IMMUNOLOGICAL INVESTIGATIONS, 2 0 ( 4 ) , 387-394 (1991)
CHEMOTACTIC CYTOKINE (IL-8 AND MCP-1) GENE EXPRESSION BY HUMAN WHOLE BLOOD
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
J. M. Ham**, S. L. Kunkel*, C. R. Dibb, T. J. Standiford, Id. W. Rolfe, and R. M. Strieter Departments of Medicine, *Pathology, and **Surgery The University of Michigan Medical Schclol Ann Arbor, Michigan, 481 00
ABSTRACT The salient features of systemic or local infla-mmation are the myriad of cellular and humoral interactions that result in elicitation of inflammatory leukocytes. In this study using specialized connective tissue, intact whole blood, w e demonstrate the gene expression of two novel chemotactic factors. The bu.ffycoat cellular expression of neutrophil chemotactic/activating factor/interleukin 8 (IL-8) and monocyte chemotactic/activating protein (MCP-I) mRNA were time a n d dose-dependent in response to either lipopolysaccharide o r zymosan stimulation. This system with the complexity of tissue provides a unique model for the determination of chemotactic cytokine gene expression.
INTRODUCTION Systemic or local inflammation is characterized by the migration of leukocytes from the vascular compartment to the interstitial space. These newly arrived leukocytes not only contribute to the pathogenesis, but also participate i n the maintenance and resolution of the inflammatory even!:. The subsequent organ/tissue injury associated with early phases of inflammation is a direct result of microvascular damage leading to hemostasis, vascular permeability, accumulation of extravascular proteinaceous fluid, intra.vascular leukocyte aggregation anld transendothelial leukocyte emigrairion (1). This latter event is complex and associated with a number of cellular and humoral mediators that remain to be fully elucidated. Recently, two novel chemotactic cytokines, that are relevant to the elicitation of leukocytes during inflammation, have been isolated, puriifieid, 387 Copyright @ 1991 by Marcel Dekker, Inc
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
3 88
HAM ET AL.
cloned, and expressed (2-4). Neutrophil chemotactic/activating factor or Interleukin 8 (IL-8) and monocyte chemotactic/activating peptide (MCP-I) have been shown to have chemotactic specificity for either neutrophils or monocytes, respectively (2-6). IL-8 and MCP-1 are derived from a number of immune and nonimmune cells (5,6). The majority of data regarding the production of either IL-8 or MCP-1 have been ascertained by in vitro studies using either primary cells or tumor lines in culture (2-6). Although these investigations have provided valuable insight as to the cellular production and regulation of IL-8 and MCP-I, they have inherent limitations due to their monocellular nature. In this study, we utilize intact human whole blood (WB), defined as specialized circulating connective tissue (7), to examine the gene expression of both IL-8 and MCP-1 from the cellular constituents of WB. Both IL-8 and MCP-1 mRNA from WB-derived buffy-coat cells were expressed in a time and dosedependent fashion after stimulation with either lipopolysaccaride (LPS) or zymosan. In addition, IL-8 antigen determined by IL-8 ELISA from WB plasma paralleled IL-8 mRNA expression. These studies demonstrate the utility of using WB as a tissue model for the evaluation of chemotactic cytokine gene expression and protein production. MATERIALS AND METHODS
Recoverv of Whole Blood: Peripheral blood was obtained from healthy human volunteers. Five mls of heparinized (50 USP units/ml) WB was aliquoted into 15 ml conical tubes and stimulated with either LPS or zymosan, then placed on a rocker and incubated at 37oC in 95% air and 5% C02. WB was centrifugated at 600g for 5 min at specified times with plasma and buffy-coat (cells) isolated for chemotactic cytokine antigenic and nucleic-acid determination, respectively. Reagents: Stock LPS (Esheuicka coli Olll:B4; Sigma) and zymosan (Sigma) were prepared at a concentration of 200 pg/ml and 10 mg/ml in RPMI, respectively. Northern blot analysis: WB buffy-coat RNA was extracted as previously described (8,9). Briefly, buffy-coats were placed in a solution of 25 mM Tris, pH 8.0 containing 4.2 M guanidine isothiocyanate, 0.5% Sarkosyl, and 0.1 M betamercaptoethanol. After homogenization, an equal volume of 100 mM Tris, pH 8.0 containing 1.0% SDS and 10 mM EDTA was added and the RNA extracted with chloroform-phenol followed by chloroform-isoamyl alcohol. The RNA was alcohol precipitated and 10 vg/lane separated by formaldehyde/ 1% agarose gels and transblotted to nitrocellulose. The baked blots were prehybridized and hybridized with 3%'-5'-end-labeled, 30-mer oligonucleotide probes. The probes were complementary to either nucleotides 256-285 of published cDNA sequence for human monocyte chemotactic and activating factor/monocyte chemoattractant protein (3,4) or nucleotides 262-291 of published cDNA sequence for neutrophil chemotactic factor (IL-8) (2). The sequence of MCP-1 probe was 5'TTG-GGT-TTG-CTT-GTC-CAG-GTG-GTC-CAT-GGA-3', while the sequence for the IL-8 probe was 5'-GTT-GGC-GCA-GTG-TGG-TCC-ACT-CTC-ATT-cAc-3'.
