Experimental Lung Research

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INTRACELLULAR STORAGE OF SURFACTANT AND PROINFLAMMATORY CYTOKINES IN CO-CULTURED ALVEOLAR EPITHELIUM AND MACROPHAGES IN RESPONSE TO INCREASING CO2 AND CYCLIC CELL STRETCH Dani-Louise Dixon a; Heather A. Barr b; Andrew D. Bersten c; Ian R. Doyle b a Department of Critical Care Medicine, Flinders University, Adelaide, Australia b Department of Human Physiology, Flinders University, Adelaide, Australia c Department of Critical Care Medicine, Flinders University and Flinders Medical Centre, Adelaide, Australia Online Publication Date: 01 January 2008 To cite this Article: Dixon, Dani-Louise, Barr, Heather A., Bersten, Andrew D. and Doyle, Ian R. (2008) 'INTRACELLULAR STORAGE OF SURFACTANT AND PROINFLAMMATORY CYTOKINES IN CO-CULTURED ALVEOLAR EPITHELIUM AND MACROPHAGES IN RESPONSE TO INCREASING CO 2 AND CYCLIC CELL STRETCH', Experimental Lung Research, 34:1, 37 - 47 To link to this article: DOI: 10.1080/01902140701807928 URL: http://dx.doi.org/10.1080/01902140701807928

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Experimental Lung Research, 34:37–47, 2008 C Informa Healthcare USA, Inc. Copyright ISSN: 0190-2148 print / 1521-0499 online DOI: 10.1080/01902140701807928

INTRACELLULAR STORAGE OF SURFACTANT AND PROINFLAMMATORY CYTOKINES IN CO-CULTURED ALVEOLAR EPITHELIUM AND MACROPHAGES IN RESPONSE TO INCREASING CO2 AND CYCLIC CELL STRETCH

Dani-Louise Dixon 2 Department of Critical Care Medicine, Flinders University, Adelaide, Australia Heather A. Barr 2 Department of Human Physiology, Flinders University, Adelaide, Australia Andrew D. Bersten 2 Department of Critical Care Medicine, Flinders University and Flinders Medical Centre, Adelaide, Australia Ian R. Doyle 2 Department of Human Physiology, Flinders University, Adelaide, Australia

2 Cell stretch stimulates both surfactant and cytokine production. The authors proposed that stretch, through these effects, modifies the pathogenesis of lipopolysaccharide-induced acute lung injury (ALI), and that this is CO2 dependent. Rat alveolar type II cells and macrophages were cocultured with lipopolysaccharide in 5%, 10%, or 20% CO2 ± stretch (30%, 60 cycles/min) for 6 hours. Intracellular TNF-α and IL-6 increased whereas secreted cytokine and surfactant decreased with increasing CO2 . Stretch independently increased intracellular TNF-α and decreased IL-6 secretion. Elevated CO2 may therefore diminish secretion of proinflammatory cytokines by alveolar cells, contributing to an explanation for protective hypercapnia in ALI. Keywords factant

acute lung injury, alveolar macrophages, alveolar type II cells, cytokines, sur-

Acute lung injury (ALI) is an inflammatory condition largely attributable to the recruitment and activation of neutrophils in response to direct or indirect lung insults, often initiated by lipopolysaccharide (LPS), a potent stimulator of inflammatory mediators. The expression and secretion of chemokines such as the neutrophil attractant interleukin (IL)-8 within the lung is arguably the key proximal step in initiating this inflammatory Received 9 August 2007; accepted 5 November 2007. This work was supported by National Health and Medical Research Council grant 229954. Address correspondence to Dr Dani-Louise Dixon, Department of Critical Care Medicine, Flinders University, PO Box 2100, Adelaide, SA 5006, Australia. E-mail: [email protected]

