INTRODUCTION

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During an acute lung inflammation as observed after intratracheal (IT) instillation of lipopolysaccharide (LPS), an important recruitment of inflammatory cells leads to an increased number of polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) (1, 2). This cell recruitment after LPS administration is secondary to the induction of proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-) (2). This early secretion of cytokines maintains and amplifies acute lung inflammation by inducing secretion of chemoattractant proteins such as - and -chemokines (1, 3, 4). Indeed, macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant (CINC) (5), and monocyte chemoattractant protein-1 (MCP-1) (6) which are secreted by AM are important for the initiation of recruitment of circulating PMN and monocytes into the airspace. The alveolar type II cells (AT_II) also take part in these inflammatory cells' recruitment by secreting interleukin-8 (IL-8), MIP-2 (7), CINC (8), and MCP-1 (9).

We hypothesized that the inflammatory response of these alveolar cells may be altered by volatile anesthetics, which may lead to respiratory complications. Indeed, volatile anesthetic agents (e.g., halothane) which are known to affect cellular metabolism (10) may be particularly involved in the genesis of postanesthetic lung infections, because these compounds are inhaled and may thus interfere with the pulmonary cells involved in the regulation of the alveolar inflammatory response to exogenous agents.

In this connection, we previously demonstrated in vitro that halothane and isoflurane decreased cytokine productions by AM (11) and surfactant biosynthesis by AT_II (12). Furthermore, halothane, at clinically relevant concentrations, inhibits the ability of PMN to kill bacteriemic culture isolates (13). The aim of this study was therefore to investigate the effect of halothane on the inflammatory pulmonary alveolar response in a model of lung inflammation induced by IT administration of LPS in rats. This was achieved by studying the effects of three different concentrations of halothane (0.5, 1, and 1.5%), i.e., three clinically relevant concentrations, on alveolar cell recruitment and cytokine production (IL-6, TNF-, MIP-2, and MCP-1) within the lungs of rats submitted to IT LPS instillation and mechanical ventilation.

Because anesthesia with halogenates requires respiratory support as mechanical ventilation which is known to induce a lung inflammatory response, rats anesthetized with halothane were compared with rats anesthetized with thiopental, an intravenous anesthetic agent, and also submitted to mechanical ventilation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

After institutional approval obtained from the local animal care and use committee, pathogen-free, male Sprague-Dawley rats weighing 220 to 250 g (Charles River, Saint-Aubin-les-Elbeuf, France) were used in these experiments. Rats were housed in standard wire-topped cages and in temperature-controlled units. Foods and water were supplied ad libitum.

Specific Experimental Groups and Procedure of Anesthesia

To study the effects of halothane, thiopental, and mechanical ventilation on intra-alveolar inflammatory response, rats were first separated in six groups according to anesthesia procedure or lung treatment by LPS. All rats were tracheotomized under ether anesthesia. During the experimental anesthesia procedure, the rats were placed under a warming light and rectal temperature was maintained close to 37° C.

LPS-induced lung inflammation was realized with 100 µg of LPS (Escherichia coli LPS B strain O26:B6; Difco, Detroit, MI) administered into lungs through the tracheal cannula during inspiration.

In the three following groups with LPS-induced lung inflammation, rats were submitted to IT instillation of LPS:

In Group Hal-1 + LPS (n = 12), anesthesia was maintained with 1% halothane delivered by Fluotec vaporizer (Cyprane, Keighley, UK) in air (O₂ = 0.25, N₂ = 0.75) and rats were mechanically ventilated (frequency = 80 · min¹, tidal volume = 3 ml, i.e., 12 ml · kg¹, zero positive-end expiratory pressure) during 4 h. The expiratory concentrations of halothane as well as the expiratory fraction of O₂ and CO₂ were measured using a volatile anesthetic analyzer (Capnomac; Datex, Helsinki, Finland). In some experiments, two other concentrations of halothane (0.5 and 1.5%, i.e., respectively 0.5 and 1.5 minimum alveolar concentration [MAC] in rat) were used to determine a dose-response effect. These represented the groups Hal-0.5 + LPS (n = 5) and Hal-1.5 + LPS (n = 5), respectively.

