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TOXICOLOGICAL SCIENCES ARTICLE NO.
46, 300 –307 (1998)
TX982557
Pulmonary Cytokine and Chemokine mRNA Levels after Inhalation of Lipopolysaccharide in C57BL/6 Mice C. J. Johnston, J. N. Finkelstein, R. Gelein, and G. Oberdo¨rster Departments of Environmental Medicine and Pediatrics, University of Rochester, Rochester, New York 14642 Received February 27, 1998; accepted July 29, 1998
Pulmonary Cytokine and Chemokine mRNA Levels after Inhalation of Lipopolysaccharide in C57BL/6 Mice. Johnston, C. J., Finkelstein, J. N., Gelein, R., and Oberdo¨rster, G. (1998). Toxicol. Sci. 46, 300 –307. Inhaled endotoxin (lipopolysaccharide, LPS) can induce acute lung injury and at high doses may lead to respiratory distress syndrome. Using a mouse model of acute lung inflammation induced by inhalation of low doses of LPS we examined the kinetics of chemokine, proinflammatory cytokine, and metallothionein. Eight-week-old C57BL/6 mice were dosed for 10 min with LPS, resulting in an estimated alveolar dose of <10 ng LPS/mouse, and euthanized 2, 6, or 24 h postexposure. Analysis of bronchoalveolar lavage fluid demonstrated increased polymorphonuclear neutrophils (PMNs) of 6.94, 32.7, and 38.8% after 2, 6, and 24 h, respectively. Examination of proinflammatory cytokine, chemokine, and Mt mRNA in the lung revealed increases for messages encoding IL-1a, IL-1b, IL-6, IFN-g, TNFa, Eotaxin, MIP-1a, MIP-1b, MIP-2, Mt, and IP-10, while messages encoding IL-12, IL-10, IFN-b, Ltn, MCP-1, TGFb1 1 2, and RANTES were unchanged from those of sham-exposed mice 2 h postexposure. By 6 h most messages had returned to near control levels. Comparison to 5 mg/kg body weight intraperitoneal injection and 5 mg/mouse intratracheal instillation 2 h postexposure demonstrated similar message responses. Our results demonstrate that low levels of LPS exposure by inhalation induce a strong PMN response and a selective cytokine response in the lung, supporting the hypothesis that PMNs may regulate inflammatory processes via cytokine and chemokine response. © 1998 Society of Toxicology.
Inhalation of cotton and other organic dusts may result in both acute and chronic lung disorders. For example, mill fever occurs with acute exposure to cotton dust (Merchant et al., 1975). In addition to respiratory symptoms and fever, acute exposure of both animals and humans to organic dusts can result in pulmonary inflammation, characterized by an influx of neutrophils into the lung (Merchant et al., 1975; Castranova et al., 1987; Ryan and Karol, 1991). Due to their complex composition, the agents within many organic dusts have not been completely defined. One of the components frequently noted is endotoxin from gram-negative bacteria. Chronic exposure of endotoxin measured in the dust from occupational and domestic settings has been related to both the risk of developing 1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.
chronic obstructive pulmonary diseases (Schwartz et al., 1995; Smid et al., 1992; Ryan et al., 1994) and the severity of domestic asthma (Michel et al., 1996). Endotoxin (lipopolysaccharide; LPS) induces acute lung injury in endotoxic sepsis and gram-negative pneumonia, two conditions marked by the activation of alveolar macrophages (AM) and massive tissue infiltration of neutrophils (PMN), which may lead to adult respiratory distress syndrome (Brigham and Meyrick, 1986). The symptoms of acute endotoxin exposure include fever, chills, dyspnea, chest tightness, coughing, and decreases in lung diffusion capacity (Pernis et al., 1961; Muittari et al., 1980a, b; Rylander et al., 1978, 1989). Airborne endotoxin concentration (0.006–0.779 mg/m3) has been correlated with decreased FEV1 in exposed humans (Castellan et al., 1987). Results from animal models also indicate that endotoxin is a potent toxicant. Inhalation of endotoxin by guinea pigs resulted in a neutrophil influx into airways (Ryan and Karol, 1991; Hudson et al., 1977). In addition, two primary inflammatory cytokines, TNF and IL-1, were induced by inhalation of endotoxin (Ryan and Karol, 1991). Cytokines including TNFa, IL-1a and b and IL-6 are secreted by macrophages (Nathan, 1987) which can autoactivate macrophages and increase the production of superoxide radicals by neutrophils (Borish et al., 1989; Sharber and Nathan, 1986). They are potent proinflammatory cytokines that trigger recruitment of adhesion molecules and chemokines (Lloyd and Oppenheim, 1992). Inflammatory cell recruitment is an important initial step in the host defense against inhaled toxicants. Recent studies have identified a growing superfamily of chemotactic peptides, termed chemokines, which play a role in the recruitment and activation of leukocytes, which have been implicated in the initiation and amplification of autoimmune inflammatory responses. These chemokines have been subdivided based on function, C-X-C chemokines such as IL-8 and MIP-2 are generally chemoattractant for neutrophils, C-C chemokines such as RANTES, MCP-1, MIP-1a, and MIP-1b are predominantly chemoattractants for monocytes and lymphocytes (Oppenheim et al., 1991; Schall, 1991; Baggiolini et al., 1994; Taub et al., 1994). Although evidence from in vitro and intratracheal instillation studies using high doses have shown that cytokines from effector cells, primarily AM (Kelly, 1990; Xing et al., 1994) and PMN (Xing et al., 1994; Lloyd and Oppenheim, 1992), are
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TABLE 1 Cellular and Biochemical Analysis of Lung Lavage Fluid 2, 6, and 24 h after a 12-min Whole-Body Exposure to Endotoxin Inhalation (;10 ng Deposited in the Alveolar Region) in 8-Week-Old C57BL/6 Mice
Untreated Sac 2 h Sac 6 h Sac 24 h
Total cells (3 106)
% AM
% PMN
% Lymph
% Viable
Protein (ng/ml)
1.10 6 0.40 0.98 6 0.26 1.83 6 0.90 2.00 6 0.49
98.95 6 0.34 91.84 6 1.99 65.88 6 14.99 60.17 6 15.04
0.24 6 0.18 6.94 6 2.05* 32.70 6 14.27* 38.81 6 14.99*
0.81 6 0.48 1.21 6 0.33 1.25 6 0.79 1.03 6 0.70
91.90 6 2.22 90.46 6 3.54 92.39 6 2.93 94.16 6 1.86
0.16 6 0.05 0.14 6 0.02 0.17 6 0.04 0.17 6 0.01
Note. Means 6 SD, n 5 4 per group. * Statistically significant compared to untreated controls (p ,0.05).