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cascade. Production of the classically pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 by both resident and chemotactic cells promote and maintain this inflammatory response. We previously demonstrated that LPS induces a variety of central and peripherally mediated changes in ventilation (V), perfusion (Q), and V/Q that occur well before neutrophil recruitment or lung injury [1]. Inevitably associated with these changes are regional variations in alveolar CO2 (PA CO2 ). The extent to which these coexisting stimuli contribute to ALI is unknown. The healthy alveolus contains 2 major cell types capable of secreting cytokines: alveolar type II cells (ATII) and alveolar macrophages (AM). Cell stretch is now accepted as the major stimulus for surfactant secretion; however, it may also be an important stimulus for cytokine synthesis and secretion from these cells. In both in vivo models of ALI as well as ALI patients, high-volume mechanical ventilation affects the expression and secretion of inflammatory mediators [2]. Although current ventilatory strategies employing lower tidal volume (VT ) ventilation successfully decrease damage to a healthy lung, they may not sufficiently allow for the significant decrease in lung volume present in the ALI lung. ALI reduces the aerated lung size dramatically (‘baby lung’) so that VT may be applied to a lung effectively 1/3 to 1/4 of the normal size, thereby applying similar stretch to the fully aerated lung with very large VT [3]. Consequent to reduced “protective” VT ventilation arterial CO2 (Pa CO2 ) and PA CO2 tensions may increase resulting in pulmonary arterial acidosis, and this may be protective against the development of ALI and acute respiratory distress syndrome (ARDS) [4, 5]. Supporting this, our previous work examining cytokine production by isolated AM and ATII cells demonstrated significant decreases in the secretion of TNF-α in high CO2 (20%) conditions, which could be abated via buffering of the culture medium [6–8]. Here, we aimed to extend our work into LPS-associated ALI by proposing that cell stretch also modifies the pathogenesis of ALI through its effects on cytokine and surfactant synthesis and secretion, and that the response is PCO2 dependant. Specifically, we aimed to determine whether both stretch-induced synthesis and secretion of surfactant, via release of the major lipid component phosphatidylcholine (PC), and the inflammatory cytokines TNF-α, IL-1β and IL-6, as well as the chemokine IL-8, by ATII cells in the presence of AM were PCO2 dependant. METHODS Isolation of Alveolar Type II Cells and Alveolar Macrophages All experiments were approved by the Flinders University Animal Ethics and Animal Welfare Committees.


CO2 and Stretch Effects on Surfactant and Cytokines

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ATII cells and AM were isolated from excised lungs of male SpragueDawley rats (200 to 260 g) as previously described [6, 9]. Briefly, rats were anaesthetized (methohexital sodium, intraperitoneal [IP], 100 mg/kg), the trachea cannulated and lungs perfused with buffered saline at 10 mL/min via the pulmonary artery. Excised lungs were then degassed and lavaged with 6 separate 8-mL volumes of saline, each instilled and withdrawn 3 times. Total lavage fluid was centrifuged at 800 × g at 4◦ C for 10 minutes and supernatant removed before resuspending the AM pellet in culture medium (Dulbecco’s modified Eagle’s medium [DMEM], 26.74 mM NaHCO3 , 10% fetal bovine serum [FBS] [v/v], and 1% [w /v] penicillin and streptomycin). Lung tissue was digested with instilled elastase (3.85 units of activity/mg protein) for 45 minutes at 37◦ C before being finely minced in 0.025% DNase and 5 mL FBS added before incubation for 2 minutes at 37◦ C. The preparation was then filtered and centrifuged at 800 × g for 5 min at 4◦ C. Macrophages were removed by adhesion in 1% DNase in DMEM containing 26.74 mM NaHCO3 , 10% FBS (v/v) for 20 min at 37◦ C. Nonadherent cells (ATII) were centrifuged at 800 × g for 10 minutes at 4◦ C and resuspended in culture medium (as above). Co-Culture Experiments AM were plated at a density of 6 × 104 cells/cm2 with or without coculture with ATII cells at 40 × 104 cells/cm2 on silastic membranes (Flexcell International, Hillsborough, NC) precoated with 5 µg/cm2 rat immunoglobulin G (IgG) (Sigma-Aldrich, St Louis, MO) and 4.33 µg/cm2 fibronectin, for 16 hours. Co-cultures were incubated overnight in culture medium containing [methyl-H3 ]choline chloride (0.5 µCi/mL). Separate cultures were then washed and incubated for 6 hours in the presence of 10 µg/mL LPS (Salmonella abortus equi; Sigma-Aldrich) in culture medium in 5%, 10%, or 20% CO2 , with normal oxygen (21%) balanced with N2 . Cultures of ATII and AM coculture or AM alone at each CO2 concentration were incubated with or without cyclic sinusoidal cell stretch at 60 cycles/min with a 30% maximum elongation of the elastomer growth surface per cycle (Flexcell Strain Unit FX3000; Flexcell International). Experiments were paired for determination of the effects of cell strain (stretch versus no stretch) and unpaired for examination of the effects of changing PCO2 . Cells were treated in duplicate per assay and each protocol repeated on 4 to 6 separate days. Following the 6-hour treatment, culture media were harvested. Adherent cells were washed with 1 mL ice-cold saline before addition of 2 mL ice-cold lysis buffer (50 mM Tris, 300 mM NaCl containing 1% (w /v) Triton X-100; pH 7.6) to each well. Harvested supernatants and lysates were centrifuged at 1000 × g at 4◦ C for 10 minutes. Both culture media and lysates were stored at −20◦ C prior to analysis.


