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Meeting Abstracts  |   April 1998
Induction of Cyclooxygenase-2 in Alveolar Macrophages after Acid Aspiration  : Selective Cyclooxygenase-2 Blockade Reduces Interleukin-6 Production
Author Notes
  • (Ohara, Kurahashi, Doshi) Research Fellow, Department of Anesthesia, University of California, San Francisco.
  • (Sawa) Assistant Research Anesthesiologist, Department of Anesthesia, University of California, San Francisco.
  • (Wiener-Kronish) Vice Chairman, Department of Anesthesia; Professor of Anesthesia and Medicine, University of California, San Francisco.
  • (Kudoh) Associate Professor of Anesthesiology, Yokohama City University School of Medicine.
  • (Gropper) Assistant Professor of Anesthesia and Physiology, University of California, San Francisco.
Article Information
Meeting Abstracts   |   April 1998
Induction of Cyclooxygenase-2 in Alveolar Macrophages after Acid Aspiration  : Selective Cyclooxygenase-2 Blockade Reduces Interleukin-6 Production
Anesthesiology 4 1998, Vol.88, 1014-1022. doi:
Anesthesiology 4 1998, Vol.88, 1014-1022. doi:
GASTRIC acid aspiration can result in acute lung injury. The mechanism causing this injury has been shown to involve proinflammatory mediators, including cytokines and lipid mediators. [1] Numerous attempts have been made to reduce lung and systemic organ injury by blocking the chemical mediators released from stimulated cells. [2–4] The alveolar macrophage, situated at the air-tissue interface in the alveoli, is the first cell to encounter injurious stimuli and to release various chemical mediators in the early stages of the inflammatory response. [5] 
The importance of prostanoids produced by prostaglandin G/H synthase, also known as cyclooxygenase, in acid aspiration has been described previously. [6,7] Two isoforms of cyclooxygenase are now known: cyclooxygenase-1 and cyclooxygenase-2. Cyclooxygenase-1 is a 65-kDa protein, constitutively expressed in most tissues, notably the kidney, stomach, vascular smooth muscle, [8] and platelets. [9] Enzyme expression remains fairly constant, and cyclooxygenase-1 is thought to be responsible for “housekeeping” functions, such as gastric protection and renal blood flow regulation via tissue-specific production of prostaglandins. [10] In 1989, a second, inducible form of the cyclooxygenase enzyme, cyclooxygenase-2, was identified. [10] Cyclooxygenase-2 is a 70-kDa protein and is 65% homologous to cyclooxygenase-1. [11] Unlike cyclooxygenase-1, however, cyclooxygenase-2 is highly inducible in certain cell types after stimulation with various substances. Cyclooxygenase-2 is responsible for the production of large amounts of prostaglandins in the early stages of the inflammatory response. Macrophages are thought to be a major source of prostaglandins produced from upregulation of cyclooxygenase-2. [12–14] 
The regulation of cytokines by prostaglandins in inflammatory states has been described. [15,16] Of the major proinflammatory cytokines, interleukin (IL)-6 appears to be most closely regulated by prostaglandins. [13,17] IL-6 is a multifunctional cytokine that has been implicated in a variety of inflammatory conditions. Prostaglandin E sub 2 (PGE2) produced by cyclooxygenase-2 has been demonstrated to be responsible for increased concentrations of IL-6 in inflammatory conditions in vivo. [13,17] 
We studied the role of cyclooxygenase-2 in alveolar macrophages after acid aspiration, and by using a specific cyclooxygenase-2 blocker we investigated the interaction between cyclooxygenase-2 and various proinflammatory cytokines early in the inflammatory response.
Methods
All animal experiments were performed in compliance with the guidelines given by the Committee on Animal Research of the University of California, San Francisco.
Acid Aspiration in Mice
Eight- to twelve-week-old male C57BL/6 mice (body weights, 25–30 g) were purchased from Simonsen Laboratories (Gilroy, CA). The mice were fed regular rodent chow and water ad libitum. Mice were anesthetized briefly with inhaled methoxyflurane (Metofane; Pitman-Moore, Inc., Mundelein, IL). The acid group was instilled with 75 micro liter hydrochloric acid solution (pH 1.15, 0.1 N, endotoxin-free; Sigma Chemical Co., St. Louis, MO) mixed with normal saline (normal saline:hydrochloric acid ratio was 1:2). The saline group was instilled with 75 micro liter of one third normal saline solution (normal saline: H2O ratio was 1:2) into one lung via a blunt end feeding needle (24 G; Popper and Sons, Inc., New Hyde Park, NY) inserted orally. [18] Mice were allowed to recover from anesthesia and breathe room air spontaneously for the experimental period. The treatment group received a specific cyclooxygenase-2 blocker, N-2(-cyclohexyloxy-4-nitrophenyl) methane-sulphonamide (NS-398; Biomol, Plymouth Meeting, PA); each mouse was given 250 micro gram intraperitoneally, 2.5 mg/ml suspended in dimethyl sulfoxide, 1 h before acid instillation. [12,19] The nontreatment group received sham treatments of 100 micro liter dimethyl sulfoxide by intraperitoneal injection.