involved, little is known about the kinetics of expression of cytokines and chemokines after inhalation of low levels of endotoxin (Michel et al., 1992; Goncalves de Mores et al., 1996; Uno et al., 1997). The primary objective of our current study was to explore the kinetics of expression of cytokines and chemokines after inhalation of LPS; secondary objectives were to compare responses of cytokines and chemokines after low-level inhalation to changes induced by intraperitoneal administration and intratracheal instillation. We focused on the lung injury model induced by inhalation since intratracheal instillation is known to result in a nonuniform deposition of the administered compound in the lung and intraperitoneal administration affects other target organs such as liver (Pritchard et al., 1985; Nelson et al., 1989) MATERIALS AND METHODS Adult male, 8-week-old mice C57BL/6 (Jackson Laboratory, Bar Harbor, ME) were housed four per cage with food and water ad libitum in a temperature- and humidity-controlled room with a 12-h light/dark cycle. Endotoxin Administration Inhalation. Twelve mice each were exposed for 10 min to air or LPS (Escherichia coli endotoxin, Sigma Chemical Co., St. Louis, MO). Aerosols were generated by using an AERO-Mist compressed air nebulizer (CIS-US, Inc., Bedford, MA); 100 ml of LPS (0.125 mg) in 2 ml saline was added to the nebulizer, which had an output airflow of 20 liter/min. Mice were exposed in a 30-liter whole-body exposure chamber in individual compartments. The LPS aerosol had a mass median aerodynamic diameter of 0.6 mm, with a geometric
standard deviation of 1.6. Using a mathematical deposition model for particles in the mouse lung (Yu and Oberdo¨rster, 1997) a dose of less than 10 ng LPS was estimated to be deposited in the alveolar region of the lung. Mice were killed at 2, 6, and 24 h postinhalation. Intraperitoneal injection. Six C57BL/6 mice were injected with saline or 5 mg/kg body weight in 0.5 ml saline. Mice were killed 2 h postinjection and lungs and livers were prepared for analysis as described above. Intratracheal instillation. Six C57BL/6 mice were anesthetized with 3% halothane and 0.1 ml saline (control) or 5 mg LPS/mouse in 100 ml saline was intratracheally instilled. Mice were killed 2 h postinstillation and lungs and livers prepared for analysis as described below. Tissue preparation. Mice were euthanized by intraperitoneal (ip) injection of Na pentobarbital (50 mg/kg) followed by exsanguination via the abdominal aorta. The lungs were excised and lavaged using 1 ml of saline, which was repeated for a total of 10 lavages. After 10 min centrifugation at 400g total lavagable cell numbers, cell viability, and cell differential were determined in the cell pellet as well as protein LDH and b-glucuronidase in the lavage supernatant. Lungs and liver were flash-frozen for RNA analysis after lavage. Ribonuclease Protection Assay Total RNA was isolated from lung tissue using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. Each frozen lung lobe (50 –100 mg) was homogenized in 1 ml of TRIzol reagent. Each final RNA pellet was resuspended in 50 ml of diethylpyrocarbonatetreated water. The RNA concentration and purity was quantified using the GeneQuant RNA/DNA Calculatory (Pharmacid Biotech, Piscataway, NJ). Quantitation of steady-state cytokine mRNA levels was performed using a previously described multicytokine ribonuclease protection assay (RPA) (Hobbs et al., 1993; Johnston et al., 1997). RNase protection assays were performed with riboprobe templates for mCK-2 (IL-12 p35, IL-12 p40, IL-10, IL-1a, IL-1b, IL-Ra, MIF, IFN-g, L32, GAPDH), mCK-3 (TNF-b, Lt-b,
TABLE 2 Cellular and Biochemical Analysis of Lung Lavage Fluid 2 h after Inhalation, Intratracheal Instillation, or Intraperitoneal Administration of Endotoxin
Treatment
Total cells (3 106)
% AM
% PMN
% Lymph
% Viable
Protein (mg/ml)
Untreated 5 mg/kg Intraperitoneal injection 5 mg/Mouse intratracheal instillation ;10 ng/mouse Deposited by inhalation
1.10 6 0.40 0.90 6 0.27 0.53 6 0.11 0.98 6 0.26
98.95 6 0.34 99.63 6 0.13 71.76 6 17.57 91.84 6 1.99
0.24 6 0.18 0.00 6 0.00 28.12 6 17.55* 6.94 6 2.05*
0.81 6 0.48 0.37 6 0.13 0.13 6 0.11 1.21 6 0.33
91.90 6 2.22 90.71 6 1.41 92.10 6 1.10 90.46 6 3.54
0.16 6 0.05 0.12 6 0.02 0.21 6 0.02 0.14 6 0.02
Note. Means 6 SD, n 5 4 (control and inhalation) or n 5 3 (ip and it injection). * Statistically significant compared to untreated controls (p ,0.05).
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JOHNSTON ET AL. TNFa, IFN-g, IFN-b, TGF-b1, TFGb2, L32, GAPDH), or mCK-5 (Ltn, RANTES, Eotaxin, MIP-1b, MIP-1a, MIP-2, IP-10, MCP-1, TCA3, L32, GAPDH) (PharMingen, San Diego, CA). The riboprobe synthesis reaction consisted of 50 ng of mCK-2, mCK-3, or mCK-5 template set; 120 mCi [a-32P]UTP (uridine triphosphate) (3000 Ci/mmol; Dupont NEN, Wilmington, DE); 5 nmol ATP (adenosin triphosphate); 5 nmol GTP (guanosine triphosphate); 5 nmol CTP (cytidine triphosphate); 150 pmol UTP; 2.5 mg yeast tRNA; 100 nmol dithiothreitol; 1 mg bovine serum albumin; 20 nmol spermidine; 10 U RNase inhibitor; and 50 U T7 RNA polymerase (Life Technologies) in 1 3 transcription buffer (40 mM Tris–HCl, pH 7.5, 6 mM MgCl2). The reaction was incubated for 90 min at 37°C and then diluted to 100 ml with DNase I buffer [50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 0.02 U/ml RQ1 DNase I (Promega, Madison, WI)]. After a 30-min incubation at 37°C, the riboprobes were purified by phenol/chloroform extraction and ethanol precipitation. The dried pellet was then resuspended in 50 ml of hybridization buffer [400 mM NaCl, 40 mM Pipes, pH 6.7, 1 mM EDTA (ethylenediaminetetraacetic acid), pH 8.0, 80% formamide (Sigma)], analyzed by scintillation counting, and then diluted to a final concentration of 2.6 3 105 cpm/ml in hybridization buffer. Ten-microgram samples of each RNA assayed were dried in a SpeedVac Concentrator (Savant, Farmingdale, NY) and resuspended in 8 ml of hybridization buffer. Two microliters of the diluted riboprobe was added to each RNA sample, heated to 80°C for 3 min, and then immediately placed at 56°C for 16 h. After solution hybridization, 100 ml of RNase cocktail [0.2 mg/ml RNase A (Sigma), 600 U/ml RNase T1 (Life Technologies), 10 mM Tris–HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA, pH 8] was added to each sample and incubated 45 min at 30°C. Eighteen microliters of proteinase K cocktail [0.67 mg/ml proteinase K (Life Technologies), 3.5% sodium dodecyl sulfate, 100 mg/ml yeast tRNA] was then added to each sample and incubated for 15 