Department of Human Physiology, School of Medicine, Flinders University of South Australia, Adelaide, Australia
Submitted 25 October 2004 ; accepted in final form 10 March 2005
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ABSTRACT |
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inflammation; hypercapnia; acute lung injury
ALI often requires mechanical ventilation, where current strategies favor low tidal volume and high end-expiratory volume. Because ventilatory-induced stretch exacerbates ALI (18), the improved morbidity and mortality, which have been demonstrated using such protective strategies, may be due to reduced mechanical trauma (7). However, such strategies can also be accompanied by elevations in CO2 (hypercapnia). Although hypercapnia can be controlled by increasing respiratory rate (33), there is evidence to suggest that hypercapnia may be beneficial in the prevention of, and recovery from, lung injury (6, 15, 16, 18, 19, 3032). However, the beneficial effect of hypercapnia is currently a topic of hot debate, since in the major study that demonstrated benefits of reduced lung stretch (33), CO2 levels did not differ between low and high ventilation. Moreover, at the cellular level, Lang et al. (20) determined that cell injury, mediated by nitric oxide activity, was enhanced in fetal rat alveolar type II epithelial cells exposed to hypercapnia. Understanding the influence of CO2 and pH on the inflammatory functions of AM is important to our understanding of the inflammatory responses in ALI, or other inflammatory lung diseases, and may contribute to the current debate concerning the role of hypercapnia in ventilatory strategies.
In the lung, the pH of the epithelial lining fluid (ELF) is normally pH 6.76.9 (26). However, the acid-base status of the ELF varies widely in health and disease, and AM may be exposed to a range of pH and CO2 levels depending on their location in the alveoli. For example, in cystic fibrosis and asthma, the pH of the ELF may fall below 6.5 (8, 13), and in tumors or abscesses, the pH can be <6.0 (25). High CO2 can lead to hypercapnic acidosis of the ELF and may also influence AM cytokine release. TNF- and IL-1
are among the best characterized of the cytokines and can be regulated as archetypal acute inflammatory cytokines. Cytokine-induced neutrophil chemoattractant factor-1 (CINC-1), a member of the IL-8 family (23), is a potent neutrophil chemotactic and activating factor (29). Therefore, in this descriptive study, we investigate the influence of CO2 and buffering pH on LPS-stimulated synthesis and/or secretion of TNF-
, IL-1
, and CINC-1 proteins in cultured rat AM.
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METHODS |
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All experiments were performed under the Flinders University Animal Ethics Committee, approval no. 385/04, and in compliance with Principles of Animal Care publication number 86-23 of the National Institutes of Health and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (6th edition).
Male Sprague-Dawley rats (mass range: 200260 g) were obtained from the Institute of Medical and Veterinary Science, Gilles Plains, Adelaide, Australia. Rats were deeply anesthetized via intraperitoneal injection of methohexital sodium (100 mg/kg body wt; Eli Lilly, Sydney, Australia), the tracheae were cannulated, and the lungs were perfused with buffered saline solution A (150 mM NaCl, 5 mM KCl, 2.5 mM sodium phosphate, 10 mM HEPES, 0.2 mM EGTA containing 0.1% glucose, pH 7.4) at 10 ml/min via the pulmonary artery. The lungs were excised, degassed for 1 min, and lavaged with six separate 10-ml volumes of saline, each volume being instilled and withdrawn three times. The lavage fluid was centrifuged at 1,000 g at room temperature for 10 min, and the pellet was resuspended in culture media [DMEM containing 23.8 mM NaHCO3, 10% FBS (vol/vol), and 1% (wt/vol) penicillin and streptomycin] at a density of 1.5 x 105 cells/ml. AM were plated at a density of 0.75 x 105 cells/cm2 in 24-well culture plates (Nalge Nunc International, Rochester, NY) precoated at 37°C for 6 h with 5 µg/cm2 rat IgG (Sigma-Aldrich, St. Louis, MO) in saline and incubated in a humidified atmosphere (pH 7.4, 95% O2, 5% CO2) at 37°C for 16 h. Nonadhered cells were removed by washing the wells with culture media.
Experimental Protocols
Cells were plated in triplicate per assay, and each protocol was repeated on five or six separate populations of isolated cells, i.e., on five or six occasions or days. The adhered cells were subjected to one of three protocols as follows.
Protocol I: gas mixture treatment. Cells were incubated for 14 h in fresh culture medium in a humidified atmosphere containing either 2.5, 5, 10, or 20% CO2, with normal oxygen (21%) and the balance comprising N2, with or without inclusion of 10 µg/ml of LPS (Salmonella abortus equi, Sigma-Aldrich). Under these conditions, the pH of the media was 7.2 at 2.5% CO2, 6.8 at 5% CO2, 6.4 at 10% CO2, and 5.8 at 20% CO2.