Harvest of Alveolar Macrophages by Bronchoalveolar Lavage
Mice were reanesthetized 2 and 4 h after acid instillation with 50 mg/kg of pentobarbital sodium. Their tracheas were exposed, and 20-gauge stub adapters were inserted and secured. Lungs were lavaged with cold 0.1% ethylenediaminetetraacetic acid/phosphate-buffered saline (PBS; 5 ml). Lavage fluid from two or three mice was pooled to obtain enough alveolar macrophages for analysis. Lavage fluid was centrifuged for 10 min at 4 [degree sign] Celsius at 1,000 x g to separate the cells from supernatant. Cells were washed and resuspended in PBS (5 ml). Alveolar macrophage numbers and viability were quantified by hemacytometer with trypan blue staining (> 90%). Macrophage concentration was adjusted to x 106cells/ml in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum and antibiotic agents. Macrophages were purified by allowing them to adhere to the bottom of the wells by incubation for 1 h in multiwell tissue culture plates. Nonadherent cells were removed by replacing the RPMI medium.
Semiquantitative Competitive Polymerase Chain Reaction for Determination of Levels of Cyclooxygenase-1, Cyclooxygenase-2, and Cytokine Genes in Messenger Ribonucleic Acid
Total ribonucleic acid (RNA) was extracted from 5 x 105freshly harvested alveolar macrophages with TRIzol reagent (Gibco BRL, Gaithersburg, MD). RNA concentrations were measured carefully by a microspectrophotometer (Spectramax; Molecular Devices, Menlo Park, CA), and 2.0 micro gram total RNA was reverse transcribed using reverse transcriptase, M-MLV (Promega, Madison, WI), and oligo-dT12-18 primers (Gibco BRL). The following specific primers for cyclooxygenase-1 and cyclooxygenase-2 were made for polymerase chain reaction (PCR):
cyclooxygenase-1
5'-primer: 5'-GGT TGA GGC ACT GGT GGA TGC CTT C-3'
3'-primer: 5'-AGA CAG ACC CGT CAT CTC CAG GGT A-3'
cyclooxygenase-2
5'-primer: 5'-ACT CAC TCA GTT TGT TGA GTC ATT C-3'
3'-primer: 5'-TTT GAT TAG TAC TGT AGG GTT AAT G-3'.
The size of the PCR products from cyclooxygenase-1 and cyclooxygenase-2 are 524 and 583 base pairs, respectively. The specificity of the PCR reaction for cyclooxygenase-1 and cyclooxygenase-2 complementary deoxyribonucleic acid (cDNA) was confirmed by digestion of PCR products by sequence-specific restriction enzymes (cyclooxygenase-1: Sac I and Sma I; cyclooxygenase-2: Pst I and Nco I). For quantitation of cyclooxygenase-1 and cyclooxygenase-2 mRNA levels, the Escherichia coli plasmid pT2M containing specific internal standards for cyclooxygenase-1 and cyclooxygenase-2 was constructed by internal deletion of sequences: The truncated fragments (320 base pairs) from cyclooxygenase-1 and cyclooxygenase-2 PCR products were incorporated into the E. coli cloning plasmid pBSII SK(+)(Stratagene, La Jolla, CA) at the EcoR I and Kpn I restriction sites and multiplied as plasmids in an E. coli strain Dh5 alpha. Plasmid solutions were adjusted to 1 x 104molecules/micro liter after double digestion by restriction enzymes (EcoR I and Kpn I) and used as internal standards for competitive PCR. For IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, inducible nitric oxide synthase (iNOS), and hypoxanthine-guanine phospho-ribosyltransferase (HPRT), a multiple cytokine competitor construct pLOC (provided by David B. Corry, University of California, San Francisco, San Francisco, CA) was used with specific cytokine primers as described. [20,21] We obtained confirmation that the cDNA samples contained the same amount of DNA by measuring the constitutively expressed HPRT gene first. The amounts of cDNA for cyclooxygenase-1, cyclooxygenase-2, and the various cytokine genes were measured based on this standardization, and the ratio of wild-type genes to competitor genes was amplified by PCR. For HPRT measurement, 1 x 105competitor molecules were added. Polymerase chain reaction was performed for 35 cycles with the following conditions: 30 s at 94 [degree sign] Celsius for denaturing DNA, 20 s at 55–60 [degree sign] Celsius annealing temperature, and 40 s at 72 [degree sign] Celsius for extension of the reaction. Products of PCR were separated by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining.
Immunocytochemical Detection of Cyclooxygenase-2 in Alveolar Macrophages
Alveolar macrophages were harvested 4 h after instillation. Cells were counted with a hemacytometer, and cell viability was calculated. After washing the cells once with PBS, they were resuspended in PBS, and 1 x 105cells were attached to glass slides by cytospin preparation. Slides were air-dried and stored at -80 [degree sign] Celsius until staining. Immunocytochemistry was performed using the avidin-biotinylated alkaline phosphatase complex method (Vectastain ABC-AP complex kit; Vector, Burlingame, CA). Frozen slides were brought to room temperature and fixed in 2% paraformaldehyde. Cells were permeabilized with 0.1% triton-X/PBS. All washing procedures were performed with high-salt PBS (2.7% NaCl/PBS). Blocking of nonspecific reactions to the secondary antibody was performed with 5% normal goat serum plus 10% normal horse serum in PBS. Slides were incubated with the primary antibody (polyclonal anti-PGHS-2, polyclonal rabbit immunoglobulin G; or control, preimmune rabbit serum; Oxford Biomedical Res., Inc., Oxford, MI) at 1/50 dilution in the blocking buffer (overnight at 4 [degree sign] Celsius). After the secondary antibody (goat anti-rabbit immunoglobulin G, biotinylated anti-rabbit immunoglobulin G; Vector) was applied (1 micro gram/ml in blocking buffer for 30 min at room temperature), slides were incubated with the avidin-biotinylated alkaline phosphatase complex and developed with the substrate (Fast Red TR/Naphthol AS-MX; Sigma). After mounting, slides were viewed at high magnification and photographed. Cells were counted at low magnification (100 cells per visual area), and the ratio of positively stained cells was calculated.