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Cellular Metabolic Activity Metabolic activity of ATII and AM in isolation and co-culture was ascertained by a colorimetric assay 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazine methosulfate (PMS) for dehydrogenase enzymatic activity (Promega, Madison, WI). Four hundred microliters of MTS/PMS tetrazolium compound was added to each well and the cells incubated in the dark with each of the gas mixtures as described in the co-culture experiments. Supernatants were sampled at 30 minutes (baseline) and 6 hours (final) by removal of 2 × 100-µL aliquots. The absorbance of colored formazan produced through bioreduction by metabolically active cells was read on an automatic plate reader at OD 490 nm. Data are presented as mean optical density. Measurement of Phosphatidylcholine Secretion via [3 H]Choline Release After treatment, lipids were extracted from aliquots of the supernatant or lysate with chloroform and methanol [10], the liquid layer dried and [3 H] measured in a Beckman Liquid Scintillation System (model LS5000 TD; Fullerton, CA) and phosphstidylcholine (PC) secretion calculated as described previously [9]. [3 H]PC levels are presented as percent [3 H]PC secreted. Cytokine Assays Supernatants and cell lysates were assayed for cytokine concentration (tumor necrosis factor [TNF]-α [PharMingen Opt EIA, San Diego, CA], interleukin [IL]-1β, IL-6 [R&D Systems, Minneapolis, MN], IL-8 [CINC-1; Peprotech, Rockhill, NJ]) using enzyme-linked immunosorbent assay (ELISA), using matched antibody pairs at concentrations recommended by the manufacturer, as described previously [7, 11]. The minimum detectable level for each assay was 62, 62, 78, and 20 pg/mL, respectively. Cytokine levels are expressed in pg/mL. Statistical Analysis All data are presented as mean ± SEM. Samples with cytokine below the minimum detectable level were assigned a value of half that limit for statistical analysis. Due to their asymmetric nature, all cytokine data were corrected by log transformation. Differences in cytokine production and percent [3 H]PC between experimental groups were determined using univariate two-way analysis of variance (ANOVA) with Tukey’s post hoc analysis. Differences between AM alone and ATII/AM coculture experiments, and between supernatant and lysate cytokine concentrations were determined



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by unpaired and paired t tests, respectively. Differences in cellular metabolic activity were determined by one-way ANOVA. A probability level of < .05 was considered to be statistically significant and all analyses were performed using SPSS for Windows 12.1 (SPSS, Chicago, IL). RESULTS Production of Cytokines and Surfactant by AM and ATII in Co-Culture in Response to Cyclic Stretch under Normal Physiologic CO2 Exposure In order to determine the effect of cyclic stretch of 30% at 60 cycles/min for 6 hours on ATII and AM in coculture at normal physiologic CO2 (5%) we examined paired cultures from n = 4–5 animals. As shown in Figure 1, cyclic stretch increased the amount of secreted TNF-α (5935 ± 632 versus 5044 ± 416 pg/mL, P = .05) and IL-6 (1711 ± 242 versus 1369 ± 233 pg/mL, P = .01) from that produced by cells incubated without stretch, contributing to