Protocol II: buffering medium pH. AM were incubated as described for protocol I. However, in this case, the DMEM was buffered to maintain a pH of 7.2 with increasing concentrations of NaHCO3 (i.e., 7.1, 14.3, 28.6, or 56.1 mM NaHCO3 during incubations with 2.5, 5, 10, and 20% CO2 gas mixtures, respectively).
Protocol III: switching gas mixture. A schematic of the experimental design for protocol III is given in Table 1. AM were incubated, as described for protocol I, for a 4-h incubation. The culture media were then collected for cytokine secretion analysis (labeled 1st 4-h incubation in Figs. 2 and 6). Fresh DMEM was then added to the cells, and the cells were incubated for a further 4 h, using the same (i.e., 2.5% then 2.5% or 20% then 20%) or a different (i.e., 2.5% then 20% or 20% then 2.5%) gas mixture. The culture media were then collected for cytokine secretion analysis (labeled 2nd 4-h incubation in Figs. 2 and 6). Note that cell lysate samples could only be collected at the end of both 4-h incubation periods (Fig. 4). A similar set of experiments was also performed where the media were not changed after the first 4-h incubation period.
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After incubation for 1, 2, 3, 4, or 8 h, the culture media were harvested and centrifuged at 8,000 g at 4°C for 10 min. The cells were immediately washed with 1 ml of ice-cold saline, and 0.5 ml of ice-cold 50 mM Tris and 0.3 M saline containing 1% (wt/vol) Triton X-100 (pH 7.6) was added to each well. The lysates were centrifuged at
14,000 g at 4°C for 15 min, and both the lysates and the culture medium were stored at 20°C for batch analysis.
Cell Viability
Nonadhered cells were removed by being washed with PBS containing 2 mM MgCl2 and counted using a hemocytometer (Improved Neubauer, B.S. 748; Weber Scientific International, Teddington, UK). In addition, adhered cells were stained with a 20% (vol/vol) methanol solution of 0.2% (wt/vol) crystal violet for 15 min. If a cell membrane is intact, the cell retains the crystal violet dye. The cells were then washed with water, dried, and solubilized with SDS (1% wt/vol), and the absorbance was read at 540 nm on a Dynatech MR5000 plate reader (Baxter Diagnostics, Billinghurst, UK). Cell numbers were calculated relative to a standard curve of optical density for cell numbers ranging from 0 to 30,000 cells/well of a 96-well plate and used to calculate the data presented in Table 3.
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Cell metabolic activity was assessed using a colorimetric assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) and phenazine methosulfate (PMS)] for dehydrogenase enzymatic activity according to the manufacturer's protocol (Promega, Madison, WI). Briefly, AM cells were plated at a density of 1.5 x 104 cells/well in 96-well plates and cultured in DMEM for 24 h. Twenty microliters of MTS and PMS were added to each well, and the cells were then incubated in the dark for 4 h with different gas mixtures as described for protocols I and II. In metabolically active cells, dehydrogenase enzymes convert MTS into a soluble formazan product. The quantity of formazan product was measured by absorbance at 490 nm (Dynatech MR5000 plate reader).
Cytokine Measurement
For each experimental protocol, cytokine content was analyzed on samples collected from as many of the five to six separate experiments as possible. Supernatant and lysate TNF- (Pharmingen Opt EIA, San Diego, CA) and supernatant IL-1
(R&D Systems, Minneapolis, MN) were measured using ELISA as per the manufacturer's protocols. For measurement of CINC-1, media samples were lyophilized and concentrated threefold by resuspension in appropriate volumes of assay diluent (PBS + 10% FBS). Lysate samples were unable to be concentrated for CINC-1 analysis due to limited sample volumes. CINC-1 was measured using an inhibition ELISA previously developed in our laboratory. Briefly, samples were incubated with a rabbit anti-rat CINC-1, NH2-terminal specific antibody (Assay Designs, Ann Arbor, MI) overnight at 4°C before being transferred to a vinyl ELISA plate (Costar, Cambridge, MA) coated with 50 ng/ml of CINC-1 (Peptide Institute, Osaka, Japan). Free antibody was captured and detected with horseradish peroxidase-conjugated sheep anti-rabbit IgG (Chemicon, Melbourne, Australia). Color development using a 3,3',5,5'-tetramethylbenzidine substrate reagent set (Pharmingen) was measured at 450 nm using a Dynatech plate reader (Dynatech Laboratories, Chantilly, VA).
Statistical Analysis
In all tables and figures, n refers to the total number of separate experiments (i.e., different populations of freshly isolated AM) in which we measured cell viability, metabolic activity, or cytokine amount. Statistical analyses were conducted using SPSS. All results are expressed as means (SD). Effects of CO2 on cell viability, metabolic activity, and cytokine production and secretion were analyzed using one- and two-way ANOVA with Dunnett's post hoc tests (using 5% CO2 as the control). Bonferroni post hoc tests were used to assess differences between incubation times.
