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Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage Naizhen Wang,* Khalilah L. Gates,* Humberto Trejo,* Silvio Favoreto, Jr.,† Robert P. Schleimer,† Jacob I. Sznajder,* Greg J. Beitel,‡ and Peter H. S. Sporn*,§,1 *Division of Pulmonary and Critical Care Medicine and †Division of Allergy-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA; ‡Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois, USA; and §Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, USA Elevated blood and tissue CO2, or hypercapnia, is common in severe lung disease. Patients with hypercapnia often develop lung infections and have an increased risk of death following pneumonia. To explore whether hypercapnia interferes with host defense, we studied the effects of elevated PCO2 on macrophage innate immune responses. In differentiated human THP-1 macrophages and human and mouse alveolar macrophages stimulated with lipopolysaccharide (LPS) and other Toll-like receptor ligands, hypercapnia inhibited expression of tumor necrosis factor and interleukin (IL)-6, nuclear factor (NF)-Bdependent cytokines critical for antimicrobial host defense. Inhibition of IL-6 expression by hypercapnia was concentration dependent, rapid, reversible, and independent of extracellular and intracellular acidosis. In contrast, hypercapnia did not down-regulate IL-10 or interferon-, which do not require NF-B. Notably, hypercapnia did not affect LPS-induced degradation of IB␣, nuclear translocation of RelA/p65, or activation of mitogen-activated protein kinases, but it did block IL-6 promoter-driven luciferase activity in mouse RAW 264.7 macrophages. Elevated PCO2 also decreased phagocytosis of opsonized polystyrene beads and heat-killed bacteria in THP-1 and human alveolar macrophages. By interfering with essential innate immune functions in the macrophage, hypercapnia may cause a previously unrecognized defect in resistance to pulmonary infection in patients with advanced lung disease.—Wang, N., Gates, K. L., Trejo, H., Favoreto, Jr., S., Schleimer, R. P., Sznajder, J. I., Beitel, G. J., Sporn, P. H. S. Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage. FASEB J. 24, 2178 –2190 (2010). www.fasebj.org ABSTRACT
Key Words: monocytes 䡠 lipopolysaccharide 䡠 cytokines 䡠 phagocytosis 䡠 lung
Elevation of the partial pressure of CO2 (PCO2) in blood and tissue, or hypercapnia, commonly occurs in patients with severe lung disease. Hypercapnia most fre2178
quently occurs as a persistent abnormality in advanced chronic obstructive pulmonary disease (COPD), which affects ⬎20 million people and is the fourth leading cause of death in the United States (1). Elevations in blood PCO2 also may develop in patients with acute respiratory distress syndrome (ARDS), for which respiratory support is typically provided using mechanical ventilation with low tidal volumes, often resulting in “permissive hypercapnia” (2). Multiple reports indicate that hypercapnia predicts an increased risk of death following hospitalization for an acute exacerbation of COPD (3– 6). Up to half of such exacerbations are caused by bacterial infection of the airways (7). Hypercapnia has also been identified as an independent risk factor for mortality in patients hospitalized with community-acquired pneumonia (8) and in patients with cystic fibrosis awaiting lung transplantation (9). In addition, patients with acute respiratory failure and hypercapnia frequently develop ventilator-associated pneumonia, a complication that increases mortality in those who require endotracheal intubation and mechanical ventilation (10). Taken together, these observations suggest that hypercapnia may increase susceptibility to and/or worsen outcomes of infection in patients with severe acute and chronic lung disease. To explore the possibility that hypercapnia interferes with host defense, we investigated the effects of elevated PCO2 on macrophage innate immune responses. Our data show that in macrophages stimulated with lipopolysaccharide (LPS) and other Toll-like receptor (TLR) ligands, normoxic hypercapnia inhibited expression of the nuclear factor (NF)-B-dependent cytokines, tumor necrosis factor (TNF), and interleukin (IL)-6, which play well-documented roles in antimicrobial host defense (11–22). Inhibition of TNF and IL-6 expression was selective, as hypercapnia did not affect LPS induction of IL-10 or interferon (IFN)-. Further1 Correspondence: Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron St., McGaw M-300, Chicago, IL 60611, USA. E-mail:
[email protected] doi: 10.1096/fj.09-136895
0892-6638/10/0024-2178 © FASEB
more, hypercapnia acted in a rapid and reversible manner, independently of extracellular and intracellular acidosis, and by inhibiting transcription without affecting NF-B activation. In addition, elevated CO2 inhibited phagocytosis by macrophages. By interfering with macrophage functions essential for antimicrobial host defense, hypercapnia may cause a previously unrecognized defect in resistance to pulmonary infection in patients with severe lung disease.
included TLR1/2, Pam3CSK4 (1 g/ml); TLR2, lyophilized heat-killed Listeria monocytogenes (1⫻108 bacteria/ml); TLR3, Poly(I:C) (25 g/ml); TLR5, flagellin (1 g/ml); TLR6/2, FSL-1 (1 g/ml); TLR7, Imiquimod (1 g/ml); TLR8, ssRNA40 (1 g/ml); and TLR9, ODN2006 (5 M).
