Isoflurane Preconditioning Reduces the Rat NR8383 Macrophage Injury Induced by Lipopolysaccharide and Interferon γ

NR8383 cells (CRL-2192), a rat pulmonary alveolar macrophage cell line, were purchased from American Type Culture Collection (Manassas, VA). Heat-inactivated fetal bovine serum and recombinant rat IFN-γ produced from Escherichia coli  were purchased from Invitrogen Corporation (Carlsbad, CA). Rabbit polyclonal antibody against iNOS was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)–based colorimetric assay kit was obtained from Roche Applied Science (Roche, IN). Isoflurane was purchased from Abbott Laboratories (North Chicago, IL). Chelerythrine chloride, calphostin C, or bisindolylmaleimide IX were obtained from Biomol (Plymouth Meeting, PA). Alexa Fluor® 488 annexin V–propidium iodide (PI) staining kit and Griess Reagent Kit were purchased from Molecular Probes (Eugene, OR). Lipopolysaccharide (E. coli  0111:B4), 0.4% trypan blue solution, aminoguanidine bicarbonate salt, and other general chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

NR8383 cells were cultured in Ham’s F12K medium containing 2 mm l-glutamine, 1.5 g/l sodium bicarbonate, 15% heat inactivated fetal bovine serum, 100 U/ml penicillin, and 100 pg/ml streptomycin in a humidified atmosphere of 95% air–5% CO²at 37°C. The medium was changed every 2 days. The cells were suspended in the culture medium at a density of 1 × 10⁵cells/ml and plated at a density of 2 × 10⁴/well on 96-well tissue culture plates for viability experiments by MTT assay or 2 × 10⁶on 100-mm-diameter dishes for Western blotting and flow cytometry experiments.

Isoflurane pretreatment of the cells was performed in an airtight chamber as we described previously.27,28Briefly, 3 h after the cells were plated, they were placed in the airtight chamber. The chamber was then gassed with 95% air–5% CO²through an isoflurane vaporizer set at 0, 1, 2, or 3% for 15 min at 37°C. The isoflurane concentrations in the gases from the outlet of the chambers were monitored with a Datex infrared analyzer (Capnomac, Helsinki, Finland) and reached the target concentrations at approximately 3 min after the onset of gassing. The chamber was sealed and the incubation was for 45 min at 37°C. At the end of the incubation time, the isoflurane concentrations in the gases from the outlet of the chamber were confirmed to be at the target concentrations. The cells were then removed from the chamber and were kept under their normal and isoflurane-free culture conditions for 30 min before they were exposed to 100 ng/ml lipopolysaccharide plus 50 U/ml IFN-γ for 24 h at 37°C. Various concentrations of aminoguanidine, an iNOS inhibitor, were added to some cells just before the application of lipopolysaccharide plus IFN-γ. These cells were not pretreated with isoflurane. To determine the involvement of protein kinase C (PKC) in the isoflurane preconditioning, the PKC inhibitors chelerythrine (10 μm), calphostin C (20 nm), or bisindolylmaleimide IX (5 nm) were added just before the isoflurane application. After the isoflurane exposure, the incubation medium was replaced with fresh medium without PKC inhibitors before the cells were exposed to lipopolysaccharide plus IFN-γ.

In a separate experiment, the isoflurane concentrations in the incubation solutions were determined by gas chromatography as we described previously.29The aqueous isoflurane concentrations at 37°C were approximately 209, 415, and 620 μm, respectively, when 1, 2, and 3% isoflurane was delivered and the samples were taken at the end of the 1-h isoflurane incubation. There was no detectable isoflurane in the incubation solution at the end of the 30-min isoflurane-free period.

Cell viability was determined by MTT assay according to the manufacturer’s procedure and as we described previously.30Briefly, 10 μl of the MTT labeling reagent was added to each well that contained 100 μl culture medium and cells. Four hours later, 100 μl of the solubilization solution was added into each well. The plate was kept overnight at 37°C. Absorbance of the samples was measured at 570 nm with the reference wavelength of 650 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA). In each experiment, the results of the MTT measurements from the controls without any treatments were set as 100%. The results from the sister cultures, subjected to various treatments, were then calculated as a percentage of the controls.