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
CHEMOTACTIC CYTOKINE (IL-8 AND MCP-1)
380
FIGURE 1. Concentration-dependent induction of WB-derived IL-8 and MCP-1 m17NA by zymosan. IA and IIA are representative Northern blots of the steady-state levels of IL-8 and MCP-1 mRNA 4 hrs post-stimulation with zymosan (0.001 to 100 pg/ml), respectively. IB and I I B represent the corrtssponding laser densitometry of the Northern blots in A . IC and IIC are the 28s and 18s rRNA for the corresponding Northern blots in A.
Blots were stringency washed and autoradiographs were quantitated by laser densitometry. Equivalent amounts of total RNA loaded per gel lane were assessed by monitoring 28s and 18s rRNA IL-8 ELISA: Antigenic IL-8 was quantitated using a doublt. ligand ELISA methold, as previously described (8). This ELISA method consistently detected IL-8 concentrations above 30 pg/ml.
RESULTS Concentration--Dependent Induction Of IL-8 a n d MCP-1 m R N A Rv W E I n initial experiments, WB was stimulated with griided doses of either LI’S or zymosan for 4 hrs with buffy-coat cells assessed for the expression of steady-state levels of IL-8 and MCP-1 mRNA. As shown by the Northern blot analyses in figure 1 IA and IIA, both I L 8 and MCI’-l mRNA were expressed over a wide concentration range of zyniosan (0.001 to 100 pg/ml). Laser densitometry of the
HAM ET AL.
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
390
FIGURE 2. Time-dependent induction of WB-derived IL-8 and MCP-I mRNA by LPS and zymosan. IA and IIIA are representative Northern blots of the kinetic induction of the steady-state levels of IL-8 mRNA by LPS and zymosan, respectively. IIA and IVA are representative Northern blots of the kinetic induction of the steadystate levels of MCP-1 mXNA by LPS and zymosan, respectively. IB, IIB, IIIB, and IVB represent the corresponding laser densitometry of the Northern blots in A . IC, IIC, IIIC, and IVC are the 28s and 18s rRNA for the corresponding Northwn blots in A . 24- represents 24 hr unstimulated control.
CHEMOTACTIC CYTOKINE ( I L - 8 AND M C P - 1 )
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
*0°
391
1
0
4
8
12
16
20
24
Time (hrs.)
B 70
Time (hrs)
FIGURE 3. Time-dependent induction of WB-derived antigenic IL-8. A and B are the kinetic induction of IL-8 antigen by LPS and zymosan, respectively.
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
392
HAM ET AL.
