.5%; eosinophils, 5.8 ± 2.0%; lymphocytes, 0.5 ± 0.3%; and monocytes, 0.06 + 0.06%). The mononuclear cell preparations consisted of 30.2 ± 2.7% monocytes; 64.0 ± 3.6% lymphocytes; 5.3 ± 1.6% neutrophils; and 0.3 ± 0.2% eosinophils. Aliquots of the cell suspensions (1 ml) were placed in sterile 1.5 ml Eppendorf tubes. The cells were stimulated with LPS alone (1 ,g/ml), with LPS plus IRAP (10 ,ug/ml), or received no addition to serve as controls. The Eppendorf tubes were then placed on a rotator in a 37C incubator containing 5% C02, analogous to the sample conditions in experiments using whole blood. After a 24 hour incubation, the samples were centrifuged at 1500g, and the cell-free supernatants were removed for analysis in the IL-8 ELISA.
was
Rapid Communication Interleukin-1 Receptor Antagonist Protein Inhibits Interleukin-8 Expression in Lipopolysaccharide-stimulated Human Whole Blood L. E. DeForge,* D. E. Tracey,t J. S. Kenney, and D. G. Remick* From the Department of Pathology,* The University of Michigan Medical School, Ann Arbor, Michigan, Hypersensitivity Diseases Research,J The Upjohn Company, Kalamazoo, Michigan, and the Department of Cellular Immunologvy, Syntex Research, Inc., Palo Alto, California
Interleukin-8 (IL-8) is a neutrophil and lymphocyte chemoattractant and activator that may play an important role in mediating events at sites of imflammation. IL-8 is produced by many cell types in response to a variety of inducers, including interleukin-i (IL-I). Studies were conducted to address the question of whether an inhibitor of IL-I action, IL-I receptor antagonist protein (IRAP), would suppress IL -8 production. L ipopolysaccharide (LPS) stimulated human whole blood was used as an ex vivo model of local cytokineproduction. Preliminary time course studies showed that plasma IL- 1a levels were fully induced by 6 hours (approximately 15 ng/ml) and persisted at this level over 24 hours. IL-8 production; in contrast, reached a plateau between 6 to 12 hours (5 ng/ml) and then increased rapidly to 17 nglml at 24 hours. Addition of IRAP was found to dose-dependently inhibitIL-8 expression at the levels of both protein and mRNA A 50% reduction in IL-8 protein release occurred at an IRAP dose of 8 Rg/ml (5 x 10- 7 mol/l) at 24 hours. A 2 hour delay in the addition of IRAP relative to LPS still permitted optimal reduction in IL-8 release, whereas delays of 4-8 hours reduced or eliminated the inhibitory effect IRAP was found to have no effect on the LPSstimulated production of IL-loa orIL-1p. In addition; experiments performed with isolated peripheral blood cells demonstrated that, whereas monocytes were the major producers of IL-8, IRAP was equally
effective in reducing IL-8 production in neutrophil and mononuclear cell suspensions. These studies further document the role of IL-I in inducing the production of IL-8 and indicate that the ability of IRAP to suppress IL-8 expression may be an important mechanism of the anti-inflammatory properties of this molecule. (AmJ Pathol 1992, 140:1045-1054)
Lipopolysaccharide (LPS) and other microbial products are known to be potent inducers of cytokine production. Interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a) are two such cytokines that serve as central, early mediators of the immuneAlnflammatory response. These molecules are capable of eliciting a diverse range of effects, including fever, hypotension, neutrophilia, endothelial cell expression of leukocyte adhesion molecules, and the production of a cascade of other mediators such as prostaglandin E2, prostacyclin, platelet-activating factor, and other cytokines.1 2 A more recently characterized property of IL-1 and TNF is their ability to stimulate the production of interleukin-8 (IL-8), a neutrophil chemoattractant/activator and a T-lymphocyte chemoattractant.34 The ability of these cytokines to induce IL-8 synthesis was first described for peripheral blood monocytes5 and has been extended to include endothelial cells, epithelial cells, fibroblasts, and keratinocytes.3'4 Although the role of IL-8 in the inflammatory response is as yet unclear, circulating levels of IL-8 have been detected in baboons injected with a lethal dose of Escherichia coli (E. co/i), LPS, or IL-1 x,6 in human volunteers injected with LPS,7 Supported by grants GM 44918 and DK 42455 and a grant-in-aid from the American Heart Association of Michigan. Accepted for publication February 20, 1992. Address reprint requests to Dr. Daniel G. Remick, Department of Pathclogy, M2210 Medical Science Building I, The University of Michigan Medical School, Ann Arbor, Ml 48109-0602.