min at 37°C. The protected RNA duplexes were purified by phenol/ chloroform extraction and ethanol precipitation, and the pellets were resuspended in 5 ml of RPA loading buffer [80% formamide, 0.53 TBE, 0.05% bromphenol blue (Sigma)]. The protected, radiolabeled RNA fragments were electrophoresed on a 5% acrylamide/8 M urea sequencing gel, and the dried gel was used to expose X-AR film (Eastman Kodak, Rochester, NY) at 280°C with intensifying screens (Quanta III; Dupont, Wilmington, DE). Membrane Hybridization For membrane hybridization, slot blots were prepared in triplicate from a single dilution of glyoxal-denatured RNA as described by Thomas (1983), except that charged nylon membranes (magnagraph 45 mm, micrometer separations) are substituted for cellulose nitrate. Each slot was sliced in thirds. One-third of each slot was hybridized to a control cDNA probe encoding L32. The other two-thirds of each set of slots were hybridized with 32P-labeled cDNA probe for Mt (provided by G. Andrews, Kansas University). Random hexamer labeling (Feinberg and Vogelstein, 1983) was used to prepare cDNA probes and blots were hybridized at 68°C for ;18 h in 13 SSPE (10 mM NaC1, 10 mM EDTA), 10% dextran sulfate, 23 Denhart’s solution [0.04% polyvinylpyrrolidone-360, 2% Ficoll, 0.4 mM EDTA, 2.0% sodium dodecyl sulfate (SDS)], with 106 cpm probe/ml hybridization buffer. Membranes were washed, 20 min each time, in 0.13 SSC (sodium chloride, sodium citrate), 0.5% SDS at room temperature with shaking, followed by four washes in the same buffer at 68°C without shaking. After washing, membranes were blot dried, wrapped in Saran Wrap, and exposed for autoradiography at 280°C using X-ray film and intensifying screens. For quantitation, the dried gels were placed against phosphorimager screens (Molecular Dynamics, Sunnyvale, CA). The intensity of each specific cytokine band was measured with a computer-linked phosphorimager using the ImageQuant software (Molecular Dynamics). To correct for RNA loading, each intensity score was normalized to the intensity of hybridization for the L32 gene.
FIG. 1. Measurement of adult C57BL/6 mouse cytokine or chemokine mRNA levels 2, 6, or 24 h postendotoxin inhalation by RNase protection assay: 10 mg total lung RNA hybridized with 5.2 3 105 cpm for 18 h at 56°C.
Statistical Analysis A one-way analysis of variance (ANOVA) was performed using the SigmaStat 2.0 software (Jandel, San Rafael, CA) to determine the significance of
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FIG. 2. Time course of increases in the relative mRNA abundance of cytokines, chemokines and Mt after endotoxin inhalation. RNase protection assays were quantified by PhosphoImager analysis. All values were normalized to constitutively expressed mRNA encoding L32. Each bar represents the mean of three mice. The asterisk indicates statistical significance relative to sham-exposed control (p ,0.05).
observed variations in lavage parameters and cytokine mRNA levels at different timepoints.
RESULTS
Analysis of lavageable cells after endotoxin inhalation showed that total cell numbers were unchanged at all postexposure time points. The mean percentage of PMNs increased to 6.94, 32.70, and 38.81%, at 2, 6, and 24 h postexposure, respectively (Table 1). There was no significant change in protein levels or LDH or b-glucuronidase. At 2 h postexposure the intratracheal (IT)-instilled group demonstrated increases in the mean percentage of PMNs to 28.12%. Protein and other lavage parameters were unchanged. No measurable changes in PMN response or any other lavage parameter were measured after ip dosing of LPS (Table 2). Cytokine and chemokine mRNAs in total lung tissue were evaluated by RNase protection assay. As shown in Fig. 1, increases in mRNA abundance were evident 2 h postinhalation for the cytokines IL-1a, IL-1b, IL-1Ra, IL-6, IFN-g, and TNFa as well as the chemokines Eotaxin, MIP-1b, MIP-1a, MIP-2, and IP-10. Also, the antioxidant Mt was increased at this time point (Fig. 2). Six hours postexposure increases persisted in messages encoding TNFa, MIP-2, IP-10, and Mt. However, increases were lower than measured 2 h postexposure (Figs. 1 and 2). Twentyfour hours postexposure all messages had returned to near control levels.Messages encoding IL-12, IL-10, MIF, TNFb, LTb, IFN-b, TGFb1, TGFb2, Ltn, RANTES, and MCP-1 were unaltered at all time points examined. Comparison of chemokine mRNAs 2 h post Inh, IT, or ip showed increased mRNA abundance for messages encoding
IL-1a, IL-1b, IL-1Ra, IL-6, TNFa, MIP-1a, MIP-1b, MIP-2, IP-10- and Mt (Figs. 3 and 4). Increases in mRNA abundance were similar after Inh, IT, and ip endotoxin exposure. Comparison of liver cytokine mRNAs 2 h post Inh, IT, or ip demonstrated that messages encoding TNFa and IL-6 were induced only after ip administration of endotoxin (Fig. 5). DISCUSSION
We utilized a model of endotoxin inhalation to compromise the respiratory system and found that extremely low doses, estimated at ,10 ng deposited in the alveolar region, caused a significant inflammatory cell response in the lung, starting at 2 h postexposure and lasting for more than 24 h. Endotoxin-induced lung injury has been well described in humans (Michel et al., 1992, 1995, 1997) and guinea pig (Gordon, 1992; Sandstrom et al., 1992) after inhalation and in rodents after intraperitoneal administration as well as after intratracheal instillation of high LPS doses (Xing et al., 1993, 1994). In this study we demonstrated that inhalation of low levels of endotoxin triggers a distinct cytokine and chemokine response in the lung, selectively inducing TNFa, IL-1a, IL-1b, MIP-2, IL-6, and Mt, but not RANTES, TGFb1,2, IL-10 or IL-12. Initiation of a PMN response correlated with cytokine induction. Furthermore, the low dose does not cause damage to the epithelial barrier function, as demonstrated by the lack of protein in the lavage samples. Intraperitoneal administration of LPS triggered a similar message response. However, there was no PMN response at the selected dose of LPS. Consequently, this route of administration may be a useful model for the priming of target cells in the lung. Intratracheal instillation also demonstrated a similar message response to inhalation of endotoxin and
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FIG. 3. Measurements of adult C57BL/6 mouse lung cytokine or chemokine mRNA levels 2 h postinhalation, intratracheal instillation, or intraperitoneal administration of endotoxin by RNase protection assay: 10 mg total lung RNA hybridized with 5.2 3 105 cpm for 18 h at 56°C.
