Regulation of Human Alveolar Macrophage- and Blood Monocyte-derived Interleukin-8 by Prostaglandin E2 and Dexamethasone Theodore J. Standiford, Steven L. Kunkel, Mark W. Rolfe, Holly L. Evanoff, Ronald M. Allen, and Robert M. Strieter Departments of Pathology and Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, Michigan

Mononuclear phagocytes are important immune effector cells that playa fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities, monocytes/macrophages produce a vast array of regulatory and chemotactic cytokines. Interleukin-S (IL-S), a potent neutrophil-activating and chemotactic peptide, is produced in large quantities by mononuclear phagocytes and may be an important mediator of local and systemic inflammatory events. In this investigation, we describe the effects of prostaglandin E2 (PGE2) and dexamethasone (Dex) on IL-S mRNA and protein expression from lipopolysaccharide (LPS)-treated human peripheral blood monocytes (PBM) and alveolar macrophages (AM). We demonstrate the dose-dependent suppression of IL~S from LPS-stimulated PBM by PGE 2 • Treatment of stimulated PBM with 10-6 M PGE2 resulted in maximal inhibition, causing 60 % suppression of both IL-S mRNA and extracellular protein levels. In contrast, PGE, (10-6 to 10-8 M) did not significantly alter IL-S mRNA or protein expression from LPS-treated AM. Treatment of LPS-stimulated PBM and AM with Dex (10-6 to 10-8 M) resulted in 75% decline in IL-S mRNA and extracellular protein from either cell population. Pretreatment of PBM with PGE 2 or Dex 1 or 2 h before LPS stimulation caused a significant suppression of steady-state IL-S mRNA levels; however, administration of either of these modulators 1 or 2 h after LPS stimulation failed to have an inhibitory effect. In a similar fashion, pretreatment with Dex before LPS stimulation resulted in a significant decrease in AM-derived steadystate IL-S mRNA levels, whereas no significant reduction in IL-S mRNA was observed from AM treated with Dex 1 to 2 h after LPS stimulation. Our findings suggest that either PGE 2 or corticosteroids may function as important immunomodulators of mononuclear phagocyte-derived IL-8. Furthermore, this regulation may be related to either the temporal activation or state of differentiation of the mononuclear phagocyte.

Many forms of acute and chronic lung injury are characterized by an initial exposure to a noxious insult, followed by a coordinated sequence of events that include inflammatory cell activation, adherence to endothelium, and subsequent migration along established chemotactic gradients. Mononuclear phagocytes play a central role in mediating these events via the expression of proximal proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin

(Received in original form March 26. 1991 and in revised form June 17, 1991) Address correspondence to: Robert M. Strieter, M.D., Department of Internal Medicine, Division of Pulmonary and Critical Care, 3916 Taubman Center/Box 0360, University of Michigan Medical School, Ann Arbor 48109-0360. Abbreviations: alveolar macrophage(s), AM; cyclic adenosine monophosphate, cAMP; dexamethasone, Dex; enzyme-linked immunosorbent assay, ELISA; interleukin, IL; lipopolysaccharide, LPS; peripheral blood monocyte(s), PBM; phosphate-buffered saline, PBS; prostaglandin E2, PGE2; tumor necrosis factor, TNF. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 75-81, 1992

(IL)-1 (1). TNF and IL-l initiate inflammation via the activation of neutrophils, induction of adherence protein expression on the surface of endothelial cells (2, 3), and by directly inducing the production of regulatory cytokines from a variety of immune and nonimmune cells (4-9). In addition, alveolar macrophages (AM) and peripheral blood monocytes (PBM) produce a vast array of chemotactic factors, including the lipid mediators leukotriene B. (LTB.) and plateletactivating factor (PAF), platelet-derived growth factor (PDGF), and the chemotactic cytokine IL-S (10, 11). IL-S has been implicated as a major neutrophil chemotactic and activating factor in several systemic inflammatory disease states (12-14) and is produced in significant quantities by both AM (15, 16) and PBM (5, 17). As mononuclear phagocytes are the predominant source of this chemotactic cytokine, the regulation of IL-S expression from these cells is of critical importance in determining the extent of a local or systemic inflammatory response. Several endogenous and exogenous agents are known to be involved in the in vitro and in vivo immunomodulation of neutrophil and macrophage function. Prostaglandin E2