In Group Thio + LPS (n = 10), rats received a single intraperitoneal injection of thiopental (60 mg · kg¹) followed by mechanical ventilation as previously described, with air only.

In Group C + LPS (n = 12), rats were allowed to awake and breathed spontaneously during 4 h.

In the three following groups without LPS-induced lung inflammation, rats were not submitted to IT LPS instillation:

In Group Hal (n = 5), anesthesia was maintained with 1% halothane delivered by Fluotec vaporizer and rats were mechanically ventilated as described previously during 4 h.

In Group Thio (n = 6), rats received a single intraperitoneal injection of thiopental (60 mg · kg¹) followed by 4 h of mechanical ventilation as described.

In the control group (Group C, n = 12), rats were directly killed by exsanguination immediately after intraperitoneal thiopental injection.

In a first step of experiments, in order to determine the effects of halothane on the early LPS-induced lung inflammatory response, the rats were killed 4 h after the onset of anesthesia. Then blood samples were collected, left lung was isolated and immediately frozen under nitrogen liquid and a bronchoalveolar lavage (BAL) was performed in the right lung. To assess the intraalveolar inflammatory response, a total and differential cell count was performed in the BALF, and inflammatory cytokines (TNF-, IL-6, MIP-2, and MCP-1) were measured in plasma, lung homogenates and BALF. Total protein concentrations were measured in BALF.

In a second step of experiments, we focused our attention on the effects of halothane on the delayed LPS-induced lung inflammatory response. The same experimental procedure was performed, as described previously, except that the rats were killed 24 h after the onset of anesthesia. During the period of time from the recovery of anesthesia to the sacrifice, the rats were housed in standard wire-topped cages and in temperature-controlled units. Foods and water were supplied ad libitum.

Preparation and Analysis of BALF, Lung Tissue, and Peripheral Blood Samples

BALF was collected after 6 times instillation and withdrawn with 6 ml phosphate-buffered saline (PBS) through a tracheal cannula. We always retrieved approximately 30 ml of BAL administered. The collected lavage fluid was centrifuged at 4° C (1,200 g for 10 min). Small aliquots of the supernatant were frozen at 20° C until cytokine and protein assays were performed. The cellular pellet was resuspended in PBS, total cell counts were done with a hemacytometer, and differential cell counts were done on cytospin monolayers of the resuspended cells stained with May-Grünwald-Giemsa stain. A minimum of 200 cells was counted per slide and the average percent of each cell type was then calculated.

A part of the left lung was homogenized with phosphate-buffered saline (PBS) (2 ml of PBS for 100 mg of lung) using an Ultra-Turrax T25 (Ika Works, Staufen, Germany). Homogenates were then centrifuged at 4° C (1,200 g for 5 min) and the supernatants stored at 20° C until the cytokine assays were performed.

Blood was drawn from aortic puncture onto heparin and centrifuged at 4° C (3,000 g for 10 min). Small aliquots of plasma were frozen at 20° C until cytokine assays were performed. In addition, a blood sample was drawn in heparinized syringe in order to measure blood gas using an automatic analyzer.

Protein Assay

Total proteins in BALF supernatants were measured with an automatic analyzer Hitachi-911 (Boehringer-Mannheim, Paris, France).

Assay for TNF- Activity

TNF- activities in plasma, BALF supernatants, and whole lung homogenates were determined with a cytotoxicity assay using murine fibroblast-like morphology (LM) cells as previously described (14). Briefly, LM cells (5 · 10⁴) were plated in each well of a 96-well cell culture plate. Each well was treated with duplicate serial dilutions of the specimens, followed by addition of actinomycin D. After incubation for 18 h at 37° C in 5% CO₂, the number of surviving cells was assessed using the colorimetric assay MTT (15). TNF- activity was normalized against human recombinant TNF-. One unit of TNF- was defined by the half-maximal proliferation of LM cells.

Assay for IL-6 Activity

Bioactive IL-6 was measured in BALF supernatants and total lungs using the IL-6-dependent B9 hybridoma cell line (16). B9 cells were cultured for 72 h in the presence of duplicate serial dilutions of the specimens in 96-well plates. B9 cell proliferation was estimated after 72 h of incubation at 37° C in 5% CO₂ using MTT colorimetric assay (15). Human recombinant IL-6 was used as an internal standard in all assays. One unit of IL-6 was defined by the half-maximal proliferation of B9 cells.