increases in lavage PMNs without changes in lavage protein. However, in contrast to inhalation the instillation method does not provide even distribution of endotoxin in the lung (Pritchard et al., 1985). The main difference between the three routes of administration is the effect on target organs, which is restricted to the lung after inhalation or intratracheal administration of endotoxin, as shown by an increase in lavageable inflammatory cells, whereas only after intraperitoneal injection did cytokines in the liver demonstrate significant up-regulation. LPS mediates many of its effects through ligand–receptor interaction, primarily through the CD14 receptor. LPS primes macrophages and PMNs, through CD14 receptor interaction, to produce reactive oxygen intermediates and induce proinflammatory cytokines (Detmers et al., 1996; Watson et al., 1994). Our observations support the notion that PMNs regulate inflammatory processes via cytokine and chemokine expression (Xing et al., 1994). Xing et al., (1993) demonstrated prior to PMN influx that alveolar macrophages appear to play a crucial role in initiating a neutrophilic response via expression of TNFa and IL-1. PMNs and alveolar macrophages have been demonstrated as a significant source of TNFa mRNA and protein activity after LPS-elicited acute lung inflammation (Xing et al., 1993, 1994). Endotoxin has also been shown to stimulate bronchial epithelial cells to release chemotactic factors for neutrophils (Koyama et al., 1991).
TNFa has been shown to stimulate PMNs to express IL-8 (Streiter et al., 1992), IL-6 (Cicco et al., 1990), IL-1b (Marucha et al., 1990), and metallothionein (Liu et al., 1991). Furthermore, TNFa has been shown to enhance adhesion of PMNs, as well as the initiation and regulation of a cytokine and antioxidant cascade (Nathan, 1987; Jordana et al., 1988; Johnston et al., 1996). IL-6 has long been known for its effect on the induction of acute-phase proteins during acute inflammation (Gauldie et al., 1987). IL-6 may act as an antiinflammatory molecule, perhaps by inhibiting expression of proinflammatory cytokines such as TNFa and enhancing neutrophil apoptosis (Afford et al., 1992). IL-10 has also been described to have potent antiinflammatory effects (Ge´rard et al., 1993; Cox, 1996) in models of systemic infection, which has been attributed to inhibition of cytokine production by macrophages (Ge´rard et al., 1993; DeWaal Malefyt et al., 1991). Our results demonstrate no alterations of this cytokine compared to sham-exposed animals, suggesting that IL-10 may play a limited role in PMN-driven responses. C-X-C chemokines, including MIP-2 and IP-10, are predominantly chemoattractants for neutrophils. MIP-2 and IP-10 mRNA were increased severalfold higher in the present study than any of the C-C chemokines. This result combined with the PMN responses may suggest that C-X-C chemokines are mainly responsible for the neutrophilic influx observed after endotoxin inhalation. There are several potential sources of
CYTOKINE AND CHEMOKINE mRNA AFTER LPS
FIG. 3—Continued
MIP-2 in the lung. Driscoll and Schlesinger (1988) have shown that ozone stimulates alveolar macrophages to release a chemotactic activity for neutrophils. Xing et al. (1994) have dem-
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onstrated increased MIP-2 mRNA in macrophages and PMNs obtained from BAL after instillation of LPS. Xu et al. (1994) demonstrated MIP-2 mRNA transcripts in mononuclear cells and within the airway wall as well as epithelial cells. These cells may likely be the sources of MIP-2 expression after endotoxin inhalation. The C-C chemokines RANTES, MCP-1, MIP-1a, MIP-1b, and Eotaxin were also examined. C-C chemokines are predominantly chemoattractant for monocytes and lymphocytes. Modest inductions of these messages in our study suggest that up to 24 h after dosing, macrophages and lymphocytes play a secondary role in the cascade of inflammatory recruitment after inhalation of low levels of endotoxin. Lymphotactin, characterized as a C chemokine, is apparently a potent chemoattractant for T lymphocytes (Kennedy et al., 1995). Ltn was not significantly induced after any time point examined. TGFb1 and b2 messages were not altered after endotoxin inhalation, which has been reported in other models of endotoxin injury (Xing et al., 1994). This is similar to results with lungs exposed to PTFE fumes (Johnston et al., 1996). TGFb mRNA expression has been demonstrated to increase after bleomycin (Hoyt and Lazo, 1988) and radiation (Finkelstein et al., 1994) and may suggest that TGFb plays a limited role in acute inflammatory pulmonary injuries compared to chronic effects such as fibrosis. However, TGFb may be induced at a later stage in the course of recovery. Mt, in addition to playing an important role in heavy metal detoxification, is reported to scavenge hydroxyl radicals and to act as an antioxidant (Thornalley and Vasak, 1985) and it has a radical scavenging capacity as a mechanism of cellular defense (Dunn et al., 1987).
FIG. 4. Comparison of relative mRNA abundance of cytokine, chemokine, or Mt 2 h after endotoxin exposure by inhalation, intratracheal instillation, or intraperitoneal administration. RNase protection assays were quantified by PhosphoImager analysis. All values were normalized to constituitively expressed mRNA encoding L32. Each bar represents the mean of three mice. The asterisk indicates statistical significance relative to sham-exposed control (p ,0.05).
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FIG. 5. Measurements of adult C57BL/6 mouse liver cytokine mRNA levels 2 h postinhalation, intratracheal instillation, or intraperitoneal administration of endotoxin by RNase protection assay; 10 mg total lung RNA hybridized with 5.2 3 105 for 18 h at 56°C.
De Waal Malefyt, R., Abrams, J., Bennet, B. et al. (1991). Interleukin-10 (IL-10) inhibits cytokine synthesis by human monocytes: An autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174, 1209 –1220. Driscoll, K. E., and Schlesinger, R. B. (1988). Alveolar macrophages stimulated neutrophil and monocyte migration: Effects of in vitro ozone exposure. Toxicol. Appl. Pharmacol. 93, 312–318. Dunn, M. A., Blaylock, T. L., and Cousins, R. J. (1987). Minireview— Metallothionein (42525A). Proc. Soc. Exp. Biol. Med. 185, 107–119.
The increased expression of Mt mRNA in our studies is indicative of an oxidative stress response after LPS exposure. The mouse model has several advantages over other animal models, such as the rat, in studying pulmonary inflammation. Inbred mouse strains and transgenic mice can be utilized to study the interactions of cytokines, inflammatory mediators, and pulmonary cells (Johnston et al., 1997, 1998). Tools for the study of mouse cytokines and antioxidants are readily available. Consequently, the model should facilitate the study of the mechanisms and the role of inflammatory responses in the pathogenesis of a variety of lung diseases (Johnston et al., 1996, 1997) including those caused by inhalation of endotoxin and particulate air pollutants. The low-dose endotoxin inhalation model should particularly be useful as a tool for studying responses of secondary stimuli in a compromised or primed organism.