Alveolar Macrophage Culture
Alveolar macrophages harvested from mice as described in the harvesting method were cultured in RPMI1640 at 37 [degree sign] Celsius in a humidified atmosphere containing 5% CO2for 24 h at a concentration of 1 x 106cells/ml, 200 micro liter/well, in 96-well tissue culture plates. When indicated, NS-398, a specific cyclooxygenase-2 blocker, was added to the culture medium at 100-micro Meter concentration at the beginning of the incubation. Culture media was collected after 24 h of incubation and stored at -80 [degree sign] Celsius until assayed for PGE2, cytokines, or nitrite. Cell viability was 90–95% at the end of the 24-h incubation period, in media with or without NS-398.
Determination of Prostaglandin E sub 2 Concentrations by Enzyme Immunoassay
Prostaglandin E2was measured in the supernatant of the primary culture medium of alveolar macrophages by PGE2enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Each sample was measured in triplicate. The specificity of this assay for PGE2is 100%. The lower detection limit was 5 pg/ml.
Measurement of Cytokines by Enzyme-linked Immunosorbent Assay
Enzyme-linked immunosorbent assays were performed for IL-1beta, IL-6 and TNF-alpha as previously described. [18] The supernatant of the alveolar macrophage culture was used for measurements. For IL-6, microtiter plates (enzyme-linked immunosorbent assay plate, Easy wash; Corning, Cambridge, MA) were coated overnight at 4 [degree sign] Celsius with capture antibody (rat anti-mouse IL-6 monoclonal antibody, MP5–20F3, rat immunoglobulin G1; PharMingen, San Diego, CA) and blocked with 3% bovine serum albumin/PBS for 2 h at room temperature. Recombinant standards and samples were then added to plates and incubated for 4 h at room temperature. After incubation with biotinylated developing antibody (rat anti-mouse IL-6 monoclonal antibody, MP5–32C11, rat immunoglobulin G2a; PharMingen) for 45 min at room temperature and alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) for 45 min at 37 [degree sign] Celsius, plates were developed with p-nitrophenyl-phosphate (Sigma 104) dissolved in 0.1 M alkaline buffer solution (2-amino-2-methyl-1-pro-panol buffer, 1.5 M, pH 10.3, Sigma 221) and read at an OD of 405 nm using a microplate reader. Plates were washed two to six times with 0.05% Tween/PBS between each procedure. Sample concentrations were calculated by comparison with a standard curve of mouse recombinant IL-6 (PharMingen). Interleukin-1beta and TNF-alpha were measured with the same methods using the following reagents: rabbit anti-mouse IL-1beta polyclonal antibody, biotinylated anti-mouse IL-1beta monoclonal antibody (1400.24.17, mouse immunoglobulin G1), recombinant mouse IL-1beta (all from Endogen, Cambridge, MA), rat anti-mouse TNF-alpha monoclonal antibody (MP6-XT22, rat immunoglobulin G1), biotinylated rabbit anti-mouse TNF-alpha polyclonal antibody, and recombinant mouse TNF-alpha (all from PharMingen). Each sample was measured in triplicate. Lower detection limits for IL-1beta, IL-6, and TNF-alpha were 50.0, 1.0, and 5.0 pg/ml, respectively.
Measurement of Metabolites of Nitric Oxide
Nitrite concentrations in the supernatant of the alveolar macrophage culture were measured by Griess reaction. The reagents were prepared immediately before assay. One hundred microliters of supernatant was mixed with 100 micro liter Griess reagent (final concentration, 1% sulfanilamide, 0.1% n-(1-naphthyl)-ethylene-diamine, 1.7% H3PO4) and incubated for 30 min in the dark. Absorbance was measured at 550 nm using a microplate reader. Concentrations were determined using a NaNO2standard (0.3–100.0 micro Meter).
Data Analysis
Results are expressed as mean +/- SD. Comparisons between groups were performed with a one-way analysis of variance, and a Bonferroni/Dunn test was used for multiple comparison. A probability value < 0.05 was considered statistically significant and is identified with an asterisk in the figures.
Results
Expression of the Cyclooxygenase-2 Gene in Alveolar Macrophages after Acid Aspiration
In the control group, mRNA levels for the cyclooxygenase-1 and cyclooxygenase-2 genes in alveolar macrophages were lower than 10% of the HPRT mRNA (Figure 1(B), rows 1 and 2). Saline instillation did not affect these levels. Two hours after acid instillation, however, a larger amount of cyclooxygenase-2 mRNA was detected; this large amount was the same at 4 h (Figure 1(B), row 2). In contrast, the cyclooxygenase-1 mRNA was not increased after acid instillation (Figure 1(B), row 1).
Figure 1. (A, upper) Interpretation of competitive polymerase chain reaction (PCR). The PCR in this figure was run with a set amount of complementary deoxyribonucleic acid (cDNA), adding increasing amounts of competitor-increasing by 10-fold going from the left lane to the right. It demonstrates the ratio of mimic to wild-type genes in each gel picture. For semiquantitation, we designated +/-, +, 2+, and 3+ for each 10-fold increase from control level and estimated the amount of gene relative to control sample level. (B, lower) Cyclooxygenase-1, -2, and cytokine messenger ribonucleic acid (mRNA) levels estimated by competitive PCR performed with 2.0 micro gram total RNA extracted from alveolar macrophages from acid-instilled mice, 2 and 4 h after instillation, with and without pretreatment with NS-398. Upper bands are PCR fragments from wild-type mRNA for cyclooxygenase-1 and -2; competitors (mimic bands) are shown in the lower portion. For cytokines and hypoxanthine-guanine phospho-ribosyltransferase, competitors are upper bands. Note the cyclooxygenase-2 induction with acid instillation; the amount of mRNA was unaffected by pretreatment with NS-398 (row 2). Cyclooxygenase-1 levels are unchanged. Interleukin-1beta was upregulated transiently 2 h after saline instillation but returned to control levels at 4 h. Interleukin-6 was upregulated at 2 and 4 h after acid instillation and decreased with cyclooxygenase-2 blockade (row 4). Tumor necrosis factor-alpha concentrations changed minimally, and iNOS mRNA levels did not change (rows 5 and 6). Each lane represents pooled macrophages from three mice. Each experiment was repeated three times. Representative results are shown in this figure. MW marker = molecular weight marker.