MIP-2 and MCP-1 Assays

The amount of MIP-2 and MCP-1 in plasma, BALF supernatants, and whole lung homogenates was determined with commercially available ELISA (Cytoscreen MIP-2/MCP-1; Biosource, Montrouge, France), according to the manufacturer's procedure. The detection limit was 1 pg · ml¹ and 8 pg · ml¹ for MIP-2 and MCP-1, respectively.

Reverse Transcriptase/Polymerase Chain Reaction (RT-PCR)

To extract RNA from the lungs, 100 mg of frozen lung tissue were homogenized with 1 ml of Trizol (GIBCO, Cergy-Pontoise, France) with an Ultra-Turrax T25 in a ribonuclease (RNase)-free tube at 4° C. The RNA isolation was performed according to the manufacturer's procedure.

After extraction, total cellular RNA was submitted to a reverse transcription; 4 µl of total RNA (0.5 µg · µl¹) and 3 µl of oligo-dT (0.8 µg · µl¹; Promega, Charbonnieres, France) were heated to 70° C for 5 min in a thermocycler (Gene Amp; Perkin-Elmer, Norwalk, CT); then, at 4° C, 2.5 µl of deoxyribonucleoside triphosphate (dNTP) (10 mM, Boehringer-Mannheim, Mannheim, Germany) and 2 µl of avian myelobastosis virus (AMV) reverse transcriptase (32 IU · µl¹; Boehringer-Mannheim) were added to a final volume of 25 µl in RNase-free water. The reverse transcription was performed in a thermocycler which heated to 42° C for 45 min and to 99° C for 5 min. The final products (complementary DNA [cDNA]) were stored at 20° C.

Two polymerase chain reactions (PCR) were performed, the first mixing MIP-2 and S14 and the second mixing TNF- and S14. The PCR of MIP-2 and TNF- was performed by mixing 2 µl of cDNA, 12.5 µl of dNTP (1 mM; Boehringer-Mannheim), 5 µl of MgCl₂ (50 mM; GIBCO), 2 µl of sense primer (12 µM, MIP-2 = 5'-GGC ACA ATC GGT ACG ATC CAG-3' or TNF- = 5'-GCC ACC ACG CTC TTC TGT CT-3'), 2 µl of anti-sense primer (12 µM, MIP-2 = 5'-ACC CTG CCA AGG GTT GAC TTC-3' or TNF- = 5'-GGG CTA CGG GCT TGT CAC T-3'), 2 µl of sense primer S14 (12 µM, S14 = 5'-ATC AAA CTC CGG GCC ACA GGA-3'), 2 µl of anti-sense primer of S14 (12 µM, S14 = 5'-GTG CTG TCA GAG GGG ATG GGG-3') to a final volume of 48 µl in RNase-free water. This mixture was heated at 80° C for 5 min, then 2 µl of Taq polymerase (1.25 µg · µl¹; GIBCO) were added. Twenty-five repeated cycles of heat denaturation (94° C for 30 s), annealing of the primers (59° C for 30 s), and extension of the annealed primers (72° C for 30 s) were performed. The optimal number of PCR cycles for each primer set was determined in preliminary experiments so that the amplification process was performed during the exponential phase of replication.

The cDNA of MIP-2 or of TNF- and S14 were size fractionated by electrophoresis through a 3% agarose gel containing 2% of Tris-buffered saline ([TBS] 1 mM; Sigma, Saint-Quentin Falavier, France) and 0.01% of Vista Green nucleic acid gel stain (Amersham Life Science, Orsay, France). The signal intensity of each band was measured under ultraviolet light with charge-coupled device (CCD) camera using an image analyzer (Gel Analyst; Iconix, Santa Monica, CA). The size of MIP-2, TNF-, and S14 were, respectively 287, 151, and 134 bp. The messsenger RNA (mRNA) of MIP-2 or of TNF- was expressed as the percentage of the mRNA of the housekeeping gene S14. This technique permitted semiquantitative analysis of the RT-PCR.