Macrophage survival was determined by trypan blue exclusion. In brief, 0.2 ml NR8383 cell suspension at a density of 1 × 10⁵/ml was mixed thoroughly with 0.2 ml trypan blue, 0.4%, for 5 min at room temperature. Trypan blue–stained or unstained cells were counted under a microscope with the use of a hemocytometer.

Macrophages (approximately 2 × 10⁶cells) in 100-mm-diameter dishes were pretreated with or without 2% isoflurane for 1 h and then exposed to 100 ng/ml lipopolysaccharide plus 50 U/ml IFN-γ for 24 h at 37°C. Cells were then stained with annexin V–fluorescein isothiocyanate conjugate and PI using an Alexa Fluor® 488 annexin V–PI staining kit according to the manufacturer’s protocol. Annexin V–fluorescein isothiocyanate conjugate labels apoptotic cells by binding to phosphatidyl serine exposed on the outer leaflet of the plasma membrane. PI is impermeant to live cells and the early phase of apoptotic cells and binds tightly to the nucleic acids in dead cells. After staining, the cells were analyzed with flow cytometry (FACSCalibur system; Becton, Dickinson and Company Biosciences, Franklin Lakes, NJ).

Western blot analysis was performed as we detailed previously.31Cells were homogenized in 25 mm Tris hydrochloride, pH 7.4, containing 1 mm EDTA, 1 mm EGTA, 0.1% (vol/vol) α-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 2 mm leupeptin, and 1 mm pepstatin A. The homogenates were centrifuged at 1,000g  for 10 min at 4°C. The supernatants were used for Western blotting and loaded at 20 μg protein per lane. The probed protein bands were visualized by the enhanced chemiluminescence reaction. The protein bands were densitometrically analyzed by an ImageQuant 5.0 densitometer (Amersham Biosciences, Piscataway, NJ).

Macrophages (approximately 2 × 10⁶cells) in 100-mm-diameter dishes were pretreated with or without 2% isoflurane for 1 h and then exposed to 100 ng/ml lipopolysaccharide plus 50 U/ml IFN-γ for 24 h at 37°C. The culture medium was collected. The concentrations of nitrite in the culture medium were measured with a Griess Reagent Kit according to the instructions. The absorbance of the samples was read at 570 nm against a standard curve using a microplate reader (Bio-Rad Laboratories).

Results are presented as mean ± SD from at least three separate experiments. Statistical analysis was performed by one-way analysis of variance followed by the Student-Newman-Keuls test for post hoc  comparison or Student t  test as appropriate. A P < 0.05 was considered statistically significant.

Cell survival measured by trypan blue exclusion test decreased as the duration of incubation with 100 ng/ml lipopolysaccharide plus 50 U/ml IFN-γ increased, and approximately 55% cells were injured when the incubation time was 24 h (fig. 1). Consistent with this result, incubation of macrophages with 100 ng/ml lipopolysaccharide plus 50 U/ml IFN-γ for 24 h reduced cell viability assessed by MTT assay to approximately 50% of control cells (fig. 2). The iNOS inhibitor aminoguanidine dose-dependently inhibited the lipopolysaccharide plus IFN-γ–reduced cell viability (fig. 2). Lipopolysaccharide plus IFN-γ also induced the expression of iNOS proteins in these macrophages that did not express iNOS under control conditions (fig. 3). Consistent with the iNOS expression data, lipopolysaccharide plus IFN-γ increased the concentration of nitrite, a stable nitric oxide oxidation product, in the culture medium (fig. 4). These results suggest that iNOS mediates the lipopolysaccharide plus IFN-γ–reduced macrophage viability.