Figure 1. (A, upper) Interpretation of competitive polymerase chain reaction (PCR). The PCR in this figure was run with a set amount of complementary deoxyribonucleic acid (cDNA), adding increasing amounts of competitor-increasing by 10-fold going from the left lane to the right. It demonstrates the ratio of mimic to wild-type genes in each gel picture. For semiquantitation, we designated +/-, +, 2+, and 3+ for each 10-fold increase from control level and estimated the amount of gene relative to control sample level. (B, lower) Cyclooxygenase-1, -2, and cytokine messenger ribonucleic acid (mRNA) levels estimated by competitive PCR performed with 2.0 micro gram total RNA extracted from alveolar macrophages from acid-instilled mice, 2 and 4 h after instillation, with and without pretreatment with NS-398. Upper bands are PCR fragments from wild-type mRNA for cyclooxygenase-1 and -2; competitors (mimic bands) are shown in the lower portion. For cytokines and hypoxanthine-guanine phospho-ribosyltransferase, competitors are upper bands. Note the cyclooxygenase-2 induction with acid instillation; the amount of mRNA was unaffected by pretreatment with NS-398 (row 2). Cyclooxygenase-1 levels are unchanged. Interleukin-1beta was upregulated transiently 2 h after saline instillation but returned to control levels at 4 h. Interleukin-6 was upregulated at 2 and 4 h after acid instillation and decreased with cyclooxygenase-2 blockade (row 4). Tumor necrosis factor-alpha concentrations changed minimally, and iNOS mRNA levels did not change (rows 5 and 6). Each lane represents pooled macrophages from three mice. Each experiment was repeated three times. Representative results are shown in this figure. MW marker = molecular weight marker.
Figure 1. (A, upper) Interpretation of competitive polymerase chain reaction (PCR). The PCR in this figure was run with a set amount of complementary deoxyribonucleic acid (cDNA), adding increasing amounts of competitor-increasing by 10-fold going from the left lane to the right. It demonstrates the ratio of mimic to wild-type genes in each gel picture. For semiquantitation, we designated +/-, +, 2+, and 3+ for each 10-fold increase from control level and estimated the amount of gene relative to control sample level. (B, lower) Cyclooxygenase-1, -2, and cytokine messenger ribonucleic acid (mRNA) levels estimated by competitive PCR performed with 2.0 micro gram total RNA extracted from alveolar macrophages from acid-instilled mice, 2 and 4 h after instillation, with and without pretreatment with NS-398. Upper bands are PCR fragments from wild-type mRNA for cyclooxygenase-1 and -2; competitors (mimic bands) are shown in the lower portion. For cytokines and hypoxanthine-guanine phospho-ribosyltransferase, competitors are upper bands. Note the cyclooxygenase-2 induction with acid instillation; the amount of mRNA was unaffected by pretreatment with NS-398 (row 2). Cyclooxygenase-1 levels are unchanged. Interleukin-1beta was upregulated transiently 2 h after saline instillation but returned to control levels at 4 h. Interleukin-6 was upregulated at 2 and 4 h after acid instillation and decreased with cyclooxygenase-2 blockade (row 4). Tumor necrosis factor-alpha concentrations changed minimally, and iNOS mRNA levels did not change (rows 5 and 6). Each lane represents pooled macrophages from three mice. Each experiment was repeated three times. Representative results are shown in this figure. MW marker = molecular weight marker.
×
Levels of Proinflammatory Cytokine Genes in Messenger Ribonucleic Acid in Alveolar Macrophages after Acid Aspiration
We found more IL-1beta gene expression in alveolar macrophages from mice instilled with either saline or acid compared with the control mice (Figure 1(B), row 3). There was a slight increase in IL-6 gene expression in alveolar macrophages transiently 2 h after saline instillation, but the level was the same as that of controls at 4 h. In the acid group, however, there was more IL-6 gene expression at 2 h after acid instillation compared with the control group, and it remained at this level after 4 h (Figure 1(B), row 4). Messenger RNA levels of TNF-alpha and iNOS genes were not different among control, saline, and acid groups (Figure 1(B), rows 5 and 6).
Intense Cyclooxygenase-2 Expression in the Cytoplasm of Alveolar Macrophages after Acid Aspiration
Two different staining patterns for cyclooxygenase-2 in alveolar macrophages were detected from immunocytochemical analysis. One pattern was the localized staining of the perinuclear membrane, suggesting minimal expression of cyclooxygenase-2 (Figure 2(A)). The other pattern was strong staining not only in the perinuclear region but also in the cytoplasmic region, indicating high expression of cyclooxygenase-2 (Figure 2(B)). Almost all macrophages showed baseline regional perinuclear cyclooxygenase-2 expression. None of the alveolar macrophages from control mice showed cytoplasmic expression of cyclooxygenase-2. In the saline group, < 1% of the cells (0.8 +/- 1.5%) showed cytoplasmic expression. Significantly more alveolar macrophages (11.3 +/- 4.4%) harvested from mice instilled with acid showed the strong cytoplasmic cyclooxygenase-2 expression (P < 0.01).