Statistical Analysis

All values were expressed as means ± SEM. Between-group differences were first assessed with analysis of variance (ANOVA) and then, in the case of global significant difference, individual group means were compared with the nonparametric Mann-Whitney U test. Correlations were performed with Spearman's rank order test. A p value < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study of the Lung Inflammatory Response at the Fourth Hour

LPS-induced lung inflammation and cell changes in the BALF. Total and differential cell counts in the BALF are shown in Table 1. Total cell count in BALF of group C was 1.082 ± 0.09 10⁶ · ml¹ with 90% of AM and 8% of PMN. No difference was observed in Hal, Thio, and C groups. As expected, intratracheal instillation of LPS in Group C + LPS induced a dramatic change in cell counts with an increase of total cell count associated with an increase of PMN. Total and differential cell counts were similar in Groups C + LPS and Thio + LPS. By contrast, differential cell count was markedly decreased in Group Hal-1 + LPS. In addition, the total number as well as the percentage of PMN was lower (55%, p < 0.001) in Group Hal-1 + LPS than in Groups C + LPS and Thio + LPS. The total number as well as the percentage of AM was higher (+200%, p < 0.001) in Group Hal-1 + LPS than in Groups C + LPS and Thio + LPS.

Blood gases. To control the adequacy of mechanical ventilation and the effects of lung inflammation, blood gases were measured. No statistical difference was observed in pH, PCO₂, HCO³, PO₂, and Sa_O2. Results are shown in Table 2.

IL-6 and TNF- production in BALF, whole lung homogenates, and plasma. The IL-6 and TNF- activities in BALF supernatants and whole lung homogenates are shown respectively in Figures 1 and 2. IL-6 and TNF- activities were not detectable in Group C both in BALF and in lung homogenates. Anesthesia procedure or mechanical ventilation induced a moderate inflammatory response assessed by the increase of both IL-6 and TNF- activities in Groups Hal and Thio without any difference between these two groups. Intratracheal instillation of LPS induced an increase of IL-6 and TNF- activities in Groups C + LPS, Hal-1 + LPS, and Thio + LPS. IL-6 and TNF- activities were similar in Groups C + LPS and Thio + LPS whereas both TNF- and IL-6 activities were decreased by 70% and 60% (p < 0.001), respectively, in Group Hal-1 + LPS as compared with groups C + LPS and Thio + LPS.

View larger version (30K): [in this window] [in a new window]   Figure 1.   IL-6 concentrations in BALF and lung homogenates at the fourth hour. Results are expressed as mean ± SEM. **p < 0.001 versus control, p < 0.001 versus C + LPS and Thio + LPS.

View larger version (32K): [in this window] [in a new window]   Figure 2.   TNF- concentrations in BALF and lung homogenates at the fourth hour. Results are expressed as mean ± SEM. **p < 0.001 versus control, p < 0.001 versus C + LPS and Thio + LPS.

The mRNA expression of TNF- in lung homogenates was under the limit of detection in Group C (Figure 3). Intratracheal instillation of LPS induced a potent expression of TNF- mRNA in Groups C + LPS, Hal-1 + LPS, and Thio + LPS without any difference between Groups C + LPS, and Thio + LPS. By contrast, TNF- mRNA expression was lower in group Hal-1 + LPS than in Groups C + LPS and Thio + LPS (p < 0.05).

View larger version (36K): [in this window] [in a new window]   Figure 3.   Expression of TNF- mRNA in lung homogenates at the fourth hour. (A) Southern-blot analysis of TNF- cDNA after RT-PCR. Results of one representative experiment out of four. (B) TNF- expression in lung homogenates expressed as TNF-/S14 ratio. Results are expressed as mean ± SEM. **p < 0.001 versus control, p < 0.001 versus C + LPS and Thio + LPS.

Whatever the group studied, neither IL-6 nor TNF- was detected in the plasma.