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RESULTS |
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Our method of AM isolation typically afforded 8.0 ± 0.5 x 106 cells (means ± SE; n = 12) of >90% purity. The %CO2 under which the cells were cultured had no effect on the adherence of the AM (data not shown). With the use of crystal violet staining, neither CO2 nor buffering significantly influenced cell viability (Table 2). However, CO2 and buffering did influence cell metabolic activity, as measured by the MTS/PMS assay. Compared with that at 5% CO2, which is considered a normal physiological CO2 level, post hoc testing showed that the cut off for a significant difference in metabolic activity is 20% CO2. At 20% CO2, metabolic activity was 60% of that at 5% CO2. Conversely, at 2.5% CO2, metabolic activity was
114% of that at 5% CO2. Buffering the culture media abated the CO2-induced alterations in metabolic activity (Table 2).
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Protocol I.
There was no appreciable TNF- secretion [35 pg/ml (SD 21) from all experimental groups] in the absence of LPS. LPS-stimulated secretion was influenced by incubation time and %CO2 in a clear, dose-responsive manner (Fig. 1). Post hoc testing showed significant differences in TNF-
secretion between the 5% CO2 group and the 2.5 and 20% CO2 groups. After 4 h, TNF-
secretion increased by
30% under 2.5% CO2 but decreased by
20% under 20% CO2 (Table 3).
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Protocol III.
The effect of CO2 on LPS-stimulated TNF- secretion in AM was reversible (Fig. 2). A similar pattern of LPS-stimulated TNF-
secretion was observed in both the nonaccumulative (1st and 2nd incubations) and accumulative gas-switching experiments (Fig. 2). It should be noted that in experiments where the culture media were changed after the first incubation, for the 2.5% CO2 concentration there was a slight increase in the amount of TNF-
secreted in the second incubation period, compared with the first incubation period, even when gas mixture was kept the same. The amount of IL-1
measured in cell supernatants was negligible in all cases (data not shown).
Lysate TNF-
Protocol I.
There was relatively little TNF- [107 pg/ml (SD 10) from all experimental groups] measured in cell lysates in the absence of LPS. In LPS-stimulated AM, lysate TNF-
was influenced by incubation time, but not CO2, in normal media (Fig. 3).
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Protocol III.
Protocol III supports observations made in protocol I, where there were no differences in lysate TNF- levels between cells incubated in 2.5 or 20% CO2 for both incubation periods in normal media. However, when cells were exposed to a change in CO2 between the first and second incubation periods, there were small, but significant, changes measured in TNF-
in the cell lysates (Fig. 4).
CINC-1 Secretion
Protocol I. There was no appreciable CINC-1 secretion in the absence of LPS. LPS-stimulated secretion was not influenced by incubation time or CO2.
Protocol II. CO2, but not incubation time, influenced CINC-1 secretion in buffered media (Fig. 5). In buffered media, CINC-1 secretion was 3540% higher in 10 and 20% CO2 groups than in the 5% CO2 groups.
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DISCUSSION |
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The aim of this study was to determine the impact of CO2 on AM cytokine release at a cellular level, and although these experiments have arisen from current physiological debate surrounding the beneficial effects of hypercapnia in ALI, the impact of hypocapnia on AM function also remains to be determined. Hence, although they are not usually observed in ventilated patients in vivo, we have also included 2.5 and 20% CO2 gas mixtures in this study. Concentrations of 2.5 and 20% CO2 may be pharmacologically relevant. Moreover, AM would be exposed to a range of pH and CO2 levels in vivo, depending on their location in the alveoli and the injury state of different regions in the lung. For the buffering experiments, a pH of 7.2 was chosen since it was the pH recorded for culture media under our lowest gas mixture (2.5% CO2). In hindsight, a pH of 6.8 (i.e., that at 5% CO2) may have been more appropriate, from a physiological perspective, to use in protocols II and III, since in vivo, the pH of the ELF has been estimated at pH 6.76.9 (26). Nevertheless, neutralizing the pH of the culture media did significantly alter cytokine patterns in response to different CO2 levels, suggesting that pH does contribute to the cytokine responses we observed here.
Cell Viability and Metabolic Activity
High CO2 reduced metabolic activity but did not significantly influence cell viability. Conversely, low CO2 increased metabolic activity. These observations are plausible, since metabolic activity is likely to be affected by changes in pH, to a greater extent than, or at least prior to, detectable changes in cell viability. Both the 60% decrease in metabolic activity observed at 20% CO2 and the 14% increase in metabolic activity observed at 2.5% CO2 were abated when the media were buffered to pH 7.2. This suggests that CO2-mediated changes in pH may play a pivotal role in the alteration of cell metabolic activity. The data cannot be completely explained by CO2-induced changes in metabolic activity, since a significant deterioration of metabolic activity was only apparent at 20% CO2. Twenty percent CO2 has also been observed as the cut-off level for significant deterioration in lung viability in vivo (17).