Figure 2. Immunocytochemical staining of alveolar macrophages showing two patterns of staining. (A) Minimal localized cyclooxygenase-2 staining of the perinuclear membrane of alveolar macrophages. (B) Strong staining found not only in the perinuclear region but also in the cytoplasmic region, demonstrating high expression of cyclooxygenase-2.
Figure 2. Immunocytochemical staining of alveolar macrophages showing two patterns of staining. (A) Minimal localized cyclooxygenase-2 staining of the perinuclear membrane of alveolar macrophages. (B) Strong staining found not only in the perinuclear region but also in the cytoplasmic region, demonstrating high expression of cyclooxygenase-2.
Figure 2. Immunocytochemical staining of alveolar macrophages showing two patterns of staining. (A) Minimal localized cyclooxygenase-2 staining of the perinuclear membrane of alveolar macrophages. (B) Strong staining found not only in the perinuclear region but also in the cytoplasmic region, demonstrating high expression of cyclooxygenase-2.
×
Alveolar Macrophages Produce High Concentrations of Prostaglandin E sub 2 and Interleukin-6 after Acid Aspiration
In 24-h culture media, we detected a trend of release of more PGE2from alveolar macrophages harvested 1 h after acid instillation compared with saline instillation (Figure 3). A significantly higher level of IL-6 production also was detected in the alveolar macrophages harvested after acid instillation compared with the saline group (Figure 4). The concentrations of IL-1beta and TNF-alpha were lower than detectable levels in the culture media from all groups of macrophages (data not shown). Low concentrations of nitric oxide metabolites (NO2sup - < 3 micro Meter) were detected in all groups of the macrophage culture, and there was no significant difference between groups (data not shown).
Figure 3. Prostaglandin E2levels in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was all-quoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. No statistical significance was found
Figure 3. Prostaglandin E2levels in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was all-quoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. No statistical significance was found
Figure 3. Prostaglandin E2levels in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was all-quoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. No statistical significance was found
×
Figure 4. Interleukin-6 concentrations in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was aliquoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. *P = 0.006 versus saline group;(dagger)P = 0.0054 versus acid group.
Figure 4. Interleukin-6 concentrations in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was aliquoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. *P = 0.006 versus saline group;(dagger)P = 0.0054 versus acid group.
Figure 4. Interleukin-6 concentrations in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was aliquoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. *P = 0.006 versus saline group;(dagger)P = 0.0054 versus acid group.
×
Selective Cyclooxygenase-2 Blockade Reduces Interleukin-6 Release from Alveolar Macrophages after Acid Aspiration
Pretreatment of mice with the selective cyclooxygenase-2 blocker, NS-398, resulted in the PGE2production from alveolar macrophages harvested after acid instillation being at the same low level as that occurring with the addition of the cyclooxygenase-2 blocker to the macrophage culture medium (Figure 3). Interleukin-6 release from alveolar macrophages harvested from mice instilled with acid was significantly reduced by pretreatment with NS-398 (Figure 4). Although cyclooxygenase-2 blockade did not change mRNA levels of the cyclooxygenase-2 gene in the alveolar macrophages harvested from mice instilled with acid, decreases of IL-6 mRNA levels were detected at 2 and 4 h after acid instillation (Figure 1(B)).
Discussion
The role of prostaglandins in acid aspiration remains controversial. [22] In canine acid aspiration models, nonsteroidal antiinflammatory drugs have been used successfully to reduce lung edema and improve pulmonary function, [7,23] whereas other investigators have found that nonsteroidal antiinflammatory drugs improved the physiologic status of the lung but did not change mortality in sepsis. [24] 
Nonspecific blockade of both cyclooxygenase-1 and -2 results in the well-known toxicities of gastric ulceration and renal and platelet dysfunction. [12] Since the cloning of cyclooxygenase-2, compounds have been developed that selectively block cyclooxygenase-2 while preserving the functions of cyclooxygenase-1. [12,19,25,26] The specific cyclooxygenase-2 inhibitor we used in our study, NS-398, is a highly selective inhibitor of cyclooxygenase-2 and forms an irreversibly inhibited complex. [19] In the acute phase of an inflammatory response (2–4 h after stimuli), the increase in prostaglandin production is entirely dependent on cyclooxygenase-2 protein synthesis. [16] In a carrageenin-induced subcutaneous air pouch model [20] and a pleurisy model in rats, [26] these specific cyclooxygenase-2 blockers were used successfully to reduce inflammation, suggesting the possibility of treating various inflammatory disorders with these new compounds without the hazards of conventional nonsteroidal antiinflammatory drugs.
In this study, significant induction of cyclooxygenase-2 was detected in alveolar macrophages after acid aspiration. First, the marked upregulation of cyclooxygenase-2 was confirmed by measurement of mRNA levels using a highly sensitive method, competitive PCR. [27] Second, intense expression of the cyclooxygenase-2 protein in alveolar macrophages after acid aspiration was demonstrated by specific cyclooxygenase-2 immunocytochemical staining. Cells with less intense cytoplasmic staining were likely lavaged from the contralateral lung, which did not receive acid. The source of increased production of PGE sub 2 was identified as cyclooxygenase-2 by selective blockade of cyclooxygenase-2 with NS-398.