FIGURE 1 Concentration of intracellular (A) TNF-α (n = 4–6), (B) IL-6 (n = 4–6), (C) IL-8 (n = 6), and (D) IL-1β (n = 4–5) at 6 hours coculture of isolated rat ATII and AM with 10 µg/mL LPS (pg/mL; mean ± SEM) in 5%, 10%, and 20% CO2 conditions with or without 30% cell stretch (60 cycles/min) analyzed by univariate one-way ANOVA on log-transformed data. (A) TNF-α CO2 × stretch interaction, P = .92; CO2 effect (5% and 10% versus 20%), P ≤ .001; cyclic stretch effect, P = .03. (B) IL-6 CO2 × stretch interaction, P = .69; CO2 effect (5% versus 20%), P = .04; cyclic stretch effect, P = .15. (C) IL-8 CO2 × stretch interaction, P = .40; CO2 effect (10% versus 20%), P = .03; cyclic stretch effect, P = .14. (D) IL-1β CO2 × stretch interaction, P = .61; CO2 effect, P = .12; cyclic stretch effect, P = .38.

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FIGURE 2 Concentration of secreted (A) TNF-α (n = 4–6), (B) IL-6 (n = 4–6), (C) IL-8 (n = 6), and (D) IL-1β (n = 4–5) at 6 hours coculture of isolated rat ATII and AM with 10 µg/ml LPS (pg/mL; mean ± SEM) in 5%, 10%, and 20% CO2 conditions with or without 30% cell stretch (60 cycles/min) analyzed by univariate two-way ANOVA on log-transformed data. (A) TNF-α CO2 × stretch interaction, P = .71; CO2 effect, P = .10; cyclic stretch effect, P = .28. (B) IL-6 CO2 × stretch interaction, P = .78; CO2 effect, P = .14; cyclic stretch effect, P = .91. (C) IL-8 CO2 × stretch interaction, P = .32; CO2 effect, P = .30; cyclic stretch effect, P = .51.

a significant increase in total TNF-α (6528 ± 681 versus 5422 ± 464, P = .04) and IL-6 (1954 ± 263 versus 1636 ± 246, P = .02). In contrast, the secreted and total IL-8 did not change (secreted: 31630 ± 3480 versus 37989 ± 8448, total: 32976 ± 3558 versus 39820 ± 8493; P > .8). There was no significant difference in intracellular amounts of any cytokine (Figure 2; P > .06). Similarly, there was no difference in the intracellular (P > .31) or total (P > .13) surfactant between cells undergoing cyclic stretch and those that did not, with only an indication of increasing surfactant secretion (13059 ± 1317 versus 9380 ± 1198, P = .06) at 6 hours of coculture (Figure 3). Interaction of CO2 with Cyclic Stretch on the Production of Cytokines by AM and ATII in Co-Culture Next we aimed to determine the effect of increasing CO2 to 10% and 20% on cytokine and surfactant production by co-cultured ATII and AM during cyclic stretch for 6 hours. Independent of stretch, there was a significant increase with increasing CO2 in both intracellular TNF-α (Figure 1A;

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FIGURE 3 Concentration of (A) intracellular and (B) secreted surfactant (n = 4–6) as a proportion of total [3 H]PC at 6 hours coculture of isolated rat ATII and AM with 10 µg/ml LPS ([3 H]PC; mean ± SEM) in 5%, 10%, and 20% CO2 conditions with or without 30% cell stretch (60 cycles/min) analyzed by univariate two-way ANOVA. (A) Intracellular [3 H]PC CO2 × stretch interaction, P = .92; CO2 effect, P = .11; cyclic stretch effect, P = .34. (B) Secreted [3 H]PC CO2 × stretch interaction, P = .79; CO2 effect, P = .06; cyclic stretch effect P = .03.

P < .001) and IL-6 (Figure 1B; P = .03), with a concomitant decrease in the percentage of TNF-α and IL-6 secreted (Table 1; P = .003 and P = .02). CO2 treatment also resulted in a biphasic effect on intracellular IL8, higher at 5% and 20% CO2 independent of stretch (Figure 1C; P = .03). Cyclic stretch increased intracellular TNF-α (Figure 1A; P = .03), whereas decreasing percent secreted IL-6 (Table 1; P = .03) independent of the CO2 effect. There was no effect of stretch or CO2 on intracellular or secreted IL-8 (Figures 1C, 2C, Table 1; P ≥ .26) or on intracellular IL-1β (Figure 1D; P ≥ .12). There was also no interactive effect of increasing CO2 and cyclic stretch on the total cytokine despite some indication of increases with CO2 TNF-α (CO2 × stretch, P = .88; CO2 , P = .06; stretch, P = .11), total IL-6 (CO2 × stretch, P = .73; CO2 , P = .09; stretch, P = .69), and total IL-8 (CO2 × stretch, P = .28; CO2 , P = .26; stretch, P = .49). There was no significant interactive effect between stretch and CO2 on any of the cytokines. Neither stretch nor increasing CO2 resulted in the TABLE 1 Proportion of Secreted Cytokines and Surfactant in Response to Increasing CO2 and 30% Cyclic Stretch at 60 cycles/min CO2 5%