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ACKNOWLEDGMENTS
Gordon, T. (1992). Dose-dependent pulmonary effects of inhaled endotoxin in guinea pig. Environ. Res. 52, 117–125.
These studies were in part supported by HEI Contract No. 95-11 and NIEHS Grants ESO1247 and ESO4872. The excellent technical assistance of Nancy Corson, Pamela Mercer, and Kiem Nguyen is gratefully acknowledged and we thank Judy Havalack for outstanding clerical assistance in the preparation of the manuscript.
Hobbs, M. V., Weigle, W. O., Noonan, D. J., et al. (1993). Patterns of cytokine gene expression by CD41T cells from young and old mice. J. Immunol. 150, 3602–3614. Hoyt, D. G., and Lazo, J. S. (1988). Alterations in pulmonary mRNA encoding procollagens fibronectin and transforming growth factor-b precede bleomy-
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Pulmonary Cytokine and Chemokine mRNA Levels after Inhalation of Lipopolysaccharide in C57BL/6 Mice C. J. Johnston, J. N. Finkelstein, R. Gelein, and G. Oberdo¨rster Departments of Environmental Medicine and Pediatrics, University of Rochester, Rochester, New York 14642 Received February 27, 1998; accepted July 29, 1998
Pulmonary Cytokine and Chemokine mRNA Levels after Inhalation of Lipopolysaccharide in C57BL/6 Mice. Johnston, C. J., Finkelstein, J. N., Gelein, R., and Oberdo¨rster, G. (1998). Toxicol. Sci. 46, 300 –307. Inhaled endotoxin (lipopolysaccharide, LPS) can induce acute lung injury and at high doses may lead to respiratory distress syndrome. Using a mouse model of acute lung inflammation induced by inhalation of low doses of LPS we examined the kinetics of chemokine, proinflammatory cytokine, and metallothionein. Eight-week-old C57BL/6 mice were dosed for 10 min with LPS, resulting in an estimated alveolar dose of <10 ng LPS/mouse, and euthanized 2, 6, or 24 h postexposure. Analysis of bronchoalveolar lavage fluid demonstrated increased polymorphonuclear neutrophils (PMNs) of 6.94, 32.7, and 38.8% after 2, 6, and 24 h, respectively. Examination of proinflammatory cytokine, chemokine, and Mt mRNA in the lung revealed increases for messages encoding IL-1a, IL-1b, IL-6, IFN-g, TNFa, Eotaxin, MIP-1a, MIP-1b, MIP-2, Mt, and IP-10, while messages encoding IL-12, IL-10, IFN-b, Ltn, MCP-1, TGFb1 1 2, and RANTES were unchanged from those of sham-exposed mice 2 h postexposure. By 6 h most messages had returned to near control levels. Comparison to 5 mg/kg body weight intraperitoneal injection and 5 mg/mouse intratracheal instillation 2 h postexposure demonstrated similar message responses. Our results demonstrate that low levels of LPS exposure by inhalation induce a strong PMN response and a selective cytokine response in the lung, supporting the hypothesis that PMNs may regulate inflammatory processes via cytokine and chemokine response. © 1998 Society of Toxicology.
Inhalation of cotton and other organic dusts may result in both acute and chronic lung disorders. For example, mill fever occurs with acute exposure to cotton dust (Merchant et al., 1975). In addition to respiratory symptoms and fever, acute exposure of both animals and humans to organic dusts can result in pulmonary inflammation, characterized by an influx of neutrophils into the lung (Merchant et al., 1975; Castranova et al., 1987; Ryan and Karol, 1991). Due to their complex composition, the agents within many organic dusts have not been completely defined. One of the components frequently noted is endotoxin from gram-negative bacteria. Chronic exposure of endotoxin measured in the dust from occupational and domestic settings has been related to both the risk of developing 1096-6080/98 $25.00 Copyright © 1998 by the Society of Toxicology. All rights of reproduction in any form reserved.
chronic obstructive pulmonary diseases (Schwartz et al., 1995; Smid et al., 1992; Ryan et al., 1994) and the severity of domestic asthma (Michel et al., 1996). Endotoxin (lipopolysaccharide; LPS) induces acute lung injury in endotoxic sepsis and gram-negative pneumonia, two conditions marked by the activation of alveolar macrophages (AM) and massive tissue infiltration of neutrophils (PMN), which may lead to adult respiratory distress syndrome (Brigham and Meyrick, 1986). The symptoms of acute endotoxin exposure include fever, chills, dyspnea, chest tightness, coughing, and decreases in lung diffusion capacity (Pernis et al., 1961; Muittari et al., 1980a, b; Rylander et al., 1978, 1989). Airborne endotoxin concentration (0.006–0.779 mg/m3) has been correlated with decreased FEV1 in exposed humans (Castellan et al., 1987). Results from animal models also indicate that endotoxin is a potent toxicant. Inhalation of endotoxin by guinea pigs resulted in a neutrophil influx into airways (Ryan and Karol, 1991; Hudson et al., 1977). In addition, two primary inflammatory cytokines, TNF and IL-1, were induced by inhalation of endotoxin (Ryan and Karol, 1991). Cytokines including TNFa, IL-1a and b and IL-6 are secreted by macrophages (Nathan, 1987) which can autoactivate macrophages and increase the production of superoxide radicals by neutrophils (Borish et al., 1989; Sharber and Nathan, 1986). They are potent proinflammatory cytokines that trigger recruitment of adhesion molecules and chemokines (Lloyd and Oppenheim, 1992). Inflammatory cell recruitment is an important initial step in the host defense against inhaled toxicants. Recent studies have identified a growing superfamily of chemotactic peptides, termed chemokines, which play a role in the recruitment and activation of leukocytes, which have been implicated in the initiation and amplification of autoimmune inflammatory responses. These chemokines have been subdivided based on function, C-X-C chemokines such as IL-8 and MIP-2 are generally chemoattractant for neutrophils, C-C chemokines such as RANTES, MCP-1, MIP-1a, and MIP-1b are predominantly chemoattractants for monocytes and lymphocytes (Oppenheim et al., 1991; Schall, 1991; Baggiolini et al., 1994; Taub et al., 1994). Although evidence from in vitro and intratracheal instillation studies using high doses have shown that cytokines from effector cells, primarily AM (Kelly, 1990; Xing et al., 1994) and PMN (Xing et al., 1994; Lloyd and Oppenheim, 1992), are
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TABLE 1 Cellular and Biochemical Analysis of Lung Lavage Fluid 2, 6, and 24 h after a 12-min Whole-Body Exposure to Endotoxin Inhalation (;10 ng Deposited in the Alveolar Region) in 8-Week-Old C57BL/6 Mice
Untreated Sac 2 h Sac 6 h Sac 24 h
Total cells (3 106)
% AM
% PMN
% Lymph
% Viable
Protein (ng/ml)
1.10 6 0.40 0.98 6 0.26 1.83 6 0.90 2.00 6 0.49
98.95 6 0.34 91.84 6 1.99 65.88 6 14.99 60.17 6 15.04
0.24 6 0.18 6.94 6 2.05* 32.70 6 14.27* 38.81 6 14.99*
0.81 6 0.48 1.21 6 0.33 1.25 6 0.79 1.03 6 0.70
91.90 6 2.22 90.46 6 3.54 92.39 6 2.93 94.16 6 1.86
0.16 6 0.05 0.14 6 0.02 0.17 6 0.04 0.17 6 0.01
Note. Means 6 SD, n 5 4 per group. * Statistically significant compared to untreated controls (p ,0.05).