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(POE,), a product of both immune (4, 18, 19) and nonimmune cells (20), has been shown to inhibit neutrophil chemotaxis, and the release of oxygen radicals and lysosomal enzymes from neutrophils (18, 21). In addition, POEz suppresses several macrophage functions, including macrophage proliferation, adherence, and migration (18). Moreover, POE z inhibits the expression of TNF and IL-l (22-27) from stimulated PBM. Endogenous corticosteroids or related synthetic analogs also appear to possess important in vitro and in vivo immunomodulatory effects, in part due to their suppressive effects on macrophage-derived cytokine synthesis. In addition to suppressing the production of TNF (22, 28, 29) and IL-l (30, 31) from cultured macrophages, exogenously administered dexamethasone (Dex) has been shown to inhibit the expression of these cytokines in vivo (32-35). Furthermore, adrenalectomy enhances the lethal effects of TNF and IL-l in a murine model of sepsis (35). Although POE, and corticosteroids would appear to be important regulators ofTNF and IL-l secretion,little is known regarding the effects of these irnmunomodulators on the expression of IL-8 from mononuclear phagocytes. In this investigation we examined the effects of POEz and Dex on the expression of IL-8 from human pulmonary macrophages and PBM. Dex causes profound suppression of lipopolysaccharide (LPS)-stimulated PBM and AM-derived IL-8 mRNA and protein expression. In contrast, POE, causes the dose-dependent inhibition of IL-8 gene expression from LPS-stimulated PBM, whereas this lipid mediator has minimal effect on the expression of IL-8 from similarly treated AM. Furthermore, the suppressive effectsof Dex and POE, are operative within a narrow time frame, as delayed addition of either POE, or Dex results in a reduction in their efficacy for inhibiting LPS-induced IL-8 mRNA accumulation.

Materials and Methods Reagents Dex was purchased from Sigma Chemical Co. (St. Louis, MO) and was solubilized in dimethyl sulfoxide at a concentration of 10-2 M. POE, was the generous gift of the Upjohn Co. (Kalamazoo, MI) and was solubilized in ethanol at a concentration of 10-' M. Human recombinant IL-8 was purchased from Peprotech (Rocky Hill, NJ). Polyclonal antihuman IL-8 antiserum used in the enzyme-linked immunosorbent assay (ELISA) was produced by immunization of rabbits with recombinant IL-8 in multiple intradermal sites with complete Freund's adjuvant. Stock cycloheximide (Sigma) was prepared at a concentration of 10 mg/ml in dimethyl sulfoxide and used at a concentration of 5 J.Iog/ml. Stock LPS (Escherichia coli 0l11:B4; Sigma) was prepared at a concentration of 200 J.Iog/ml in sterile RPMI-1640 (Whitaker Biomedical Products, Whitaker, CA) containing 1 mM glutamine, 25 mM Hepes, 100 V/ml penicillin, and 100 J.Iog/ml streptomycin (Hazelton Research Products, Denver, PA) (complete media). Recovery and Isolation of Human PBM and AM Nine normal, nonsmoking volunteers consented to undergo venopuncture and flexible fiberoptic bronchoscopy with bronchoalveolar lavage by standard techniques. Volunteers