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and in patients with septic shock.8 Local production of IL-8 has been documented in synovial fluid,9 psoriatic plaques,10 burn-damaged skin,11 and in the sputum of trauma patients with nosocomial pneumonia.12 These studies suggest that IL-8 may potentially be involved in mediating events in these diverse inflammatory states. The IL-1 receptor antagonist protein (IRAP, also termed IL-ira) is a naturally occurring molecule that binds specifically to IL-1 receptors with no agonist effects.13 First characterized in the supernatants of human monocytes cultured on adherent IgG,14 this molecule has been cloned and expressed.15'16 Its availability has made possible the re-examination of the specific role of IL-1 in a variety of inflammatory processes. Notably, several studies using in vivo models of septic shock and local inflammation have shown that IRAP treatment markedly reduced the infiltration of inflammatory cells to tissue sites. 17-21 This effect may be accounted for by the known ability of IRAP to inhibit both neutrophil release from bone marrow-' and the expression of leukocyte adhesion proteins by endothelial cells.16, Alternatively, reduced neutrophil infiltration could potentially arise from an IRAPmediated reduction in IL-8 expression. The potential of IRAP to decrease IL-8 gene expression was evaluated using LPS-stimulated human whole blood as an ex vivo model of local cytokine production. Whole blood represents a readily available, intact human tissue that has been successfully used in several different studies.23'0 Moreover, this model obviates potential artifacts arising from isolation procedures and cellular adherence and also maintains the cells within a physiologic environment where cellular interactions are preserved. These studies demonstrated that IRAP dose-dependently reduced the expression of both IL-8 protein and mRNA by approximately 50% with no effect on the LPS-induced production of IL-1a or IL-1 3.
Materials and Methods
Experimental Design Experiments were performed essentially as described.23 Briefly, blood from normal male volunteers was drawn into heparinized syringes (20 U heparin/ml blood). A total white blood cell (WBC) count was obtained using a Coulter counter (Coulter Electronics, Hialeah, FL). Aliquots of blood (1 ml) were placed in sterile 1.5 ml Eppendorf tubes (Brinkmann Instruments, Westbury, NY) and stimulated with either LPS alone or LPS combined with IRAP. In all experiments, blood was adjusted to 1 jig/ml LPS (E. coil Qi 11i:B4, Sigma Chemical Co., St. Louis, MO) using a 200 ,ug/ml stock solution prepared in RPMI-1640 media (Gibco Laboratories, Grand Island,
NY). Relative to the LPS stimulus, human recombinant IRAP (produced at the Upjohn Company, Kalamazoo, MI) was added either concomitantly or delayed by 2-8 hours. A stock solution of IRAP (5 mg/ml, 99.9% pure as assessed by Coomassie blue staining subsequent to sodium dodecylsulfate polyacrylamide gel electrophoresis) was diluted in RPMI + 10% sterile glycerol (suggested by the manufacturer as a means of preventing loss of the protein on surfaces) and added to the blood in concentrations ranging between 0.1 ng/ml to 100 jig/ml (6 x 10-12-8 X 10-6 mol/l). Controls demonstrated that the final glycerol concentration of 0.2% had no effect on the extent of cytokine release. Lastly, some samples received no additions and served as controls for spontanteous cytokine production. The closed Eppendorf tubes containing the aliquots of blood were placed on a rotator and incubated at 370C for 4-24 hours in an atmosphere containing 5% CO2. The samples were then removed from the incubator and centrifuged at 600g for 5 minutes. The plasma was removed, diluted 1:5 (a convenient starting dilution for the cytokine assays) in RPMI containing 1 % fetal calf serum (FCS), and frozen at -200C for later analysis. In some instances, the buffy coat was also removed, and the contaminating red blood cells were lysed with 0.82% NH4CI. The WBC pellet was then solubilized in 0.3 ml of an RNA extraction buffer (25 mmolA TRIS, pH 8.0, 4.2 mol/l guanidine isothiocyanate, 0.5% N-laurosarcosine, and 0.1 mol/l 2-mercaptoethanol), and the samples were stored at - 700C for later RNA extraction.