Recently, the interactions between prostaglandins produced by cyclooxygenase-2 and various inflammatory cytokines or nitric oxide have been examined in vivo and in vitro. [16,17,28–30] In this model, despite the fact that an increased mRNA level of IL-1beta was detected in alveolar macrophages after acid aspiration, the relation of IL-1beta released from alveolar macrophages to cyclooxygenase-2 upregulation was unclear, as similar upregulation also was observed in alveolar macrophages from mice instilled with saline. In addition, no detectable concentrations of IL-1beta were measured in the macrophage culture medium. We found a close relationship between cyclooxygenase-2 and IL-6, however; increased production of IL-6 in alveolar macrophages after acid aspiration was prevented by cyclooxygenase-2 blockade. The roles of TNF-alpha [1,31] and nitric oxide in acid aspiration, a chemical injury, remain controversial. [32] These mediators are implicated in lung injury from bacteria; however, we did not detect upregulation of TNF-alpha or iNOS even with our extremely sensitive competitive PCR method. We conclude that these mediators are not involved in the early phase of inflammation in our model of acid aspiration. Others have shown in a rabbit model of acid lung injury that IL-8 is a major contributor in the inflammatory process. [3] The homologous protein for IL-8 has not been identified in the mouse. Mediators of the chemokine family (KC, MIP-2, and others), however, may be released from alveolar macrophages to play a role in acute lung injury.
Using a murine model of acid aspiration, we have demonstrated that acid aspiration induces cyclooxygenase-2 in alveolar macrophages. We have demonstrated that IL-6 production from alveolar macrophages was significantly increased after acid aspiration. Using a specific cyclooxygenase-2 inhibitor, we also have shown that cyclooxygenase-2-derived prostanoids were responsible for induction of macrophage IL-6 production. It is well established that IL-6 plays a major role in the propagation of inflammatory responses, such as fever induction in the hypothalamus and induction of the acute phase response in the liver. [33,34] Interleukin-6-deficient mice were found to have impaired fever induction and acute phase response to inflammatory stimuli. [35] In addition, IL-6 production correlates with clinical outcome in the systemic inflammatory response syndrome. [36,37] In this model, the upregulation of IL-6 was reduced by selective cyclooxygenase-2 blockade. This result is consistent with previous reports using a rat arthritis model [17] and peritonitis model [13] in that induction of cyclooxygenase-2 is responsible for the increase in PGE2concentrations, which in turn induces IL-6.
We conclude that cyclooxygenase-2 is strongly induced in alveolar macrophages after acid aspiration, and specific blockade of cyclooxygenase-2 may be used in decreasing production of inflammatory mediators such as PGE2and IL-6, which may cause tissue injury.
The authors thank Richard Shanks for technical assistance.
REFERENCES
Goldman G, Welbourn R, Kobzik L, Valeri CR, Shepro D, Hechtman HB: Tumor necrosis factor-alpha mediates acid aspiration-induced systemic organ injury. Ann Surg 1990; 212:513-20.
Kudoh I, Ohtake M, Nishizawa H, Kurahashi K, Hattori S, Okumura F, Pittet JF, Wiener-Kronish JP: The effect of pentoxyfylline on acid-induced alveolar epithelial injury. Anesthesiology 1995; 82:531-41.
Folkesson HG, Matthay MA, Hebert CA, Broaddus VC: Acid aspiration-induced lung injury in rabbits is mediated by interleukin-8-dependent mechanisms. J Clin Invest 1995; 90:107-16.
Goldman G, Welbourn R, Kobzik L, Valeri CR, Shepro D, Hechtman HB: Neutrophil adhesion receptor CD18 mediates remote but not localized acid aspiration injury. Surgery 1995; 117:83-9.
Cavaillon JM: Cytokines and macrophages. Biomed Pharmacother 1994; 48:445-53.
Matthay MA, Rosen GD: Acid aspiration induced lung injury-New insights and therapeutic options. Am J Respir Crit Care Med 1996; 154:277-8.
Utsunomiya T, Krausz MM, Dunham B, Valeri CR, Levine L, Shepro D, Hechtman HB: Modification of inflammatory response to aspiration with ibuprofen. Am J Physiol 1982; 243:H903-10.
Smith WL, Meade EA, DeWitt DL: Interactions of PGH synthase isozymes-1 and -2 with NSAIDs. Ann N Y Acad Sci 1994; 744:50-7.
Funk CD, Funk LB, Kennedy ME, Pong AS, Fitzgerald GA: Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression and gene chromosomal assignment. FASEB J 1991; 5:2304-12.
Hla T, Neilson K: Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci U S A 1992; 89:7384-8.
Williams CS, DuBois RN: Prostaglandin endoperoxide synthase: Why two isoforms? Am J Physiol 1996; 270:G393-400.
Masferrer JL, Zweifel BS, Manning PT, Hauser SD, Leahy KM, Smith WG, Isakson PC, Seibert K: Selective inhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory and nonulcerogenic. Proc Natl Acad Sci U S A 1994; 91:3228-32.
Hinson RM, Williams JA, Shacter E: Elevated interleukin 6 is induced by prostaglandin E sub 2 in a murine model of inflammation: Possible role of cyclooxygenase-2. Proc Natl Acad Sci U S A 1996; 93:4885-90.
Davies P, MacIntyre DE: Prostaglandins and inflammation, Inflammation: Basic Principles and Clinical Correlates. Edited by Gallin JI, Goldstein IM, Snyderman R. New York, Raven Press, 1992, pp 123-38.
Crofford LJ, Tan B, McCarthy CJ, Hla T: Involvement of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis Rheum 1997; 40:226-36.
Peplow PV: Actions of cytokines in relation to arachidonic acid metabolism and eicosanoid production. Prostaglandins Leukot Essent Fatty Acids 1996; 54:303-17.
Anderson GD, Hauser SD, McGarity KL, Bremer ME, Isakson PC, Gregory SA: Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J Clin Invest 1996; 97:2672-9.