denied symptoms of recent upper respiratory tract infection in the preceding 6 wk, and none were taking any medications. Briefly, subjects were given 1.0 mg atropine intramuscularly followed by the removal of 100 ml of venous blood in heparinized syringes. Supraglottic structures were then anesthetized with topical 4% xylocaine. The bronchoscope was wedged in two separate segments of the right middle lobe, and 300 ml normal saline was instilled in 30-ml aliquots with an average return of 200 ml. The recovered BAL fluid was filtered through sterile gauze and centrifuged at 600 x g for 8 min. The cells were resuspended in complete media. This procedure yielded 30.3 ± 3.9 x 106 cells with the following differential: 94.7 ± 1.2% AM, 4.1 ± 1.1 % lymphocytes, 0.4 ± 0.2% neutrophils, and 0.6 ± 0.3% eosinophils. The cells were> 95 % viable as assessed by trypan blue exclusion. Cells were washed two additional times and resuspended in complete serum-free media. AM were adherence-purified in 35-mm plastic culture dishes (Costar, Cambridge, MA) at a final concentration of 106 AM/plate in 1 ml of complete media. Venous blood was mixed 1:1 with 0.9% saline, and mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation (22). Isolated mononuclear cells were washed twice, and PBM were adherence-purified in plastic culture dishes at a concentration of 106 monocytes/ml of complete serum-free media. Percentages of plated AM and PBM that were adherent to plastic exceeded 90% for both cell populations. IL-8 ELISA Extracellular immunoreactive IL-8 was quantitated using a modification of a double ligand method as previously described (36). Briefly, flat-bottomed 96-well microtiter plates (Nunc Immuno-Plate I 96-F) were coated witih 50 J.Iollwell of rabbit anti-Hi-S antibody (1 ng/ml in 0.6 M NaC!, 0.26 M H3B04 , and 0.08 N NaOH [pH 9.6]) for 16 h at 4 0 C and then washed with phosphate-buffered saline (PBS) (pH 7.5), 0.05% Tween 20 (wash buffer). Microtiter plate nonspecific binding sites were blocked with 2 % bovine serum albumin in PBS and incubated for 90 min at 37 C. Plates were rinsed 4 times with wash buffer, and diluted (neat, 1:5, and 1:10) cell-free supernatants (50 J.Iol) in duplicate were added, followed by incubation for 1 h at 37 C. Plates were washed 4 times, followed by the addition of 50 J.IoI/well biotinylated rabbit anti-IL-8 antibody (3.5 ng/ml in PBS [pH 7.5], 0.05% Tween 20, and 2% fetal calf serum), and incubated for 30 min at 3r c. Plates were washed 4 times, streptavidinperoxidase conjugate (Bio-Rad Laboratories, Richmond, CA) was added, and the plates were incubated for 30 min at 37 C. Plates were again washed 4 times, and chromogen substrate (Bio-Rad) was added. The plates were then incubated at room temperature to the desired extinction, and the reaction was terminated with 50 J.Iollwell of 3 M H2S04 solution. Plates were read at 490 nm in an ELISA reader. Standards were 1/2 log dilutions of recombinant IL-8, from 1 pg/ml to 100 ng/ml. This ELISA method consistently detected IL-8 concentrations greater than 10 pg/ml. 0

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Northern Blot Analysis Total cellular RNA from AM and PBM was isolated using a modification of Chirgwin and associates (37) and Jonas and

Standiford, Kunkel, Rolfe et al.: Regulation of Mononuclear Phagocyte-derived IL-8 by PGEz and Dex

co-workers (38). Briefly, cells were overlaid with 1 ml of a solution consisting of 25 mM Tris (pH 8.0), containing 4.2 M guanidine isothiocyanate, 0.5% Sarkosyl, and 0.1 M 2-mercaptoethanol. After homogenization, the above suspension was added to an equal volume of 100 mM Tris (pH 8.0), containing 10 mM EDTA and 1.0% sodium dodecyl sulfate. The mixture was then extracted with chloroform-phenol and chloroform-isoamyl alcohol. The RNA was alcohol-precipitated, and the pellet dissolved in DEPC H,o. RNA was separated by Northern blot analysis using formaldehyde, 1% agarose gels, transblotted to nitrocellulose, baked, prehybridized, and hybridized with a 3'P 5'-end labeled oligonucleotide probe. The human IL-8 oligonucleotide probe was complementary to nucleotides 262 through 291 and has the sequence 5'-GTT-GGC-GCA-G TG-TGG-TCC-ACT-CTCAAT-CAC-3' (39). Blots were washed, and autoradiographs quantitated using laser densitometry (Ultrascan XS; LKB Instruments, Houston, TX). Equivalent amounts of total RNA/well were assessed by monitoring 28S and 18S ribosomal RNA. Statistical Analysis Data were analyzed by a Macintosh II computer using Statview II statistical package (Abacus Concepts, Inc., Berkeley, CA). Data are expressed as mean ± SEM and compared using a two-tailed Student's t test with the Bonferroni correction for multiple comparisons. Data were considered statistically significant at P < 0.05.