TNF- and IL-1
We demonstrate that high CO2 attenuates secretion of TNF- from LPS-stimulated AM but has no effect on lysate TNF-
in AM incubated in normal medium. Conversely, low CO2 enhanced both lysate and secreted TNF-
in LPS-stimulated AM cultures. The increases we observed in TNF-
secretion at low CO2 may be due, at least in part, to increases in cell metabolic rate, since metabolic activity also increased in AM incubated at low CO2. Buffering the media abated the inhibitory effects of high CO2 on TNF-
secretion, suggesting that this inhibitory response to CO2 is modulated by extracellular pH (pHe). Studies by Bidani et al. (4) and Heming et al. (10, 11) support this finding. The small increase in lysate TNF-
levels we observed in AM incubated in buffered media, at high CO2, could be related to the retention of synthesized TNF-
, in a similar manner to that suggested by Bidani et al. (4) and Heming et al. (10). We found no appreciable IL-1
in the cell culture media, regardless of the presence or absence of LPS, or CO2. This finding is consistent with the observation that IL-1
lacks the signal sequence necessary to translocate the protein to the endoplasmic reticulum for secretion (1).
CINC-1
Incubation time did not influence CINC-1 secretion, suggesting that CINC-1 release is immediate and rapid after LPS-induced AM activation. We observed no effect of CO2 on the release of CINC-1 from AM incubated in normal media, suggesting that neither CO2 nor CO2-induced changes in pHe influence CINC-1 secretion in these cells. However, given the pH-mediated effects CO2 had on AM metabolic activity and TNF- secretion, as well as the intrinsic role CO2 is known to play in cellular acid-base status (21), this conclusion seems unlikely. We suggest that CO2 plays a role in regulating CINC-1 release independently of pH, since in buffered media, where the pH was kept constant, increases in CO2 significantly enhanced CINC-1 secretion from AM. In AM incubated in normal media, the stimulatory effect of this non-pH-mediated mechanism may have been counteracted by decreases in metabolic rate. Nevertheless, from a physiological perspective, the ELF is thought to have little buffering capacity (26). Hence, normal in vivo patterns of CINC-1 secretion from AM in response to hypercapnia are more likely to mimic those of cells grown in normal culture media where high CO2 has no effect on CINC-1 secretion. However, since increases in CINC-1 could lead to an accumulation of inflammatory cells into the lung, our observations in buffered media could explain, at least in part, the reduction in the protective effects of hypercapnic acidosis with buffering, previously observed in vivo (15).
Possible Mechanisms for CO2-Mediated Cytokine Responses in AM
The effects of CO2 on TNF- secretion in AM were abated when the media were buffered, indicating that the effects of CO2 on TNF-
are, at least in part, pH mediated. Both TNF-
and IL-1
production in LPS-activated monocytes have been reported to increase in concert with a rise in intracellular pH (pHi), mediated via the influx of Na+ by Na+/H+ ion exchangers (27). Similarly, hypoxia-induced decreases in pHi are associated with enhanced activation of NF-
B and increased expression of intracellular adhesion molecule (35). Hypercapnia, probably via changes in cellular acid-base status (2), increases nitric oxide production and the nitration of proteins (20). Furthermore, hypercapnic acidosis can attenuate LPS-stimulated NF-
B activation and the lung's inflammatory response by the inhibition of I
B-
degradation (32) and endogenous xanthine oxidase (30), respectively. Either one, or all, of these mechanisms may contribute to the effects of CO2 described in this study. Further studies should identify more precise mechanisms of CO2-induced responses in primary cultures of rat AM.
In conclusion, this study provides evidence for differential effects of CO2 on TNF- and CINC-1 in rat AM. Buffering experiments demonstrate that cellular acid-base status mediates the effects of CO2 on cytokine secretion in AM, at least in part, by modulating metabolic activity. Because TNF-
is a key cytokine in the initiation of lung inflammation (3), it is possible that the 50% decrease we observed in TNF-
secretion in response to high CO2 could lead to reductions in lung injury in ventilated patients. However, caution must be taken before concluding that hypercapnia is beneficial in ALI, because the effects of CO2 on inflammatory mediators are differential and may differ between lung cell types. In AM, the increase in CINC-1 in response to CO2, albeit in the presence of high bicarbonate, may be cause for concern. Moreover, many other regulatory mechanisms and cell interactions occur in vivo that may modify the AM responses we observed here in vitro.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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