involved, little is known about the kinetics of expression of cytokines and chemokines after inhalation of low levels of endotoxin (Michel et al., 1992; Goncalves de Mores et al., 1996; Uno et al., 1997). The primary objective of our current study was to explore the kinetics of expression of cytokines and chemokines after inhalation of LPS; secondary objectives were to compare responses of cytokines and chemokines after low-level inhalation to changes induced by intraperitoneal administration and intratracheal instillation. We focused on the lung injury model induced by inhalation since intratracheal instillation is known to result in a nonuniform deposition of the administered compound in the lung and intraperitoneal administration affects other target organs such as liver (Pritchard et al., 1985; Nelson et al., 1989) MATERIALS AND METHODS Adult male, 8-week-old mice C57BL/6 (Jackson Laboratory, Bar Harbor, ME) were housed four per cage with food and water ad libitum in a temperature- and humidity-controlled room with a 12-h light/dark cycle. Endotoxin Administration Inhalation. Twelve mice each were exposed for 10 min to air or LPS (Escherichia coli endotoxin, Sigma Chemical Co., St. Louis, MO). Aerosols were generated by using an AERO-Mist compressed air nebulizer (CIS-US, Inc., Bedford, MA); 100 ml of LPS (0.125 mg) in 2 ml saline was added to the nebulizer, which had an output airflow of 20 liter/min. Mice were exposed in a 30-liter whole-body exposure chamber in individual compartments. The LPS aerosol had a mass median aerodynamic diameter of 0.6 mm, with a geometric
standard deviation of 1.6. Using a mathematical deposition model for particles in the mouse lung (Yu and Oberdo¨rster, 1997) a dose of less than 10 ng LPS was estimated to be deposited in the alveolar region of the lung. Mice were killed at 2, 6, and 24 h postinhalation. Intraperitoneal injection. Six C57BL/6 mice were injected with saline or 5 mg/kg body weight in 0.5 ml saline. Mice were killed 2 h postinjection and lungs and livers were prepared for analysis as described above. Intratracheal instillation. Six C57BL/6 mice were anesthetized with 3% halothane and 0.1 ml saline (control) or 5 mg LPS/mouse in 100 ml saline was intratracheally instilled. Mice were killed 2 h postinstillation and lungs and livers prepared for analysis as described below. Tissue preparation. Mice were euthanized by intraperitoneal (ip) injection of Na pentobarbital (50 mg/kg) followed by exsanguination via the abdominal aorta. The lungs were excised and lavaged using 1 ml of saline, which was repeated for a total of 10 lavages. After 10 min centrifugation at 400g total lavagable cell numbers, cell viability, and cell differential were determined in the cell pellet as well as protein LDH and b-glucuronidase in the lavage supernatant. Lungs and liver were flash-frozen for RNA analysis after lavage. Ribonuclease Protection Assay Total RNA was isolated from lung tissue using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. Each frozen lung lobe (50 –100 mg) was homogenized in 1 ml of TRIzol reagent. Each final RNA pellet was resuspended in 50 ml of diethylpyrocarbonatetreated water. The RNA concentration and purity was quantified using the GeneQuant RNA/DNA Calculatory (Pharmacid Biotech, Piscataway, NJ). Quantitation of steady-state cytokine mRNA levels was performed using a previously described multicytokine ribonuclease protection assay (RPA) (Hobbs et al., 1993; Johnston et al., 1997). RNase protection assays were performed with riboprobe templates for mCK-2 (IL-12 p35, IL-12 p40, IL-10, IL-1a, IL-1b, IL-Ra, MIF, IFN-g, L32, GAPDH), mCK-3 (TNF-b, Lt-b,
TABLE 2 Cellular and Biochemical Analysis of Lung Lavage Fluid 2 h after Inhalation, Intratracheal Instillation, or Intraperitoneal Administration of Endotoxin
Treatment
Total cells (3 106)
% AM
% PMN
% Lymph
% Viable
Protein (mg/ml)
Untreated 5 mg/kg Intraperitoneal injection 5 mg/Mouse intratracheal instillation ;10 ng/mouse Deposited by inhalation
1.10 6 0.40 0.90 6 0.27 0.53 6 0.11 0.98 6 0.26
98.95 6 0.34 99.63 6 0.13 71.76 6 17.57 91.84 6 1.99
0.24 6 0.18 0.00 6 0.00 28.12 6 17.55* 6.94 6 2.05*
0.81 6 0.48 0.37 6 0.13 0.13 6 0.11 1.21 6 0.33
91.90 6 2.22 90.71 6 1.41 92.10 6 1.10 90.46 6 3.54
0.16 6 0.05 0.12 6 0.02 0.21 6 0.02 0.14 6 0.02
Note. Means 6 SD, n 5 4 (control and inhalation) or n 5 3 (ip and it injection). * Statistically significant compared to untreated controls (p ,0.05).