Results The Effect of PGE z and Dex on IL-8 Production from LPS-stimulated AM and PBM To determine if PGEz or Dex altered the expression of antigenic IL-8 from stimulated mononuclear phagocytes, and whether differences existed in the regulation of IL-8 synthesis between two different mononuclear phagocyte populations, we treated isolated PBM and AM with graded doses of PGEz or Dex and assessed them for extracellular IL-8 as determined by ELISA. Cells were preincubated for 1 h in the presence or absence of PGEz or Dex (10-6 to 10-' M), treated with LPS (100 ng/ml) , and cell-free supernatants were harvested 24 h after stimulation with LPS (n = 9). This concentration of LPS was used in all experiments as it represented the dose at which half-maximal expression ofIL-8 occurred (data not shown). As shown in Figure 1, untreated PBM generated 19.7 ± 5.0 ng/106 cells of IL-8, whereas untreated AM constitutively produced 192.6 ± 47.2 ng/lQ6 cells of extracellular IL-8. We have previously observed significant production of IL-8 from PBM and especially AM upon adherence in the absence of other stimuli (40). The above findings were unaltered by the addition of polymyxin B, a potent inhibitor of LPS (data not shown), which suggested that the expression of IL-8 from these adherent cells was not secondary to LPS contamination. When both mononuclear phagocyte populations were treated with LPS, substantial upregu1ation of IL-8 production was observed, as PBM produced 418.4 ± 112.1 ng/l O' cells and AM produced 453.5 ± 95.2 ng/106 cells. Pretreatment of PBM with PGE z before LPS addition resulted in the dose-dependent suppression of IL-8 expression, with significant in-

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hibition occurring at PGEz concentrations of 10-6 M (59% suppression, P = 0.03) and 10-7 M (48 % suppression, P = 0.04). Interestingly, AM were less responsive to the suppressive effects of PGEz, as no significant reduction of IL-8 synthesis occurred at PGEz concentrations of 10-6 to 10-' M. Pretreatment of cells with Dex (10-6 M), however, caused significant suppression of IL-8 secretion from both PBM and AM, resulting in a 73% reduction in PBM-derived IL-8 (P = 0.02) and a 61% reduction in AM-derived IL-8 (P = 0.002). The concentrations of PGEz, Dex, LPS, or their vehicles used in these studies did not alter the viability of either macrophage population studied (data not shown). The Effect of PGE z and Dex on IL-8 mRNA Expression from LPS-treated AM and PBM To determine whether PGEz or Dex exert their suppressive effects at the level of IL-8 mRNA, we next examined the effect of these immunomodulators on IL-8 mRNA expression from AM and PBM. The two mononuclear phagocyte populations were cultured at a concentration of 106 cells/ml (1 X lQ6 cells/plate for AM,S X 106 cells/plate for PBM), preincubated for 1 h with PGEz (10-6 to 10-' M) or Dex (10-6 to 10-' M), stimulated with LPS, and total cellular RNA was isolated 4 h after LPS stimulation (n = 3). Total

78

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 61992

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RNA was harvested at 4 h, as maximal IL-8 mRNA accumulation from PBM and AM occurs at this time point. As shown in Figure 2, PGE, (10-6 M) caused a 49 ± 4.6% reduction in steady-state IL-8 mRNA levels expressed from LPS-treated PBM, whereas lower concentrations of PGE, were less effective in reducing steady-state IL-8 mRNA levels. When PBM preincubated with PGE2 (10-6 M) were challenged with higher concentrations of LPS (l and 10 ",g/ml), however, no significant suppression of IL-8 mRNA expression from these cells was observed (data not shown). Furthermore, PG~ (10-6 to 10-8 M) failed to significantly alter steady-state IL-8 mRNA levels from stimulated AM" as PG~ (10-6 M) resulted in only a 14 ± 3.8% reduction in AM-derived IL-8 mRNA. Dex caused profound inhibition of IL-8 mRNA levels expressed from PBM, even at concentrations as low as 10-8 M. IL-8 mRNA expression from AM was also inhibited by Dex in a dose-dependent fashion, but not to the same extent as observed in similarly treated PBM

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(Figure 3). Dex maintained its inhibitory influence on IL-8 mRNA expression from PBM and AM even when these cells were challenged with greater concentrations of LPS (l and 10 ",g/ml, data not shown). The Effeet of Delayed Addition of PGE, or Dex on IL-8 mRNA Expression from. LPS-treated AM and PBM The above-mentioned experiments demonstrated that Dex, and to a lesser extent PGE2, suppressed IL-8 mRNA and protein secretion from LPS-treated PBM and AM. We next performed studies to determine whether similar inhibitory effects on IL-8 mRNA expression would be seen when Dex or PGE2 was administered in a time-dependent fashion before or after LPS stimulation. PBM were treated with either Dex or PGE, 2 h before LPS or 1 or 2 h after treatment with LPS, and total cellular RNA was isolated 4 h after LPS stimulation. Because PG~ had little effect on AM-derived IL-8 mRNA levels, we examined only the effects of pre-