MIP-2 production in BALF, whole lung homogenates, and plasma. Low concentrations of MIP-2 were detected in the BALF supernatants and the lung homogenates in groups C, Hal, and Thio without any difference between these three groups (Figure 4). Intratracheal instillation of LPS induced an increase of MIP-2 concentration in Groups C + LPS, Hal-1 + LPS, and Thio + LPS. MIP-2 concentrations were similar in Groups C + LPS and Thio + LPS. By contrast, MIP-2 concentration was lower (60%, p < 0.001) in Group Hal-1 + LPS than in Group C + LPS.

View larger version (32K): [in this window] [in a new window]   Figure 4.   MIP-2 concentrations in BALF and lung homogenates at the fourth hour. Results are expressed as mean ± SEM. **p < 0.001 versus control, p < 0.001 versus C + LPS and Thio + LPS.

Although low MIP-2 protein levels were detectable, the mRNA expression of MIP-2 in lung homogenates was under the limit of detection in Group C (Figure 5). Intratracheal instillation of LPS induced an expression of MIP-2 mRNA in Groups C + LPS, Hal-1 + LPS, and Thio + LPS without any difference between Groups C + LPS and Thio + LPS. In agreement with MIP-2 protein level, MIP-2 mRNA expression was lower in Group Hal-1 + LPS than in Groups C + LPS and Thio + LPS (p < 0.05).

View larger version (41K): [in this window] [in a new window]   Figure 5.   Expression of MIP-2 mRNA in lung homogenates at the fourth hour. (A) Southern-blot analysis of MIP-2 cDNA after RT-PCR. Results of one representative experiment out of three. (B) MIP-2 expression in lung homogenates expressed as MIP-2/S14 ratio. Results are expresed as mean ± SEM. **p < 0.001 versus control,  p < 0.001 versus C + LPS and Thio + LPS.

The PMN counts and the MIP-2 concentrations in the BALF were strongly correlated (r = 0.73, p < 0.05) (Figure 6) for the whole study population. This correlation was also found in each group and especially in the Group Hal-1 + LPS (r = 0.77, p < 0.05).

View larger version (9K): [in this window] [in a new window]   Figure 6.   Correlation between absolute PMN counts and MIP-2 concentrations in BALF at the fourth hour. Correlation was studied in the different groups taken as a whole.

MIP-2 was not detected in the plasma of the six groups studied.

MCP-1 production in BALF, lung homogenates, and plasma. Low concentrations of MCP-1 were detected in the BALF supernatants and the whole lung homogenates in Groups C, Hal, and Thio without any difference between these three groups (Figure 7). Intratracheal instillation of LPS induced an increase of the concentrations of MCP-1 in Groups C + LPS, Hal-1 + LPS, and Thio + LPS without any difference between groups.

View larger version (27K): [in this window] [in a new window]   Figure 7.   MCP-1 concentrations in BALF and lung homogenates at the fourth hour. Results are expressed as mean ± SEM. **p < 0.001 versus control.

No correlation was found between the AM counts and the MCP-1 concentrations in the BALF of the Groups C, Hal, Thio, C + LPS, Hal-1 + LPS, and Thio + LPS whatever the group studied (p > 0.05).

No MCP-1 was detected in the plasma of the six groups studied.

Protein concentrations in BALF fluids. Total protein concentrations in BALF were similar in Groups C, Hal, and Thio. Intratracheal LPS induced an increase of total protein concentrations to 1.71 ± 0.22, 1.92 ± 0.21, and 1.55 ± 0.13 g · L¹ in Groups C + LPS, Thio + LPS, and Hal-1 + LPS, respectively, without any difference between groups.

Halothane dose-response effects. The lung inflammatory response was not modified up to 0.5% of halothane whereas at 1 and 1.5% of halothane, the PMN counts in the BALF as well as the cytokine concentrations (TNF-, IL-6, and MIP-2) decreased in a dose-dependent manner as shown in Figure 8. By contrast, the MCP-1 and the total protein concentrations were not modified even with 1.5% of halothane.

View larger version (50K): [in this window] [in a new window]   Figure 8.   Dose effects of halothane at the fourth hour. PMN counts and cytokine concentrations were measured in BALF in the different groups of rats. Results are expressed as the percentage of the control group C + LPS ± SEM. **p < 0.001 versus Thio + LPS,  p < 0.001 versus Hal-0.5 + LPS.