TNF-α IL-6 IL-8 3 HPC

CO2 10%

CO2 20%

No stretch

Stretch

No stretch

Stretch

No stretch

Stretch

P*

93.1 ± 0.8 87.4 ± 0.6 95.1 ± 1.8 12.7 ± 1.6

90.9 ± 0.4 83.1 ± 1.8 95.8 ± 0.4 16.2 ± 2.3

90.9 ± 1.2 82.4 ± 4.7 96.8 ± 0.3 8.6 ± 1.1

89.0 ± 1.2 77.2 ± 3.6 95.5 ± 0.5 9.6 ± 0.7

84.9 ± 3.5 79.8 ± 2.7 95.2 ± 0.4 7.6 ± 0.8

77.7 ± 4.9 71.7 ± 1.7 95.3 ± 0.4 7.2 ± 0.3

.003 .020 .480 .001

Note. Data are % total, mean ± SEM. Analyzed by univariate two-way ANOVA. TNF-α: CO2 × stretch interaction, p = .51; stretch effect, P = .12. IL-6: CO2 × stretch interaction P = .82; stretch effect, P = .03. IL-8: CO2 × stretch interaction, P = .43; stretch effect, P = .78. Surfactant: CO2 × stretch interaction, P = .32; stretch effect, P = .17. *CO2 effect: TNF-α: 5% and 10% versus 20%; IL-6: 5% versus 20%; surfactant: 5% versus 10% and 20%.


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secretion of detectable amounts of IL-1β. For all other cytokines, there was significantly more secreted than remained intracellularly after 6 hours in coculture (Table 1; P ≤ .001). Secretion of PC in Response to Stretch and Increasing CO2 Increasing CO2 (5% versus 10% versus 20%) significantly decreased the proportion of surfactant released by the cocultures (Table 1; P ≤ .001). There was no additive effect of increasing CO2 and stretch on the intracellular (Figure 3A) or secreted amount of surfactant as measured by [3 H]PC (Figure 3B) or on the total surfactant produced (CO2 × stretch, P = .93; CO2 , P = .23; stretch, P = .24). Effect of ATII and AM Co-culture Compared with AM Alone In order to delineate the contribution of each cell type to the production of cytokines at increasing CO2 during 6 hours of cyclic stretch in parallel experiments, we incubated AM alone at 5%, 10%, and 20% CO2 with or without stretch. Unfortunately cell yields for ATII prevented a similar analysis of their cytokine production in the absence of AM. For all cytokines, there was a significant contribution by ATII cells in coculture to the total cytokine produced above that of AM alone (TNF-α 26%, IL-6 1264%, IL-1β 61%, IL-8 587%; P ≤ .001). Whether this is an additive concentration or synergistic increase cannot be definitively determined here due to the absence of ATII cultured in isolation. However, our previous results from AM and ATII alone demonstrating a 12.5% contribution by ATII to the calculated total TNF-α suggests an increase in coculture above the contribution of each cell type alone [8]. As reported previously [7], the only significant effect of either stretch or increasing CO2 on AM alone was a decrease in the secreted TNF-α at 20% CO2 (P ≤ .001; data not shown). Cellular Metabolic Activity There was no difference in the baseline (30 minutes) metabolic activity between CO2 treatments for either isolated AM or ATII, or for AM and ATII coculture (P > .13; data not shown). At the completion of the treatment protocol (6 hours), the isolated AM incubated at 5% CO2 demonstrated significantly elevated metabolic activity via increased optical density, than either 10 or 20% CO2 (Table 2; P ≤ .05). No differences in metabolic activity of ATII in isolation or AM and ATII co-culture were apparent (P > .13). DISCUSSION The specific mechanism underlying the protective effect of hypercapnic acidosis, particularly as it relates to an observed inflammatory inhibition,