Sawa T, Corry DB, Gropper MA, Ohara M, Kurahashi K, Wiener-Kronish JP: IL-10 improves lung injury and survival in Pseudomonas aeruginosa pneumonia. J Immunol 1997; 159:2858-66.
Ouellet M, Percival MD: Effect of inhibitor time-dependency on selectivity towards cyclooxygenase isoforms. Biochem J 1995; 306:247-51.
Corry DB, Folkesson HG, Warnock ML, Erle DJ, Matthay MA, Wiener-Kronish JP, Locksley RM: Interleukin 4, but not interleukin 5 or eosinophils, are required in a murine model of acute airway hyperreactivity. J Exp Med 1996; 183:109-17.
Reiner SL, Zheng S, Corry DB, Locksley RM: Constructing polycompetitor cDNA for quantitative PCR. J Immunol Methods 1993; 165:37-46.
Mayers I, Breen PH, Gottlieb S, Long R, Wood LD: The effects of indomethacin on edema and gas exchange in canine acid aspiration. Respir Physiol 1987; 69:149-60.
Wu W, Halebian PH, Hariri RJ, Cabrales SX, Shires GT, Barie PS: Differential effects of cyclo-oxygenase and thromboxane synthetase inhibition on ventilation-perfusion relationships in acid aspiration-induced acute lung injury. J Trauma 1992; 33:561-7.
Bernard GR, Wheeler AP, Russell JA, Schein R, Summer WR, Steinberg KP, Fulkerson WJ, Wright PE, Christman BW, Dupont WD, Higgins SB, Swindell BB: The effects of ibuprofen on the physiology and survival of patients with sepsis. The ibuprofen in sepsis study group. N Engl J Med 1997; 336:912-8.
Vane J: Towards a better aspirin. Nature 1994; 367:215-6.
Nakatsugi S, Terada T, Yoshimura T, Horie Y, Furukawa M: Effects of nimesulide, a preferential cyclooxygenase-2 inhibitor, on carrageenan-induced pleurisy and stress-induced gastric lesions in rats. Prostaglandins Leukot Essent Fatty Acids 1996; 55:395-402.
Siebert PD, Larrick JW: Competitive PCR. Nature 1992; 359:557-8.
Dinarello CA: Role of interleukin-1 and tumor necrosis factor in systemic response to infection and inflammation, Inflammation: Basic Principles and Clinical Correlates. Edited by Gallin JI, Goldstein IM, Snyderman R. New York, Raven Press, 1992, pp 211-32.
Di Rosa M, Ialenti A, Ianaro A, Sautebin L: Interaction between nitric oxide and cyclooxygenase pathways. Prostaglandins Leukot Essent Fatty Acids 1996; 54:229-38.
Williams G, Giroir BP: Regulation of cytokine gene expression: Tumor necrosis factor, interleukin-1, and the emerging biology of cytokine receptors. New Horiz 1995; 3:276-87.
Fantuzzi G, Dinarello C: The inflammatory response in interleukin-1beta-deficient mice: Comparison with other cytokine-related knockout mice. J Leukoc Biol 1996; 59:489-93.
Strohmaier W, Schlag G: Experimental models in surfactant research, Pathophysiology of Shock, Sepsis and Organ Failure. Edited by Schlag G, Redl H. New York, Springer-Verlag, 1994, pp 757-71.
Akira S, Kishimoto T: IL-6 and NF-IL6 in acute-phase response and viral infection. Immunol Rev 1992; 127:25-50.
Van Snick J: Interleukin-6: Overview. Annu Rev Immunol 1990; 8:253-78.
Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, Kishimoto T, Zinkernagel R, Bluethmann H, Kohler G: Impaired immune and acute-phase response in interleukin-6-deficient mice. Nature 1994; 368:339-42.
Damas P, Ledoux D, Nys M, Vrindts Y, De Groote D, Franchimont P, Lamy M: Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann Surg 1992; 215:356-62.
Drost AC, Burleson DG, Cioffi WG Jr, Jordan BS, Mason AD Jr, Pruitt BA Jr: Plasma cytokines following thermal injury and their relationship with patient mortality, burn size, and time postburn. J Trauma 1993; 35:335-9.
Figure 1. (A, upper) Interpretation of competitive polymerase chain reaction (PCR). The PCR in this figure was run with a set amount of complementary deoxyribonucleic acid (cDNA), adding increasing amounts of competitor-increasing by 10-fold going from the left lane to the right. It demonstrates the ratio of mimic to wild-type genes in each gel picture. For semiquantitation, we designated +/-, +, 2+, and 3+ for each 10-fold increase from control level and estimated the amount of gene relative to control sample level. (B, lower) Cyclooxygenase-1, -2, and cytokine messenger ribonucleic acid (mRNA) levels estimated by competitive PCR performed with 2.0 micro gram total RNA extracted from alveolar macrophages from acid-instilled mice, 2 and 4 h after instillation, with and without pretreatment with NS-398. Upper bands are PCR fragments from wild-type mRNA for cyclooxygenase-1 and -2; competitors (mimic bands) are shown in the lower portion. For cytokines and hypoxanthine-guanine phospho-ribosyltransferase, competitors are upper bands. Note the cyclooxygenase-2 induction with acid instillation; the amount of mRNA was unaffected by pretreatment with NS-398 (row 2). Cyclooxygenase-1 levels are unchanged. Interleukin-1beta was upregulated transiently 2 h after saline instillation but returned to control levels at 4 h. Interleukin-6 was upregulated at 2 and 4 h after acid instillation and decreased with cyclooxygenase-2 blockade (row 4). Tumor necrosis factor-alpha concentrations changed minimally, and iNOS mRNA levels did not change (rows 5 and 6). Each lane represents pooled macrophages from three mice. Each experiment was repeated three times. Representative results are shown in this figure. MW marker = molecular weight marker.