Northern blots demonstrated that significant levels of either IL-8 and MCP-1 mRNA were induced by as little as 1 pg/ml and 10 pg/ml of zymosan (Figure 1 IB and IIB), respectively. A maximal expression in WB buffy-coat-derived IL-8 and MCP-1 mRNA expression was reached at a concentration of 100 pg/ml of zymosan. Unstimulated WB buffy-coat cells, however, did not constitutively express IL-8 or MCP-1 mRNA. Half maximal expression of IL-8 or MCP-1 mRNA by LPS stimulation occurred at 100 ng/ml (data not shown). Time-Dependent Induction Of IL-8 And MCP-1 mRNA Bv WB. In order to determine the time-dependent expression of either WB buffy-coat-derived steady-state levels of IL-8 or MCP-1 mRNA by LPS or zymosan, WB was stimulated with either 500 ng/ml LFS or 100 pg/ml zymosan. As shown in Figure 2 IA and IIIA, WB treated with either LPS or zymosan expressed IL-8 mRNA by 0.5 hr. Maximal IL-8 steady-state mRNA was seen post-stimulation with either zymosan or LPS by 2 or 24 hrs, respectively. In contrast, maximal steady-state levels of MCP-1 mRNA were not seen until 4 and 24 hrs poststimulation with either LPS or zymosan (figure 2 IIA and IVA), respectively. WB-Derived Antigenic IL-8. Since WB buffy-coat cells were demonstrated to express IL-8 mRNA in both a dose and time-dependent manner, we next examined WB-derived plasma for the kinetic antigenic production of IL-8 (Figure 3, A and B). WB was stimulated with either LPS (500 ng/ml) or zymosan (100 pg/ml) and WB-derived plasma isolated at the specified times. Antigenic production of IL-8 was determined by IL-8 ELISA, and was demonstrated to be first detectable by 2 and 4 hrs post-stimulation with either LPS or zymosan, respectively. Maximal antigenic IL-8 was noted at 24 hrs post-stimulation with either LPS or zymosan. LPS (500 ng/ml) stimulation for 24 hrs resulted in a 2.5 to 3-fold greater production of antigenic IL-8 as compared to treatment with zymosan (100 pg/ml). Antibodies used in this ELISA were specific to IL-8, since they failed to respond to either CTAP-111, NAP-2, GRO/MGSA, or platelet-factor-4 by Western blot analysis (data not shown). DISCUSSION
Systemic or local inflammation is associated with a spectrum of diseases from ARDS, sepsis, shock, or multiple trauma to pneumonia or abscess formation, yet all these entities have in common the presence of activated tissue leukocytes. During acute inflammation neutrophilic accumulation initially exceeds the emigration of monocytes, but as the inflammatory process evolves monocyte elicitation becomes the dominate inflammatory leukocyte (1). This dynamic shift in characteristic of inflammatory leukocytes is secondary to the production of numerous leukocyte chemotactic factors. IL-8 and MCP-1 belong to
CHEMOTACTIC CYTORINE (IL-8 AND M C P - 1 )
393
a novel class of chemotactic cytokines that possess specific cheniotactic/activating activity for leukocytes (5,6). Although our laboratory an'd others have shown that both IL-8 and MCP-1 are produced by a variety c'ells (5,6), these studies h'ave
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
been limited to rnonocellular systems that fail to exploit the intricacy of a tissue environment. To further understand the molecular and cellular regulation. o f these chemotactic cytokines in vivo the development of an in nitro tissue model would be a logical transition. We have previously demonstrated that WB, a specialized connective tissue, is an unique medium in which to assess cytoltine production (9). Under normal conditions i n vivo, WB's role is unparalleled for its fluidic ability tcl provide interorgan/ tissue communication, whereas duriiig inflammation the microvascular (environment favors hemostasis with associated sequen tia!, leukocyte aggregation, margination, adherence, and diapedesis (1). It is under these conditions that WB-derived chemotactic cytokine generation is relevant to the subsequent migration of inflammatory leukocytes. In this study, we describe the dose and time-dependent expression of both IL-8 and MCP-1 by WB in response to either products of gram-negative bacteria or yeast, LPS and zymosan, respectively. Although indirectly, LPS or zymosan could ccinceivably mediate their response by complement activation, recombinant C5a stimulation done in this system was unable to induce IL-8 or MCP-1 mFLVA (data not shown). The use of WB with the complexity of tissue has an 'enormous potential for thiz evaluation of chemotactic cytokine regulation and response to pharmacologic agents. The assessment of a pharmacologic agent's influence in chemotactic cytokine expression by this i n vitvo tissue system may have significant implications as to its potential efficacy in vivo. ACKNOWLEDGEMENTlS
This work was supported in part by NIH grants HL31693, HL35276, HL02401, and 13K38149. Additional support was provided by the Council far Tobacco Researlch, the American Heart Association of Florida, and the American Lung Association. Dr. Strieter is a RJR Nabisco Research Scholar. REFERENCES
1. 2.