Pretreatment of macrophages with 2% isoflurane, but not 1% or 3% isoflurane, at 30 min before the application of lipopolysaccharide plus IFN-γ significantly improved the viability of the cells (fig. 5). The cell viability assessed by MTT assay was 51.5 ± 7.2 and 61.5 ± 6.8%, respectively, of the control in the absence and presence of 2% isoflurane pretreatment (P < 0.05, n = 9). Of note, the viability of cells exposed to 1, 2, and 3% isoflurane alone was 99% ± 21, 97% ± 22, and 94% ± 18% of the control (P > 0.05, n = 36), suggesting that isoflurane does not affect cell viability under control conditions. Consistent with the cell viability results, only 2% isoflurane pretreatment significantly decreased the lipopolysaccharide plus IFN-γ–induced expression of iNOS proteins (fig. 3). The expression of β-actin after application of 1, 2, and 3% isoflurane with lipopolysaccharide plus IFN-γ was 98 ± 11, 100 ± 16, and 99 ± 16%, respectively, of that with lipopolysaccharide plus IFN-γ alone (P > 0.05, n = 9), indicating that the effects of 2% isoflurane pretreatment on iNOS expression after the application of lipopolysaccharide plus IFN-γ is specific. Isoflurane at 2% also decreased lipopolysaccharide plus IFN-γ–induced increase of nitrite production (fig. 4).

To determine whether cell death was via  apoptosis or necrosis under our experimental conditions, we stained the cells with annexin V and PI and then sorted the cells by flow cytometry. There were 14 ± 3 and 24 ± 10% cells that were PI- and annexin V–positive staining cells, respectively, under control conditions (cells that were positive staining for both PI and annexin V contributed to both percentages). Incubation of macrophages with lipopolysaccharide plus IFN-γ for 24 h increased the PI-positive staining cells to 47 ± 2% (n = 8, P < 0.05 compared with the control) and annexin V–positive staining cells to 45 ± 26% (n = 8, P < 0.05 compared with the control). These results suggest that lipopolysaccharide plus IFN-γ increased macrophage necrosis and apoptosis. Pretreatment of macrophages with 2% isoflurane before the application of lipopolysaccharide plus IFN-γ significantly reduced the lipopolysaccharide plus IFN-γ–induced increase of PI-positive staining cells but not the annexin V–positive staining cells (fig. 6), indicating that isoflurane preconditioning reduces the macrophage necrosis but not the apoptosis after lipopolysaccharide plus IFN-γ application.

The PKC inhibitors chelerythrine, calphostin C, and bisindolylmaleimide IX abolished the 2% isoflurane preconditioning–induced protection in macrophages stimulated by lipopolysaccharide plus IFN-γ, although these inhibitors did not affect the viability of macrophages after the application of lipopolysaccharide plus IFN-γ only (fig. 7). These results suggest a role of PKC in the isoflurane preconditioning–induced protection in macrophages.

Isoflurane has been shown to induce a preconditioning effect in various organs and cell types against ischemia–reperfusion injury.22,24–26Because inflammation contributes to the ischemia–reperfusion injury and is a pathophysiologic process involved in many diseases, it is important to determine whether isoflurane induces a preconditioning effect in cells participating in inflammation. Our study showed that isoflurane induced a preconditioning effect in macrophages, cells that travel around the whole body and involve in the host defense and inflammation.

It has been a well-known phenomenon that proinflammatory cytokines and bacterial products trigger iNOS expression and nitric oxide production in various cells, including macrophages.31Nitric oxide can then produce proinflammatory and cell-destructive effects. Our results showed that stimulation of NR8383 cells with lipopolysaccharide plus IFN-γ for 24 h induced a significant amount of iNOS protein expression and decreased the cell viability in rat macrophages. This decreased cell viability was dose-dependently reversed by aminoguanidine, an iNOS inhibitor. These results indicate that lipopolysaccharide plus IFN-γ–induced rat NR8383 macrophage injury is iNOS dependent.

It has been established that nitric oxide, lipopolysaccharide alone, and lipopolysaccharide plus IFN-γ induce macrophage apoptosis.15,32–34Consistent with this idea, our study showed that lipopolysaccharide plus IFN-γ increased the annexin V–positive staining cells. Annexin V binds to phosphatidyl serine exposed on the surface of the plasma membrane, a characteristic of cell apoptosis. Our study also showed that lipopolysaccharide plus IFN-γ increased the PI-positive staining cells. PI is a highly polar fluorescent dye and penetrates damaged plasma membrane to bind to DNA. It has been used as a marker to stain necrotic cells. Therefore, our results suggest that lipopolysaccharide plus IFN-γ increases macrophage necrosis. To support our finding, lipopolysaccharide has been shown to induce macrophage death via  caspase-independent autophagy.32 