Figure 1. (A, upper) Interpretation of competitive polymerase chain reaction (PCR). The PCR in this figure was run with a set amount of complementary deoxyribonucleic acid (cDNA), adding increasing amounts of competitor-increasing by 10-fold going from the left lane to the right. It demonstrates the ratio of mimic to wild-type genes in each gel picture. For semiquantitation, we designated +/-, +, 2+, and 3+ for each 10-fold increase from control level and estimated the amount of gene relative to control sample level. (B, lower) Cyclooxygenase-1, -2, and cytokine messenger ribonucleic acid (mRNA) levels estimated by competitive PCR performed with 2.0 micro gram total RNA extracted from alveolar macrophages from acid-instilled mice, 2 and 4 h after instillation, with and without pretreatment with NS-398. Upper bands are PCR fragments from wild-type mRNA for cyclooxygenase-1 and -2; competitors (mimic bands) are shown in the lower portion. For cytokines and hypoxanthine-guanine phospho-ribosyltransferase, competitors are upper bands. Note the cyclooxygenase-2 induction with acid instillation; the amount of mRNA was unaffected by pretreatment with NS-398 (row 2). Cyclooxygenase-1 levels are unchanged. Interleukin-1beta was upregulated transiently 2 h after saline instillation but returned to control levels at 4 h. Interleukin-6 was upregulated at 2 and 4 h after acid instillation and decreased with cyclooxygenase-2 blockade (row 4). Tumor necrosis factor-alpha concentrations changed minimally, and iNOS mRNA levels did not change (rows 5 and 6). Each lane represents pooled macrophages from three mice. Each experiment was repeated three times. Representative results are shown in this figure. MW marker = molecular weight marker.
Figure 1. (A, upper) Interpretation of competitive polymerase chain reaction (PCR). The PCR in this figure was run with a set amount of complementary deoxyribonucleic acid (cDNA), adding increasing amounts of competitor-increasing by 10-fold going from the left lane to the right. It demonstrates the ratio of mimic to wild-type genes in each gel picture. For semiquantitation, we designated +/-, +, 2+, and 3+ for each 10-fold increase from control level and estimated the amount of gene relative to control sample level. (B, lower) Cyclooxygenase-1, -2, and cytokine messenger ribonucleic acid (mRNA) levels estimated by competitive PCR performed with 2.0 micro gram total RNA extracted from alveolar macrophages from acid-instilled mice, 2 and 4 h after instillation, with and without pretreatment with NS-398. Upper bands are PCR fragments from wild-type mRNA for cyclooxygenase-1 and -2; competitors (mimic bands) are shown in the lower portion. For cytokines and hypoxanthine-guanine phospho-ribosyltransferase, competitors are upper bands. Note the cyclooxygenase-2 induction with acid instillation; the amount of mRNA was unaffected by pretreatment with NS-398 (row 2). Cyclooxygenase-1 levels are unchanged. Interleukin-1beta was upregulated transiently 2 h after saline instillation but returned to control levels at 4 h. Interleukin-6 was upregulated at 2 and 4 h after acid instillation and decreased with cyclooxygenase-2 blockade (row 4). Tumor necrosis factor-alpha concentrations changed minimally, and iNOS mRNA levels did not change (rows 5 and 6). Each lane represents pooled macrophages from three mice. Each experiment was repeated three times. Representative results are shown in this figure. MW marker = molecular weight marker.
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Figure 2. Immunocytochemical staining of alveolar macrophages showing two patterns of staining. (A) Minimal localized cyclooxygenase-2 staining of the perinuclear membrane of alveolar macrophages. (B) Strong staining found not only in the perinuclear region but also in the cytoplasmic region, demonstrating high expression of cyclooxygenase-2.
Figure 2. Immunocytochemical staining of alveolar macrophages showing two patterns of staining. (A) Minimal localized cyclooxygenase-2 staining of the perinuclear membrane of alveolar macrophages. (B) Strong staining found not only in the perinuclear region but also in the cytoplasmic region, demonstrating high expression of cyclooxygenase-2.
Figure 2. Immunocytochemical staining of alveolar macrophages showing two patterns of staining. (A) Minimal localized cyclooxygenase-2 staining of the perinuclear membrane of alveolar macrophages. (B) Strong staining found not only in the perinuclear region but also in the cytoplasmic region, demonstrating high expression of cyclooxygenase-2.
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Figure 3. Prostaglandin E2levels in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was all-quoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. No statistical significance was found
Figure 3. Prostaglandin E2levels in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was all-quoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. No statistical significance was found
Figure 3. Prostaglandin E2levels in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was all-quoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. No statistical significance was found
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Figure 4. Interleukin-6 concentrations in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was aliquoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. *P = 0.006 versus saline group;(dagger)P = 0.0054 versus acid group.
Figure 4. Interleukin-6 concentrations in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was aliquoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. *P = 0.006 versus saline group;(dagger)P = 0.0054 versus acid group.
Figure 4. Interleukin-6 concentrations in 24-h alveolar macrophage culture supernatant. Macrophages were freshly harvested from mice instilled with acid or saline, and cell concentration was adjusted to 1 x 106cells/ml. Cells were suspended in media, and 2 x 105cells/200 micro liter media of each sample was aliquoted into 96-well microplates. Values are mean +/- SD. The experiment was repeated three times. *P = 0.006 versus saline group;(dagger)P = 0.0054 versus acid group.
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