M. I. Cybulsky, M. K. W. Chan, and H. Z. Mov
CHEMOTACTIC CYTOKINE (IL-8 AND MCP-1) GENE EXPRESSION BY HUMAN WHOLE BLOOD
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
J. M. Ham**, S. L. Kunkel*, C. R. Dibb, T. J. Standiford, Id. W. Rolfe, and R. M. Strieter Departments of Medicine, *Pathology, and **Surgery The University of Michigan Medical Schclol Ann Arbor, Michigan, 481 00
ABSTRACT The salient features of systemic or local infla-mmation are the myriad of cellular and humoral interactions that result in elicitation of inflammatory leukocytes. In this study using specialized connective tissue, intact whole blood, w e demonstrate the gene expression of two novel chemotactic factors. The bu.ffycoat cellular expression of neutrophil chemotactic/activating factor/interleukin 8 (IL-8) and monocyte chemotactic/activating protein (MCP-I) mRNA were time a n d dose-dependent in response to either lipopolysaccharide o r zymosan stimulation. This system with the complexity of tissue provides a unique model for the determination of chemotactic cytokine gene expression.
INTRODUCTION Systemic or local inflammation is characterized by the migration of leukocytes from the vascular compartment to the interstitial space. These newly arrived leukocytes not only contribute to the pathogenesis, but also participate i n the maintenance and resolution of the inflammatory even!:. The subsequent organ/tissue injury associated with early phases of inflammation is a direct result of microvascular damage leading to hemostasis, vascular permeability, accumulation of extravascular proteinaceous fluid, intra.vascular leukocyte aggregation anld transendothelial leukocyte emigrairion (1). This latter event is complex and associated with a number of cellular and humoral mediators that remain to be fully elucidated. Recently, two novel chemotactic cytokines, that are relevant to the elicitation of leukocytes during inflammation, have been isolated, puriifieid, 387 Copyright @ 1991 by Marcel Dekker, Inc
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
3 88
HAM ET AL.
cloned, and expressed (2-4). Neutrophil chemotactic/activating factor or Interleukin 8 (IL-8) and monocyte chemotactic/activating peptide (MCP-I) have been shown to have chemotactic specificity for either neutrophils or monocytes, respectively (2-6). IL-8 and MCP-1 are derived from a number of immune and nonimmune cells (5,6). The majority of data regarding the production of either IL-8 or MCP-1 have been ascertained by in vitro studies using either primary cells or tumor lines in culture (2-6). Although these investigations have provided valuable insight as to the cellular production and regulation of IL-8 and MCP-I, they have inherent limitations due to their monocellular nature. In this study, we utilize intact human whole blood (WB), defined as specialized circulating connective tissue (7), to examine the gene expression of both IL-8 and MCP-1 from the cellular constituents of WB. Both IL-8 and MCP-1 mRNA from WB-derived buffy-coat cells were expressed in a time and dosedependent fashion after stimulation with either lipopolysaccaride (LPS) or zymosan. In addition, IL-8 antigen determined by IL-8 ELISA from WB plasma paralleled IL-8 mRNA expression. These studies demonstrate the utility of using WB as a tissue model for the evaluation of chemotactic cytokine gene expression and protein production. MATERIALS AND METHODS
Recoverv of Whole Blood: Peripheral blood was obtained from healthy human volunteers. Five mls of heparinized (50 USP units/ml) WB was aliquoted into 15 ml conical tubes and stimulated with either LPS or zymosan, then placed on a rocker and incubated at 37oC in 95% air and 5% C02. WB was centrifugated at 600g for 5 min at specified times with plasma and buffy-coat (cells) isolated for chemotactic cytokine antigenic and nucleic-acid determination, respectively. Reagents: Stock LPS (Esheuicka coli Olll:B4; Sigma) and zymosan (Sigma) were prepared at a concentration of 200 pg/ml and 10 mg/ml in RPMI, respectively. Northern blot analysis: WB buffy-coat RNA was extracted as previously described (8,9). Briefly, buffy-coats were placed in a solution of 25 mM Tris, pH 8.0 containing 4.2 M guanidine isothiocyanate, 0.5% Sarkosyl, and 0.1 M betamercaptoethanol. After homogenization, an equal volume of 100 mM Tris, pH 8.0 containing 1.0% SDS and 10 mM EDTA was added and the RNA extracted with chloroform-phenol followed by chloroform-isoamyl alcohol. The RNA was alcohol precipitated and 10 vg/lane separated by formaldehyde/ 1% agarose gels and transblotted to nitrocellulose. The baked blots were prehybridized and hybridized with 3%'-5'-end-labeled, 30-mer oligonucleotide probes. The probes were complementary to either nucleotides 256-285 of published cDNA sequence for human monocyte chemotactic and activating factor/monocyte chemoattractant protein (3,4) or nucleotides 262-291 of published cDNA sequence for neutrophil chemotactic factor (IL-8) (2). The sequence of MCP-1 probe was 5'TTG-GGT-TTG-CTT-GTC-CAG-GTG-GTC-CAT-GGA-3', while the sequence for the IL-8 probe was 5'-GTT-GGC-GCA-GTG-TGG-TCC-ACT-CTC-ATT-cAc-3'.