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CO2 and Stretch Effects on Surfactant and Cytokines TABLE 2 Metabolic Activity of Cells in Response to Increasing CO2 at 6 Hours of Culture

AM ATII AM+ATII

CO2 5%

CO2 10%

CO2 20%

P∗

0.607 ± 0.07∗∗ 1.719 ± 0.17 2.207 ± 0.50

0.407 ± 0.05 1.610 ± 0.05 1.637 ± 0.05

0.371 ± 0.03 1.479 ± 0.13 1.530 ± 0.12

.01 .35 .13

Note. Data are optical density at 490 nm, mean ± SEM. by one-way ANOVA. ∗∗ Versus 10% and 20% CO (P < .05) using Tukey’s post hoc analysis. 2 ∗ Analyzed

remains elusive. Similarly, the counter intuitive benefits of cell stretch in terms of surfactant release and decreased oxidative stress [12, 13], opposed to those of alveolar epithelial injury and release of inflammatory cytokines [2, 14], require significant elucidation. We report a pattern of elevated intracellular storage of proinflammatory cytokines as well as surfactant by ATII and AM in coculture under high CO2 and stretch conditions. Our previous study [7] examining the effect of cyclic stretch and CO2 on isolated AM demonstrated a decrease in secreted TNF-α with a concomitant decrease in total cellular metabolic activity, with both stretch and CO2 at 20%. The results from our current study reflect these and further indicate that this effect is contributed to in a coculture environment, perhaps synergistically. In addition, the current study highlights a significant parallel effect of diminished IL-6 and surfactant secretion in this coculture environment. Surfactant produced by ATII cells can inhibit AM activity [15]. However, production of all cytokines was substantially elevated in coculture. These results suggest a CO2 -dependent mechanism of increased cellular storage and/or a decrease in secretion of cellular products that is independent of any significant change in cellular metabolic activity. Although the current study gave no indication of an interactive or additive effect of stretch with increasing CO2 on any of the measured parameters, it does support previous findings regarding an increase in surfactant in response to high levels of stretch at physiologically normal CO2 [7, 12]. Surfactant release in response to a substantial single stretch, such as a sigh or volume-recruitment maneuvre, has been hypothesized to be a significant contributing factor to protective pulmonary mechanisms. The decrease in release of surfactant in response to increasing CO2 , as demonstrated here, may therefore appear counterintuitive if hypercapnia is to be considered a protective mechanism. However, recent findings demonstrating decreased release of surfactant by ATII cells under high-level stretch continued for greater than 30 minutes indicate a similar time-dependent mechanism of intracellular storage under prolonged stress [16]. Therefore, the cumulative effect of potential stressors on the release of surfactant by ATII needs to be elucidated in vivo.