79

Standiford, Kunkel, Rolfe et al.: Regulation of Mononuclear Phagocyte-derived IL-8 by PGE 2 and Dex

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treatment and post-treatment with Dex. As shown in Figure 4, 2 h pretreatment of PBM with PGE 2 (10-6 M) or Dex (10-6 M) resulted in a significant reduction in IL-8 mRNA, with inhibitory effects still being observed when these two modulators were administered 1 h after LPS. When treatment of PBM with Dex was delayed 2 h after LPS stimulation, only a modest reduction in steady-state mRNA levels was noted. PGE 2 had no effect on PBM-derived IL-8 mRNA levels when this substance was given 2 h after LPS. In a similar fashion, 1- or 2-h preincubation of AM with Dex (10-6 M) resulted in substantial suppression in IL-8 mRNA levels, whereas Dex, when administered concomitantly or following exposure to LPS, failed to suppress AM-derived IL-8 mRNA expression (Figure 5).

Discussion Mononuclear phagocytes are multifunctional effector cells that are essential to the maintenance of normal immune re-

c Figure 5. The time-dependent effect of Dex (10-·M) on LPS (100 ng/ml)-stimulated AM-derived IL-8 mRNA. Panel A: Representative Northern blot autoradiograph representing the kinetics of Dex treatment from LPS (100 ng/ml)-stimulated AM (n = 2). Panel B: Laser densitometry analysis of the autoradiograph. Panel C: The corresponding 18S and 28S rRNA of the autoradiograph in panel A, demonstrating equivalent loading oftotal RNA. 2°- = 2 h before LPS; 1°_ = I h before LPS; 0 = concomitant treatment; 1°+ = 1 h after LPS; 2°+ = 2 h after LPS.

sponses. Not only do these cells serve as major phagocytic and antigen-presenting cells of the immune system, but they also produce a vast array of regulatory and chemotactic cytokines (10, 11). Several of these cytokines, including IL-8, are likely critically important in host defense, and the overexuberant production of these cytokines may have important immunopathologic consequences in a variety of disease states (12-14, 41). The major cellular sources of IL-8 are blood monocytes (5, 17) and, in the lung, the AM (15, 16). Hence, the regulation of mononuclear phagocyte-derived IL-8 production may dramatically influence the evolution of an inflammatory response, and the understanding of mechanisms involved is critical in designing future therapeutic interventions. In this investigation, we described the inhibition of IL-8 mRNA and protein expression from PBM by PGE 2 and Dex. Dex similarly suppressed IL-8 expression from AM, whereas treatment of these cells with PGE, failed to significantly alter AM-derived IL-8 at either the mRNA or protein levels. IL-8 is an 8-kD peptide that is produced by a wide variety of immune and nonimmune cells. In addition to macrophages, neutrophils (42), endothelial cells (6), epithelial cells (9), and fibroblasts (7) also produce IL-8. IL-8 has been found to have significant neutrophil-activating activity, as this peptide enhances neutrophil oxygen radical generation, enzyme release, and 5-lipoxygenase activity (41). Furthermore, IL-8 is chemotactic for neutrophils, lymphocytes, and basophils (41, 43). Although a number of studies have