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
CHEMOTACTIC CYTOKINE (IL-8 AND MCP-1)
380
FIGURE 1. Concentration-dependent induction of WB-derived IL-8 and MCP-1 m17NA by zymosan. IA and IIA are representative Northern blots of the steady-state levels of IL-8 and MCP-1 mRNA 4 hrs post-stimulation with zymosan (0.001 to 100 pg/ml), respectively. IB and I I B represent the corrtssponding laser densitometry of the Northern blots in A . IC and IIC are the 28s and 18s rRNA for the corresponding Northern blots in A.
Blots were stringency washed and autoradiographs were quantitated by laser densitometry. Equivalent amounts of total RNA loaded per gel lane were assessed by monitoring 28s and 18s rRNA IL-8 ELISA: Antigenic IL-8 was quantitated using a doublt. ligand ELISA methold, as previously described (8). This ELISA method consistently detected IL-8 concentrations above 30 pg/ml.
RESULTS Concentration--Dependent Induction Of IL-8 a n d MCP-1 m R N A Rv W E I n initial experiments, WB was stimulated with griided doses of either LI’S or zymosan for 4 hrs with buffy-coat cells assessed for the expression of steady-state levels of IL-8 and MCP-1 mRNA. As shown by the Northern blot analyses in figure 1 IA and IIA, both I L 8 and MCI’-l mRNA were expressed over a wide concentration range of zyniosan (0.001 to 100 pg/ml). Laser densitometry of the
HAM ET AL.
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
390
FIGURE 2. Time-dependent induction of WB-derived IL-8 and MCP-I mRNA by LPS and zymosan. IA and IIIA are representative Northern blots of the kinetic induction of the steady-state levels of IL-8 mRNA by LPS and zymosan, respectively. IIA and IVA are representative Northern blots of the kinetic induction of the steadystate levels of MCP-1 mXNA by LPS and zymosan, respectively. IB, IIB, IIIB, and IVB represent the corresponding laser densitometry of the Northern blots in A . IC, IIC, IIIC, and IVC are the 28s and 18s rRNA for the corresponding Northwn blots in A . 24- represents 24 hr unstimulated control.
CHEMOTACTIC CYTOKINE ( I L - 8 AND M C P - 1 )
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
*0°
391
1
0
4
8
12
16
20
24
Time (hrs.)
B 70
Time (hrs)
FIGURE 3. Time-dependent induction of WB-derived antigenic IL-8. A and B are the kinetic induction of IL-8 antigen by LPS and zymosan, respectively.
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
392
HAM ET AL.