Study of the Lung Inflammatory Response at the 24th Hour

Cell changes in the BALF. Total and differential cell counts in the BALF at the 24th hour are shown in Table 3. Total cell count in BALF of Group C was 0.661 ± 0.284 10⁶ · ml¹ with 65% of AM and 33% of PMN. No difference was observed in C, Thio, and Hal groups. The total cell count was higher at the 24th hour than at the fourth hour in Groups C + LPS, Hal-1 + LPS, and Thio + LPS. By contrast to the fourth hour, total and differential cell counts were similar in the three groups C + LPS, Hal-1 + LPS, and Thio + LPS.

Cytokine production in BALF and lung homogenates. The IL-6, TNF-, MIP-2, and MCP-1 concentrations in BALF supernatants and lung homogenates at the 24th hour are shown in Figure 9. Cytokine levels in Groups C + LPS, Hal-1 + LPS, and Thio + LPS remained elevated at the 24th hour. However, these cytokine levels were lower than those measured 4 h after the IT LPS.

View larger version (30K): [in this window] [in a new window]   Figure 9.   IL-6 (A), TNF- (B), MIP-2 (C ), and MCP-1 (D) concentrations in BALF and lung homogenates at the 24th hour. Results are expressed as mean ± SEM. *p < 0.05 and **p < 0.001 versus control, p < 0.05 versus C + LPS.

TNF-, MIP-2, and MCP-1 concentrations were similar in Groups C + LPS, Hal-1 + LPS, and Thio + LPS. In contrast, IL-6 concentrations were increased by 4- to 5-fold (p < 0.05) in groups mechanically ventilated (Hal-1 + LPS and Thio + LPS), as compared with the group in spontaneous ventilation (C + LPS).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The main result of our study was that halothane reduced the early LPS-induced intra-alveolar inflammatory response in rat, in a dose-dependent manner. Indeed, anesthesia with halothane concentrations  1% decreased lung inflammatory cytokine production and intra-alveolar PMN recruitment after 4 h of halothane exposure. The decrease of PMN was tightly correlated with the decrease of MIP-2 (a potent chemoattractant cytokine for PMN). These alterations were transient but were not the result of nonspecific effects of anesthesia or mechanical ventilation because no modification of lung inflammatory response was observed in thiopental-anesthetized and mechanically ventilated rats. Our results also demonstrated that, without LPS-induced lung inflammation, halothane and thiopental did not modify the lung inflammatory status, whereas tracheotomy or mechanical ventilation induced a minor lung inflammation.

To study the effects of halothane on lung alveolar inflammatory response, experiments were performed in the absence or in the presence of LPS-induced lung inflammation.

In rats breathing spontaneously with tracheotomy cannula, during 4 h and not submitted to LPS instillation, tracheotomy per se induced a minor lung inflammatory response. Indeed, lung concentrations of TNF- and IL-6 increased 5- to 10-fold compared with control rats (data not shown). However, this inflammatory response remained limited because cell recruitment was not altered and because only low concentrations of MIP-2 and MCP-1 in lung homogenates as well as in the BALF were measured, as compared with control rats.

The concentrations of inflammatory cytokines and chemokines measured in rats tracheotomized, anesthetized, and submitted to mechanical ventilation were similar to those of rats tracheotomized and breathing spontaneously, whatever the anesthetics used. These results suggested that the low levels of lung inflammation observed in the thiopental- and halothane-anesthetized rats were induced partly by the tracheotomy. The role of mechanical ventilation in the lung inflammatory response cannot be excluded and mechanical ventilation is likely involved in the increase of IL-6 that we observed 24 h after the onset of anesthesia. However, the mechanical ventilation conditions used in our study (i.e., a low tidal volume of 12 ml · kg¹ without positive-end expiratory pressure) seem not sufficient to induce a ventilatory injury or a major effect on lung inflammation as shown by other studies (17, 18). Indeed, although Tremblay and coworkers have demonstrated a large increase of TNF-, IL-6, and MIP-2 concentrations in the BALF of ex vivo lungs for tidal volume of 40 ml · kg¹, they only showed a moderate increase of these cytokines when using 15 ml · kg¹, without positive end-expiratory pressure. Therefore, in the present study, neither halothane nor thiopental altered the small lung inflammatory response induced by both tracheotomy and mechanical ventilation.