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992

characterized the cellular sources and immunologic activities of IL-8, little has been reported concerning the regulation of IL-8 gene expression. We have previously identified IL-4 as a potent inhibitor ofIL-8 gene expression from PBM (44), though this lymphokine did not alter the expression of IL-8 mRNA from stimulated nonimmune cells (45). Mukaida and colleagues described the suppression of IL-8 mRNA accumulation from LPS-stimulated PBM by Dex, which was felt to be mediated through a specific glucocorticoid-binding site within the IL-8 genome (46). The effect of Dex on IL-8 secretion from macrophages, or its effect on IL-8 gene expression from other cellular sources, has not been described. The molecular mechanisms by which Dex and PGEz suppress IL-8 gene expression remain unknown. Dex reduced IL-8 mRNA transcripts expressed from both PBM and AM, suggesting suppression at the level of gene transcription. We do not know whether this inhibition represents decreased mRNA synthesis or accelerated mRNA decay. We suspect the former explanation as Dex has been shown to inhibit the transcription of other cytokine mRNA species, including TNF, IL-l, and IL-6 (23,27,30, 31,47). We cannot exclude a minor post-transcriptional regulatory effect, as Dex has been shown to have additional post-transcriptional inhibitory effects on TNF and IL-l gene expression (27,30). The suppressive effects of Dex were not dependent upon the de novo synthesis of a protein intermediate, as cycloheximide did not alter the Dex-induced reduction in IL-8 mRNA (data not shown). Mechanisms by which PGEz inhibits PBM-derived IL-8 gene expression are less clear. PGE z, like Dex, dosedependently reduces PBM-derived IL-8 mRNA levels. This is similar to the effect of this arachidonic acid metabolite on TNF mRNA expression from peritoneal macrophages (23), which is reduced secondary to direct inhibition of TNF gene transcription. In contrast, PGEz does not significantly alter monocyte-derived IL-l,6 mRNA levels or cell-associated IL-l{3, but appears to inhibit extracellular IL-l,6 by inhibiting cytokine secretion (25,26). Both the transcriptional and posttranscriptional effects of PGEz appear to be mediated by increased levels of cyclic adenosine monophosphate (cAMP), as other agents that increased intracellular cAMP, including forskolin (adenyl cyclase activator) and dibutyryl cAMP, had similar suppressive effects on TNF and IL-l production (24, 25, 27). The suppression of monocyte-derived IL-8 gene expression by PGEz may not be mediated by alterations in intracellular levels of cAMP, as Mukaida has described marginal induction of IL-8 mRNA accumulation by forskolin (46). Preliminary studies in this laboratory are in agreement with this observation, as treatment of LPS-stimulated PBM with either dibutyryl cAMP (10-5 M) or forskolin (10-5 M) did not alter or may have induced modest increases in IL-8 mRNA and protein production (data not shown). Furthermore, the treatment of LPS-challenged AM or PBM with both Dex and PGEz concomitantly did not further suppress IL-8 mRNA levels as compared with Dex pretreatment alone (data not shown), suggesting that these two agents may exert their inhibitory effects by similar molecular mechanisms. Our findings suggest that Dex, and especially PGEz, have lesser immunomodulatory effects on IL-8 expression from AM as compared with PBM. This is consistent with previous investigations that have demonstrated disparity in

both the production of cytokines and regulation of cytokine expression from the more terminally differentiated AM as compared with freshly isolated PBM. Significant alterations in TNF, IL-l, and IL-6 production occur with in vitro maturation of monocytes (48-50). Furthermore, AM and PBM display substantial differences in their ability to express these cytokines. Disparity in the regulation of cytokine production also exists, as we have previously shown that PGEz and Dex have profound inhibitory effects on TNF biosynthesis from PBM, whereas these substances exerted minimal influence on TNF secretion from AM (22). In addition, we have observed that IL-4 inhibits IL-8 gene expression from PBM but does not alter the expression of IL-8 mRNA from AM or monocytes aged in culture (unpublished observations). These findings suggest that PBM and newly recruited lung macrophages may be more amenable to both endogenous and exogenous regulatory signals, whereas in the process of terminal differentiation, AM become less responsive to such immunomodulators. We have demonstrated that preincubation of cells with either PGEz (PBM) or Dex (PBM and AM) before exposure to a primary stimulus can suppress IL-8 mRNA accumulation. The treatment of mononuclear phagocytes with these agents after LPS stimulation, however, failed to suppress PBM- or AM-derived IL-8 mRNA expression. Our findings are consistent with observations from studies investigating the effectsof delayed addition of Dex on TNF production (27, 28). These investigators found that if treatment ofPBM with Dex was delayed 2 to 8 h after LPS exposure, the inhibitory effect of Dex on TNF biosynthesis was significantly abrogated. These findings are important in the clinical context because attempts to modulate in vivo cytokine cascade pharmacologically generally occur after exposure to the inciting pathologic event. This, in part, may explain why systemically administered corticosteroids fail to significantly alter the clinical outcome of patients with the sepsis syndrome (51). Furthermore, knowing that regulation of cytokine expression can occur only within a narrow temporal window, and that AM are relatively refractory to the immunomodulatory effects of exogenous and endogenous regulators, it is not surprising that neither PGE z nor Dex has been shown to be of benefit in the treatment of patients with the adult respiratory distress syndrome (52, 53). Acknowledgments: This work was supported in part by an American Lung Association Research Grant, by Grants HL-02401, HL-31693, HL-35276, and DK38149 from the National Institutes of Health, and by the Council for Tobacco Research. Dr. Strieter is an RIR Nabisco Research Scholar.

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Standiford, Kunkel, Rolfe et al.: Regulation of Mononuclear Phagocyte-derived IL-8 by PGE 2 and Dex

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