White Blood Cell Viability Aliquots of blood with and without LPS stimulation were incubated as described earlier for various time intervals up to 32 hours. The buffy coat was then isolated, subjected to NH4CI lysis twice to completely clear red blood cell contamination, and resuspended in 1 ml RPMI containing 1 % fetal calf serum (FCS), 1 00 U/mI penicillin, 100 ,ug/ml streptomycin, 1 mmol/l L-glutamine, 25 mmol/l Hepes, and 50 ,umoVI 2-mercaptoethanol. Triplicate aliquots of the cell suspension (200 RI) were placed in 96well plates, and 20 ,ul MTT-tetrazolium (5 mg/ml, Sigma) was added. This reagent is metabolized to a dark blue end product only by live cells and thereby provides a rapid, accurate means of determining cell viability in a large number of samples.31 The plates were incubated for 4 hours at 370C in a humidified atmosphere containing 5% CO2. One hundred and fifty microliters of the supernatant was then removed, 1 00 RI of 0.04 N HCI/ isopropanol was added to solubilize the blue crystals, and the plates were allowed to stand overnight protected from light. The absorbance at 550 nm was measured using a Bio-Tek microplate reader (Bio-Tek Instruments, Inc.,
IL-1 Receptor Antagonist Protein Inhibits IL-8 Gene Expression
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Winooski, VT). The triplicate values obtained at each time point for each blood donor were averaged, and the data for the five individual blood donors was then compiled.
Isolated Cell Studies Mononuclear cells were isolated from whole blood using Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and washed three times with RPMI + 1% FCS. The neutrophil/red blood cell pellets were resuspended in 20 ml Hanks' balanced salt solution (HBSS) and the red blood cells were sedimented by adding an equal volume of 3% dextran T500 (Pharmacia, Piscataway, NJ) in 0.9% NaCI. After standing at room temperature for 20 minutes, the neutrophil-rich upper layer was subjected to hypotonic lysis twice to clear residual red blood cell contamination. The mononuclear cells and neutrophils were resuspended at concentrations of 1 x 106 cells/ml and 5 x 106 cells/ml, respectively, in RPMI supplemented with 10% FCS, 100 U/ml pencillin, 100 ,ug/ml streptomycin, 1 mmol/l L-glutamine, and 25 mmol/l Hepes. Cytospin preparations were made of these final cell suspensions. These were subsequently stained with Diff-Quik (Scientific Products, McGaw Park, IL), and 300-cell differential cell counts were performed. The composition of the neutrophil suspensions (mean ± SEM from cell preparations of 6 different blood donors) was: neutrophils, 93.5 + 1.5%; eosinophils, 5.8 ± 2.0%; lymphocytes, 0.5 ± 0.3%; and monocytes, 0.06 + 0.06%). The mononuclear cell preparations consisted of 30.2 ± 2.7% monocytes; 64.0 ± 3.6% lymphocytes; 5.3 ± 1.6% neutrophils; and 0.3 ± 0.2% eosinophils. Aliquots of the cell suspensions (1 ml) were placed in sterile 1.5 ml Eppendorf tubes. The cells were stimulated with LPS alone (1 ,g/ml), with LPS plus IRAP (10 ,ug/ml), or received no addition to serve as controls. The Eppendorf tubes were then placed on a rotator in a 37C incubator containing 5% C02, analogous to the sample conditions in experiments using whole blood. After a 24 hour incubation, the samples were centrifuged at 1500g, and the cell-free supernatants were removed for analysis in the IL-8 ELISA.
was