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JOHNSTON ET AL. TNFa, IFN-g, IFN-b, TGF-b1, TFGb2, L32, GAPDH), or mCK-5 (Ltn, RANTES, Eotaxin, MIP-1b, MIP-1a, MIP-2, IP-10, MCP-1, TCA3, L32, GAPDH) (PharMingen, San Diego, CA). The riboprobe synthesis reaction consisted of 50 ng of mCK-2, mCK-3, or mCK-5 template set; 120 mCi [a-32P]UTP (uridine triphosphate) (3000 Ci/mmol; Dupont NEN, Wilmington, DE); 5 nmol ATP (adenosin triphosphate); 5 nmol GTP (guanosine triphosphate); 5 nmol CTP (cytidine triphosphate); 150 pmol UTP; 2.5 mg yeast tRNA; 100 nmol dithiothreitol; 1 mg bovine serum albumin; 20 nmol spermidine; 10 U RNase inhibitor; and 50 U T7 RNA polymerase (Life Technologies) in 1 3 transcription buffer (40 mM Tris–HCl, pH 7.5, 6 mM MgCl2). The reaction was incubated for 90 min at 37°C and then diluted to 100 ml with DNase I buffer [50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 0.02 U/ml RQ1 DNase I (Promega, Madison, WI)]. After a 30-min incubation at 37°C, the riboprobes were purified by phenol/chloroform extraction and ethanol precipitation. The dried pellet was then resuspended in 50 ml of hybridization buffer [400 mM NaCl, 40 mM Pipes, pH 6.7, 1 mM EDTA (ethylenediaminetetraacetic acid), pH 8.0, 80% formamide (Sigma)], analyzed by scintillation counting, and then diluted to a final concentration of 2.6 3 105 cpm/ml in hybridization buffer. Ten-microgram samples of each RNA assayed were dried in a SpeedVac Concentrator (Savant, Farmingdale, NY) and resuspended in 8 ml of hybridization buffer. Two microliters of the diluted riboprobe was added to each RNA sample, heated to 80°C for 3 min, and then immediately placed at 56°C for 16 h. After solution hybridization, 100 ml of RNase cocktail [0.2 mg/ml RNase A (Sigma), 600 U/ml RNase T1 (Life Technologies), 10 mM Tris–HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA, pH 8] was added to each sample and incubated 45 min at 30°C. Eighteen microliters of proteinase K cocktail [0.67 mg/ml proteinase K (Life Technologies), 3.5% sodium dodecyl sulfate, 100 mg/ml yeast tRNA] was then added to each sample and incubated for 15 min at 37°C. The protected RNA duplexes were purified by phenol/ chloroform extraction and ethanol precipitation, and the pellets were resuspended in 5 ml of RPA loading buffer [80% formamide, 0.53 TBE, 0.05% bromphenol blue (Sigma)]. The protected, radiolabeled RNA fragments were electrophoresed on a 5% acrylamide/8 M urea sequencing gel, and the dried gel was used to expose X-AR film (Eastman Kodak, Rochester, NY) at 280°C with intensifying screens (Quanta III; Dupont, Wilmington, DE). Membrane Hybridization For membrane hybridization, slot blots were prepared in triplicate from a single dilution of glyoxal-denatured RNA as described by Thomas (1983), except that charged nylon membranes (magnagraph 45 mm, micrometer separations) are substituted for cellulose nitrate. Each slot was sliced in thirds. One-third of each slot was hybridized to a control cDNA probe encoding L32. The other two-thirds of each set of slots were hybridized with 32P-labeled cDNA probe for Mt (provided by G. Andrews, Kansas University). Random hexamer labeling (Feinberg and Vogelstein, 1983) was used to prepare cDNA probes and blots were hybridized at 68°C for ;18 h in 13 SSPE (10 mM NaC1, 10 mM EDTA), 10% dextran sulfate, 23 Denhart’s solution [0.04% polyvinylpyrrolidone-360, 2% Ficoll, 0.4 mM EDTA, 2.0% sodium dodecyl sulfate (SDS)], with 106 cpm probe/ml hybridization buffer. Membranes were washed, 20 min each time, in 0.13 SSC (sodium chloride, sodium citrate), 0.5% SDS at room temperature with shaking, followed by four washes in the same buffer at 68°C without shaking. After washing, membranes were blot dried, wrapped in Saran Wrap, and exposed for autoradiography at 280°C using X-ray film and intensifying screens. For quantitation, the dried gels were placed against phosphorimager screens (Molecular Dynamics, Sunnyvale, CA). The intensity of each specific cytokine band was measured with a computer-linked phosphorimager using the ImageQuant software (Molecular Dynamics). To correct for RNA loading, each intensity score was normalized to the intensity of hybridization for the L32 gene.
FIG. 1. Measurement of adult C57BL/6 mouse cytokine or chemokine mRNA levels 2, 6, or 24 h postendotoxin inhalation by RNase protection assay: 10 mg total lung RNA hybridized with 5.2 3 105 cpm for 18 h at 56°C.
Statistical Analysis A one-way analysis of variance (ANOVA) was performed using the SigmaStat 2.0 software (Jandel, San Rafael, CA) to determine the significance of
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FIG. 2. Time course of increases in the relative mRNA abundance of cytokines, chemokines and Mt after endotoxin inhalation. RNase protection assays were quantified by PhosphoImager analysis. All values were normalized to constitutively expressed mRNA encoding L32. Each bar represents the mean of three mice. The asterisk indicates statistical significance relative to sham-exposed control (p ,0.05).
observed variations in lavage parameters and cytokine mRNA levels at different timepoints.
RESULTS
Analysis of lavageable cells after endotoxin inhalation showed that total cell numbers were unchanged at all postexposure time points. The mean percentage of PMNs increased to 6.94, 32.70, and 38.81%, at 2, 6, and 24 h postexposure, respectively (Table 1). There was no significant change in protein levels or LDH or b-glucuronidase. At 2 h postexposure the intratracheal (IT)-instilled group demonstrated increases in the mean percentage of PMNs to 28.12%. Protein and other lavage parameters were unchanged. No measurable changes in PMN response or any other lavage parameter were measured after ip dosing of LPS (Table 2). Cytokine and chemokine mRNAs in total lung tissue were evaluated by RNase protection assay. As shown in Fig. 1, increases in mRNA abundance were evident 2 h postinhalation for the cytokines IL-1a, IL-1b, IL-1Ra, IL-6, IFN-g, and TNFa as well as the chemokines Eotaxin, MIP-1b, MIP-1a, MIP-2, and IP-10. Also, the antioxidant Mt was increased at this time point (Fig. 2). Six hours postexposure increases persisted in messages encoding TNFa, MIP-2, IP-10, and Mt. However, increases were lower than measured 2 h postexposure (Figs. 1 and 2). Twentyfour hours postexposure all messages had returned to near control levels.Messages encoding IL-12, IL-10, MIF, TNFb, LTb, IFN-b, TGFb1, TGFb2, Ltn, RANTES, and MCP-1 were unaltered at all time points examined. Comparison of chemokine mRNAs 2 h post Inh, IT, or ip showed increased mRNA abundance for messages encoding
IL-1a, IL-1b, IL-1Ra, IL-6, TNFa, MIP-1a, MIP-1b, MIP-2, IP-10- and Mt (Figs. 3 and 4). Increases in mRNA abundance were similar after Inh, IT, and ip endotoxin exposure. Comparison of liver cytokine mRNAs 2 h post Inh, IT, or ip demonstrated that messages encoding TNFa and IL-6 were induced only after ip administration of endotoxin (Fig. 5). DISCUSSION
We utilized a model of endotoxin inhalation to compromise the respiratory system and found that extremely low doses, estimated at ,10 ng deposited in the alveolar region, caused a significant inflammatory cell response in the lung, starting at 2 h postexposure and lasting for more than 24 h. Endotoxin-induced lung injury has been well described in humans (Michel et al., 1992, 1995, 1997) and guinea pig (Gordon, 1992; Sandstrom et al., 1992) after inhalation and in rodents after intraperitoneal administration as well as after intratracheal instillation of high LPS doses (Xing et al., 1993, 1994). In this study we demonstrated that inhalation of low levels of endotoxin triggers a distinct cytokine and chemokine response in the lung, selectively inducing TNFa, IL-1a, IL-1b, MIP-2, IL-6, and Mt, but not RANTES, TGFb1,2, IL-10 or IL-12. Initiation of a PMN response correlated with cytokine induction. Furthermore, the low dose does not cause damage to the epithelial barrier function, as demonstrated by the lack of protein in the lavage samples. Intraperitoneal administration of LPS triggered a similar message response. However, there was no PMN response at the selected dose of LPS. Consequently, this route of administration may be a useful model for the priming of target cells in the lung. Intratracheal instillation also demonstrated a similar message response to inhalation of endotoxin and
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FIG. 3. Measurements of adult C57BL/6 mouse lung cytokine or chemokine mRNA levels 2 h postinhalation, intratracheal instillation, or intraperitoneal administration of endotoxin by RNase protection assay: 10 mg total lung RNA hybridized with 5.2 3 105 cpm for 18 h at 56°C.