Our results showed that 2% isoflurane induced a preconditioning effect in macrophages. Pretreatment with this concentration of isoflurane also decreased the lipopolysaccharide plus IFN-γ–induced iNOS expression and the nitrite production. These results suggest that isoflurane preconditioning reduces iNOS expression to protect the macrophages from lipopolysaccharide plus IFN-γ–induced injury. Interestingly, 2% isoflurane pretreatment attenuated the lipopolysaccharide plus IFN-γ–induced macrophage necrosis but did not affect the lipopolysaccharide plus IFN-γ–induced macrophage apoptosis. Isoflurane preconditioning has been shown to reduce cell apoptosis in other cells, such as myocardium and brain cells,22,25after ischemia. Future studies are needed to determine why isoflurane preconditioning did not reduce lipopolysaccharide plus IFN-γ–induced macrophage apoptosis.

Our results showed that chelerythrine, calphostin C, and bisindolylmaleimide IX abolished the 2% isoflurane pretreatment–induced protection in macrophages. Chelerythrine, bisindolylmaleimide IX, and calphostin C are structurally different PKC inhibitors. Therefore, our results strongly suggest a role of PKC in the isoflurane preconditioning effects on macrophages. Consistent with our results, PKC may play a role in the isoflurane preconditioning effect against cytokine-induced cell injury in smooth muscle cells and endothelial cells.26It has been shown that volatile anesthetics may directly activate PKC,35and many of isoflurane pharmacologic effects on various cells and tissues may be mediated by PKC.28,36In addition, although PKC activation can increase lipopolysaccharide- and cytokine-induced iNOS expression in macrophages,37activation of certain PKC isozymes, such as PKC-ϵ, also can lead to the inhibition of iNOS expression in macrophages stimulated by lipopolysaccharide, IFN-γ, or other cytokines.38,39Therefore, these previous findings are consistent with our identified mechanisms that isoflurane preconditioning reduces lipopolysaccharide plus IFN-γ–induced iNOS overexpression to provide protection in macrophages and that activation of PKC may play a role in this effect.

We observed that 2% isoflurane, but not 1% or 3% isoflurane, induced a preconditioning effect. This bell-shaped response has been reported previously in other biologic effects of isoflurane.27,36The mechanisms for loss of protection by a higher concentration of isoflurane are not clear. However, the phenomenon may not be due to the isoflurane toxicity because various concentrations of isoflurane alone did not affect the viability of macrophages in our study. Because 3% isoflurane pretreatment also did not inhibit lipopolysaccharide plus IFN-γ–induced iNOS overexpression and activation of different PKC isozymes may result in increase or decrease of iNOS expression in the macrophage stimulated by lipopolysaccharide or cytokines,37,38it is possible that 3% isoflurane may activate PKC isozymes that can increase iNOS expression and, therefore, cancel out the inhibition of iNOS expression by isoflurane pretreatment via  other PKC isozymes. Activation of selective PKC isozymes by different concentrations of isoflurane was proposed to explain the biphasic effects of isoflurane on contraction in skinned pulmonary arterial strips.36 

The antiinflammatory effects of isoflurane when it is present during the application of proinflammatory reagents have been shown in many studies.11,18,19Recently, isoflurane has been revealed to induce a preconditioning effect against lipopolysaccharide-induced inflammation and injury in lungs and vasculature in rats and mice.12,18Our findings that isoflurane pretreatment reduces iNOS expression in macrophages and improves the viability of these cells after the challenge of lipopolysaccharide plus IFN-γ may help to explain the results of these previous in vivo  studies, especially the results from pulmonary study because our macrophages are derived from lungs. Our findings also may have implications for clinical medicine and experimental biology because isoflurane is frequently used in animal studies and in patients with ongoing pulmonary infection and inflammation.

In summary, we have shown that lipopolysaccharide plus IFN-γ causes an iNOS-dependent macrophage injury. Isoflurane preconditioning induces a protection against lipopolysaccharide plus IFN-γ–induced macrophage necrosis. This protection may be mediated by PKC. Considering that isoflurane is a commonly used anesthetic in clinical practice and that animal studies and surgery often induces pulmonary inflammation and infection, our study may have broad implications.