Northern blots demonstrated that significant levels of either IL-8 and MCP-1 mRNA were induced by as little as 1 pg/ml and 10 pg/ml of zymosan (Figure 1 IB and IIB), respectively. A maximal expression in WB buffy-coat-derived IL-8 and MCP-1 mRNA expression was reached at a concentration of 100 pg/ml of zymosan. Unstimulated WB buffy-coat cells, however, did not constitutively express IL-8 or MCP-1 mRNA. Half maximal expression of IL-8 or MCP-1 mRNA by LPS stimulation occurred at 100 ng/ml (data not shown). Time-Dependent Induction Of IL-8 And MCP-1 mRNA Bv WB. In order to determine the time-dependent expression of either WB buffy-coat-derived steady-state levels of IL-8 or MCP-1 mRNA by LPS or zymosan, WB was stimulated with either 500 ng/ml LFS or 100 pg/ml zymosan. As shown in Figure 2 IA and IIIA, WB treated with either LPS or zymosan expressed IL-8 mRNA by 0.5 hr. Maximal IL-8 steady-state mRNA was seen post-stimulation with either zymosan or LPS by 2 or 24 hrs, respectively. In contrast, maximal steady-state levels of MCP-1 mRNA were not seen until 4 and 24 hrs poststimulation with either LPS or zymosan (figure 2 IIA and IVA), respectively. WB-Derived Antigenic IL-8. Since WB buffy-coat cells were demonstrated to express IL-8 mRNA in both a dose and time-dependent manner, we next examined WB-derived plasma for the kinetic antigenic production of IL-8 (Figure 3, A and B). WB was stimulated with either LPS (500 ng/ml) or zymosan (100 pg/ml) and WB-derived plasma isolated at the specified times. Antigenic production of IL-8 was determined by IL-8 ELISA, and was demonstrated to be first detectable by 2 and 4 hrs post-stimulation with either LPS or zymosan, respectively. Maximal antigenic IL-8 was noted at 24 hrs post-stimulation with either LPS or zymosan. LPS (500 ng/ml) stimulation for 24 hrs resulted in a 2.5 to 3-fold greater production of antigenic IL-8 as compared to treatment with zymosan (100 pg/ml). Antibodies used in this ELISA were specific to IL-8, since they failed to respond to either CTAP-111, NAP-2, GRO/MGSA, or platelet-factor-4 by Western blot analysis (data not shown). DISCUSSION
Systemic or local inflammation is associated with a spectrum of diseases from ARDS, sepsis, shock, or multiple trauma to pneumonia or abscess formation, yet all these entities have in common the presence of activated tissue leukocytes. During acute inflammation neutrophilic accumulation initially exceeds the emigration of monocytes, but as the inflammatory process evolves monocyte elicitation becomes the dominate inflammatory leukocyte (1). This dynamic shift in characteristic of inflammatory leukocytes is secondary to the production of numerous leukocyte chemotactic factors. IL-8 and MCP-1 belong to
CHEMOTACTIC CYTORINE (IL-8 AND M C P - 1 )
393
a novel class of chemotactic cytokines that possess specific cheniotactic/activating activity for leukocytes (5,6). Although our laboratory an'd others have shown that both IL-8 and MCP-1 are produced by a variety c'ells (5,6), these studies h'ave
Immunol Invest Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
been limited to rnonocellular systems that fail to exploit the intricacy of a tissue environment. To further understand the molecular and cellular regulation. o f these chemotactic cytokines in vivo the development of an in nitro tissue model would be a logical transition. We have previously demonstrated that WB, a specialized connective tissue, is an unique medium in which to assess cytoltine production (9). Under normal conditions i n vivo, WB's role is unparalleled for its fluidic ability tcl provide interorgan/ tissue communication, whereas duriiig inflammation the microvascular (environment favors hemostasis with associated sequen tia!, leukocyte aggregation, margination, adherence, and diapedesis (1). It is under these conditions that WB-derived chemotactic cytokine generation is relevant to the subsequent migration of inflammatory leukocytes. In this study, we describe the dose and time-dependent expression of both IL-8 and MCP-1 by WB in response to either products of gram-negative bacteria or yeast, LPS and zymosan, respectively. Although indirectly, LPS or zymosan could ccinceivably mediate their response by complement activation, recombinant C5a stimulation done in this system was unable to induce IL-8 or MCP-1 mFLVA (data not shown). The use of WB with the complexity of tissue has an 'enormous potential for thiz evaluation of chemotactic cytokine regulation and response to pharmacologic agents. The assessment of a pharmacologic agent's influence in chemotactic cytokine expression by this i n vitvo tissue system may have significant implications as to its potential efficacy in vivo. ACKNOWLEDGEMENTlS
This work was supported in part by NIH grants HL31693, HL35276, HL02401, and 13K38149. Additional support was provided by the Council far Tobacco Researlch, the American Heart Association of Florida, and the American Lung Association. Dr. Strieter is a RJR Nabisco Research Scholar. REFERENCES
1. 2.
M. I. Cybulsky, M. K. W. Chan, and H. Z. Mov