In rats breathing spontaneously and submitted to IT LPS instillation, in order to mimic a localized acute lung inflammation, an intra-alveolar recruitment of PMN, a rapid secretion of TNF- and IL-6 associated with an increased secretion and expression of MIP-2 within the lung at the fourth hour were observed. This inflammatory response was restricted to the lung because plasma cytokine (TNF-, MIP-2, and MCP-1) concentrations remained undetectable. These data confirmed that, in this model, the inflammatory response was compartmentalized to the airspace as previously described (19).

As shown recently by Xing and coworkers, PMN recruitment and cytokine production (TNF-, MIP-2, IL-6) peaked at the fourth hour in a similar murine model (20). Schmal and coworkers have demonstrated that the amount of MIP-2 in BALF peaks at the fourth hour after an LPS-induced lung inflammation in rats (21). Therefore, our experimental model allowed us to study the lung inflammatory response at the time period of maximal cytokine response with the use of 4 h volatile anesthetics exposure, which still remained clinically relevant.

The similar response observed 4 h after a LPS challenge, between thiopental-anesthetized and mechanically ventilated rats and rats breathing spontaneously suggested that neither mechanical ventilation nor thiopental modified this LPS-induced lung inflammation. By contrast, halothane anesthesia, using concentrations  1%, decreased PMN recruitment, cytokine (IL-6, TNF-) and chemokine (MIP-2) concentrations in the BALF and in the lung homogenates as well as MIP-2 mRNA level steady-state in the lung.

As lung concentrations of total protein and of MCP-1 were not modified in halothane-anesthetized rats, the observed decreased secretion of MIP-2, IL-6, and TNF- could not be attributed to a global protein synthesis inhibition. Rannels and coworkers and Wartell and coworkers have shown that halothane decreased the total lung protein synthesis for concentrations higher than 2% and for time exposures longer than 6 h (22, 23). The discrepancy with these studies may be explained by differences in time exposure or by halothane concentrations, or both. In line with this hypothesis, we have demonstrated that in vitro total protein synthesis by both AT_II cells in primary culture (24) and rat alveolar macrophages (11) was not inhibited by 1% halothane exposure during 4 h but was decreased by using halothane concentrations higher than 1.5%.

The decrease of MIP-2 and IL-6 concentrations within the lung might have been induced by the decrease of TNF- which is well-known to participate in their secretion (2). However, in our study, halothane might have directly decreased MIP-2 secretion by acting at least in part at the mRNA level because a decrease of MIP-2 steady-state mRNA level was observed. In agreement with our study, Bruce has previously shown that halothane decreased total mRNA (25).

In agreement with other studies (1, 2, 4, 21), we found a tight correlation between MIP-2 concentrations and PMN counts in the BALF whatever the groups studied. Therefore, our results suggested that the decrease of MIP-2 concentration was involved in the reduced intra-alveolar PMN recruitment in halothane-anesthetized rats.

The decrease of PMN recruitment induced by halothane  1% was associated with an increase of AM. In our study, this increase of AM could not be explained by a modification of MCP-1, which is also known to be a monocytes/macrophages chemoattractant. In agreement with our study, Zhang and coworkers, in another model of chronic lung inflammation, were unable to demonstrate a correlation between AM and MCP-1, suggesting the role of different -chemokines (26). One other possible explanation was that our samples were collected before peak secretion of MCP-1. Indeed, in vitro, the MCP-1 secretion by LPS-stimulated monocytes peaked 16 h after LPS administration (27) and in vivo monocytes recruitment into the skin of rat peaked 12 h after skin MCP-1 injection (28).

Several alveolar cells (AM, AT_II, PMN) might be altered by volatile anesthetics and involved in the reduced cytokine production induced by halothane. Among them, AM, the most potent source of cytokines within lung represent a potential target of inhaled anesthetics. Kotani and coworkers recently reported that halothane augments the expression of MIP-2 and IL-6 mRNA by AM in rats submitted to mechanical ventilation, but they did not observe any modification in the total and differential cell counts in BAL (29). As opposed to Kotani, in a preliminary in vitro study, our group has shown that IL-6 and TNF- production by AM was decreased by 1% halothane exposure (11). This discrepancy might be explained by differences in experimental procedure (in vivo versus in vitro) and in time exposure (2 h versus 4 h). Moreover, in the absence of LPS stimulation (as in the study of Kotani and coworkers) we observed a moderate inflammatory response in rats tracheotomized, anesthetized, and mechanically ventilated. However we did not find, in the present study, any difference between halothane and thiopental groups.