increases in lavage PMNs without changes in lavage protein. However, in contrast to inhalation the instillation method does not provide even distribution of endotoxin in the lung (Pritchard et al., 1985). The main difference between the three routes of administration is the effect on target organs, which is restricted to the lung after inhalation or intratracheal administration of endotoxin, as shown by an increase in lavageable inflammatory cells, whereas only after intraperitoneal injection did cytokines in the liver demonstrate significant up-regulation. LPS mediates many of its effects through ligand–receptor interaction, primarily through the CD14 receptor. LPS primes macrophages and PMNs, through CD14 receptor interaction, to produce reactive oxygen intermediates and induce proinflammatory cytokines (Detmers et al., 1996; Watson et al., 1994). Our observations support the notion that PMNs regulate inflammatory processes via cytokine and chemokine expression (Xing et al., 1994). Xing et al., (1993) demonstrated prior to PMN influx that alveolar macrophages appear to play a crucial role in initiating a neutrophilic response via expression of TNFa and IL-1. PMNs and alveolar macrophages have been demonstrated as a significant source of TNFa mRNA and protein activity after LPS-elicited acute lung inflammation (Xing et al., 1993, 1994). Endotoxin has also been shown to stimulate bronchial epithelial cells to release chemotactic factors for neutrophils (Koyama et al., 1991).
TNFa has been shown to stimulate PMNs to express IL-8 (Streiter et al., 1992), IL-6 (Cicco et al., 1990), IL-1b (Marucha et al., 1990), and metallothionein (Liu et al., 1991). Furthermore, TNFa has been shown to enhance adhesion of PMNs, as well as the initiation and regulation of a cytokine and antioxidant cascade (Nathan, 1987; Jordana et al., 1988; Johnston et al., 1996). IL-6 has long been known for its effect on the induction of acute-phase proteins during acute inflammation (Gauldie et al., 1987). IL-6 may act as an antiinflammatory molecule, perhaps by inhibiting expression of proinflammatory cytokines such as TNFa and enhancing neutrophil apoptosis (Afford et al., 1992). IL-10 has also been described to have potent antiinflammatory effects (Ge´rard et al., 1993; Cox, 1996) in models of systemic infection, which has been attributed to inhibition of cytokine production by macrophages (Ge´rard et al., 1993; DeWaal Malefyt et al., 1991). Our results demonstrate no alterations of this cytokine compared to sham-exposed animals, suggesting that IL-10 may play a limited role in PMN-driven responses. C-X-C chemokines, including MIP-2 and IP-10, are predominantly chemoattractants for neutrophils. MIP-2 and IP-10 mRNA were increased severalfold higher in the present study than any of the C-C chemokines. This result combined with the PMN responses may suggest that C-X-C chemokines are mainly responsible for the neutrophilic influx observed after endotoxin inhalation. There are several potential sources of
CYTOKINE AND CHEMOKINE mRNA AFTER LPS
FIG. 3—Continued
MIP-2 in the lung. Driscoll and Schlesinger (1988) have shown that ozone stimulates alveolar macrophages to release a chemotactic activity for neutrophils. Xing et al. (1994) have dem-
305
onstrated increased MIP-2 mRNA in macrophages and PMNs obtained from BAL after instillation of LPS. Xu et al. (1994) demonstrated MIP-2 mRNA transcripts in mononuclear cells and within the airway wall as well as epithelial cells. These cells may likely be the sources of MIP-2 expression after endotoxin inhalation. The C-C chemokines RANTES, MCP-1, MIP-1a, MIP-1b, and Eotaxin were also examined. C-C chemokines are predominantly chemoattractant for monocytes and lymphocytes. Modest inductions of these messages in our study suggest that up to 24 h after dosing, macrophages and lymphocytes play a secondary role in the cascade of inflammatory recruitment after inhalation of low levels of endotoxin. Lymphotactin, characterized as a C chemokine, is apparently a potent chemoattractant for T lymphocytes (Kennedy et al., 1995). Ltn was not significantly induced after any time point examined. TGFb1 and b2 messages were not altered after endotoxin inhalation, which has been reported in other models of endotoxin injury (Xing et al., 1994). This is similar to results with lungs exposed to PTFE fumes (Johnston et al., 1996). TGFb mRNA expression has been demonstrated to increase after bleomycin (Hoyt and Lazo, 1988) and radiation (Finkelstein et al., 1994) and may suggest that TGFb plays a limited role in acute inflammatory pulmonary injuries compared to chronic effects such as fibrosis. However, TGFb may be induced at a later stage in the course of recovery. Mt, in addition to playing an important role in heavy metal detoxification, is reported to scavenge hydroxyl radicals and to act as an antioxidant (Thornalley and Vasak, 1985) and it has a radical scavenging capacity as a mechanism of cellular defense (Dunn et al., 1987).
FIG. 4. Comparison of relative mRNA abundance of cytokine, chemokine, or Mt 2 h after endotoxin exposure by inhalation, intratracheal instillation, or intraperitoneal administration. RNase protection assays were quantified by PhosphoImager analysis. All values were normalized to constituitively expressed mRNA encoding L32. Each bar represents the mean of three mice. The asterisk indicates statistical significance relative to sham-exposed control (p ,0.05).
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These studies were in part supported by HEI Contract No. 95-11 and NIEHS Grants ESO1247 and ESO4872. The excellent technical assistance of Nancy Corson, Pamela Mercer, and Kiem Nguyen is gratefully acknowledged and we thank Judy Havalack for outstanding clerical assistance in the preparation of the manuscript.
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