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Despite strong evidence for neutrophil-mediated injury in ALI, no clear effect of either stretch or CO2 on IL-8 was observed in this current coculture study. It may therefore be hypothesized that although alveolar epithelial cells may contribute to the pool of this neutrophil chemoattractant in the lung during the stress of high VT , that the endothelium may be the major source of the necessary chemotactic gradient for the influx of vast numbers of neutrophils into the alveolus, and that production by the epithelium, as well as the resident macrophages, remains secondary. The complete lack of detectable IL-1β in the supernatant of either cocultured cells or AM alone is difficult to explain, as mature forms of IL1-β are secreted by both macrophages and alveolar epithelial cells after stimulation with LPS in other in vitro systems [17, 18]. IL-1 cytokines are unusual in that the intracellular precursors do not contain a recognizable hydrophobic secretory signal sequence allowing secretion of the protein by classical secretory pathways involving the endoplasmic reticulum/Golgi system. The absence of secreted IL-1β may therefore be demonstrative of inhibitory effects of the culture conditions employed in this study on complex secretory mechanisms such as inhibition of proteases such as the IL-1β convertase (ICE). Such complex systems may also contribute to the general profile of cytokine retention as observed with TNF-α and IL-6 in this system through similar inhibition of secretory factors such as the metalloproteases, ADAM 17 and ADAM 19. The results presented here suggest that both cyclic stretch and increasing CO2 play roles in the regulation of inflammatory cytokines and surfactant by ATII cells in the presence of AM but that this regulation is not additive. It is, however, apparent that elevated CO2 may diminish the secretion of some proinflammatory cytokines by alveolar epithelium and macrophages, potentially contributing to the protective paradigm of hypercapnia in ALI. REFERENCES [1] Davidson KG, Bersten AD, Barr HA, Dowling KD, Nicholas TE, Doyle IR: Endotoxin induces respiratory failure and increases surfactant turnover and respiration independent of alveolocapillary injury in rats. Am J Respir Crit Care Med. 2002;165:1516–1525. [2] Plotz FB, Slutsky AS, van Vught AJ, Heijnen CJ: Ventilator-induced lung injury and multiple system organ failure: a critical review of facts and hypotheses. Intensive Care Med. 2004;30:1865–1872. [3] Gattinoni L, Pesenti A: The concept of “baby lung.” Intensive Care Med. 2005;31:776–784. [4] Laffey JG, Engelberts D, Kavanagh BP: Buffering hypercapnic acidosis worsens acute lung injury. Am J Respir Crit Care Med. 2000;161:141–146. [5] Laffey JG, Tanaka M, Engelberts D, Luo X, Yuan S, Tanswell AK, Post M, Lindsay T, Kavanagh BP: Therapeutic hypercapnia reduces pulmonary and systemic injury following in vivo lung reperfusion. Am J Respir Crit Care Med. 2000;162:2287–2294. [6] Lang CJ, Dong P, Hosszu EK, Doyle IR: Effect of CO2 on LPS-induced cytokine responses in rat alveolar macrophages. Am J Physiol Lung Cell Mol Physiol. 2005;289:L96–L103. [7] Lang CJ, Barnett EK, Doyle IR: Stretch and CO2 modulate the inflammatory response of alveolar macrophages through independent changes in metabolic activity. Cytokine. 2006;21;33:346–351.



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[8] Hosszu EK, Nicholas TE, Doyle IR: PCO2 does not influence stretch-mediated surfactant lipid secretion, but alters TNF-α secretion from isolated LPS-stimulated rat alveolar type 2 cells. Am J Resp Crit Care Med. 2004;169:A738. [9] Edwards YS, Sutherland LM, Power JH, Nicholas TE, Murray AW: Osmotic stress induces both secretion and apoptosis in rat alveolar type II cells. Am J Physiol. 1998;275(4 Pt 1):L670–L678. [10] Bligh FF, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem. 1959;37:911–917. [11] Bryan D-L, Hart P, Forsyth KD, Gibson RA: Modulation of respiratory syncytial virus induced prostaglandin E2 production by n-3 LCPUFA in human respiratory epithelium. Lipids. 2005;40:1007–1011. [12] Bersten AD, Doyle IR, Davidson KG, Barr HA, Nicholas TE, Kermeen F: Surfactant composition reflects lung overinflation and arterial oxygenation in patients with acute lung injury. Eur Respir J. 1998;12:301–308. [13] McAdams RM, Mustafa SB, Shenberger JS, Dixon PS, Henson BM, DiGeronimo RJ: Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2006;291:L166–11L174. [14] Belperio JA, Keane MP, Lynch JP 3rd, Strieter RM: The role of cytokines during the pathogenesis of ventilator-associated and ventilator-induced lung injury. Semin Respir Crit Care Med. 2006;27:350–364. [15] Kanj RS, Kang JL, Castranova V: Interaction between primary alveolar macrophages and primary alveolar type II cells under basal conditions and after lipopolysaccharide or quartz exposure. J Toxicol Environ Health A. 2006;69:1097–1116. [16] Arold SP, Bartolak-Suki E, Suki B: Long-term cyclic stretch inhibits surfactant secretion in alveolar type II epithelial cells. Am J Resp Crit Care Med. 2006;173:A759. [17] Shogi T, Oono H, Nakagawa M, Miyamoto A, Ishiguro S, Nishio A: Effects of a low extracellular magnesium concentration and endotoxin on IL-1beta and TNF-alpha release from, and mRNA levels in, isolated rat alveolar macrophages. Magn Res. 2002;15:147–152. [18] Haddad JJ, Land SC: Amiloride blockades lipopolysaccharide-induced proinflammatory cytokine biosynthesis in an IκB-α/NF-κB-dependent mechanism. Am J Respir Cell Mol Biol. 2002;26:114– 126.