AT_II epithelial cells might also be altered by halothane because this halogenate decreased their surfactant biosynthesis (12). AT_II cells act in the lung cytokine network by secreting IL-6 (30) and chemokines (8, 9), and the effects of halothane upon cytokine secretion of these cells might be considered. We have recently demonstrated that halothane altered the capacity of primary culture of rat AT_II cells to secrete in vitro IL-6 and MIP-2 without modification of either MCP-1 secretion or total protein synthesis (24). As observed in the present study, the effect of halothane was time- and dose-dependent, was transient, and completely reversed 20 h after exposure. Moreover, viability (assessed by lactate dehydrogenase [LDH] release) was not affected, suggesting the absence of a direct toxic effect of halothane at the concentration used. Therefore, the diminished capacity of both AM and AT_II cells to secrete cytokines after an inflammatory stimulus and halothane exposure might explain, at least in part, the effect of halothane on the early inflammatory response.

During acute lung inflammation, an increased number of PMN in addition to AM and AT_II cells participate actively in chemokine secretion (31). Welch has shown that halothane reversibly inhibited human neutrophil bacterial killing (13) and it remains to determine if halothane might also affect their cytokine secretion.

Because only the early inflammatory response was decreased in our study, whether halothane has clinical effects long after the anesthesia period has to be determined. Indeed, a postinjury immunodepression is observed in several surgical conditions and halothane may represent an additional suppressive effect leading to perioperative infections. The reduced PMN recruitment and cytokine production observed in our study might have deleterious consequences. Several studies in animals with respiratory tract infections suggest that the local production of proinflammatory cytokines importantly contributes to host defense against pneumonia. In murine models of either gram-positive or gram-negative pneumonia, mortality was significantly earlier and higher in anti-TNF-treated mice (32). Similar results were obtained in IL-6-deficient mice with pneumococcal pneumonia (33). In addition, treatment of granulocytopenic mice with low doses of TNF- and/or IL-1, or both, diminished mortality and enhanced pulmonary clearance of Pseudomonas aeruginosa (34). Hence, an inhibition of the proinflammatory cytokine production by halothane might further contribute to perioperative infections.

The reduced PMN recruitment per se might have two opposite effects. It might be deleterious because some studies have shown that the decrease of lung PMN recruitment induced by the neutralization of MIP-2 using MIP-2 antibodies increased the early mortality in mice (35). At the opposite, decreasing PMN recruitment may also be beneficial because it has been shown that the inhibition of PMN recruitment in a ventilatory-induced lung injury model improved gas exchange (36). To our knowledge, there is no in vivo study that has analyzed the effects of volatile anesthetics on mortality in a model of either LPS-induced lung inflammation or bacterial pneumonia. Shayevitz and coworkers, in a murine model of multiple organ dysfunction syndrome, have shown that halothane and isoflurane attenuated histologic lung inflammation (37). Another study in mice infected by influenza A virus has shown that halothane reduced cell recruitment into the lungs. In both studies no effect on mortality was found (38). Using a model of acid-induced lung injury, Nader-Djalal and coworkers did not show any difference in mortality whatever the drug used to anesthetize the rats (halothane, isoflurane, or ketamine) (39). None of these studies allows us to draw conclusions regarding the beneficial or deleterious effect of halothane in vivo in lung inflammation. The mortality we observed in our study was low and the same between groups, whatever the anesthetics used. However, it remains to determine in humans the clinical consequences of this transient reduction of lung inflammatory response induced by halothane.

In conclusion, our study shows that halothane ( 1%) delivered during 4 h by mechanical ventilation, but not mechanical ventilation per se, alters the early LPS-induced lung inflammation in rat, suggesting a specific effect of halothane on this response.