MATERIALS AND METHODS
RNA isolation and quantitative real-time-PCR (qPCR)
Macrophages
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was reverse-transcribed to cDNA using MultiScribe™ MuLV reverse transcriptase (Applied Biosystems, Foster City, CA, USA). Specific mRNAs were quantitated by real-time PCR using a 7500 Fast Real-Time PCR system (Applied Biosystems). Reaction mixtures contained 100 ng of reverse-transcribed cDNA, TaqMan universal master mix, gene-specific primers, and the FAM-labeled MGB probe for each individual gene (Applied Biosystems). Amplification parameters were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s alternating with 60°C for 1 min. Expression of the housekeeping gene, eukaryotic translation elongation factor-1␣1 (EEF1A1), was used as a reference, and the fold change of target genes was calculated by the ⌬⌬CT method.
Human monocytic leukemia THP-1 cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured at 1 ⫻ 106 cells/ml in RPMI 1640 [supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM lglutamine, 1 mM sodium pyruvate, 20 M -mercaptoethanol, 100 U/ml penicillin, and 100 g/ml streptomycin]. THP-1 cells were differentiated by exposure to 5 nM phorbol myristate acetate (PMA) for 48 h. Mouse monocyte-macrophage RAW 264.7 cells (ATCC) were cultured at 5 ⫻ 105 cells/ml in DMEM (supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 g/ml streptomycin). Mouse alveolar macrophages were obtained from 8- to 9-wkold C57BL/6 mice by whole lung lavage via a tracheostomy, following sacrifice of the animal. Human alveolar macrophages were obtained by bronchoalveolar lavage from an uninvolved area of lung in subjects undergoing bronchoscopy for clinical diagnosis of noninfectious focal lung lesions. The study protocols were approved by the Northwestern University Animal Care and Use Committee and Institutional Review Board; all human subjects provided informed consent. Human and mouse alveolar macrophages (both ⱖ98% pure, following adherence to plastic and removal of nonadherent cells) were cultured at 0.75 ⫻ 105 and 0.5 ⫻ 105 cells/ml, respectively, in RPMI 1640 with supplements as above. Normocapnia and hypercapnia exposures Normocapnia exposures were carried out under standard incubator conditions, in a humidified atmosphere of 5% CO2 (PCO2 36 mmHg)/95% air at 37°C. Hypercapnia exposures were carried out by incubation in humidified atmospheres of 9% CO2 (PCO2 64 mmHg), 12.5% CO2 (PCO2 88 mmHg), or 20% CO2 (PCO2 140 mmHg), in each case with 21% O2 and the balance N2, at 37°C. Culture medium was presaturated with CO2 by incubation in the appropriate gas mixture overnight, then added to cell cultures as indicated in each experiment. Incubator CO2 concentrations were maintained using a PRO-CO2 carbon dioxide controller (BioSpherix, Lacona, NY, USA). We measured pH, PCO2, and PO2 of culture medium with a pHOx Plus blood gas analyzer (Nova Biomedical, Waltham, MA, USA). Stimulation with LPS and other TLR ligands Cells were quiesced by incubation overnight in RPMI 1640 containing 0.5% FBS prior to stimulation with LPS or other TLR ligands, which were added to cultures in medium presaturated with CO2 at the desired concentration. Ultra Pure Escherichia coli K12 LPS (InvivoGen, San Diego, CA, USA) was added to cells at a final concentration of 1 or 100 ng/ml, as indicated. Other TLR ligands (all from InvivoGen) CO2 INHIBITS MACROPHAGE INNATE IMMUNE RESPONSES
Cytotoxicity assay Cytotoxicity was determined by measurement of LDH release using the Cytotoxicity Detection Kit (Roche, Indianapolis, IN, USA).
Cytokine assays Cytokines secreted from cultured THP-1 cells, and human or mouse alveolar macrophages were assayed by cytometric bead array (CBA) assay (BD Biosciences, San Jose, CA, USA). Determination of nitrite production RAW 264.7 macrophages were stimulated with LPS in the absence or presence of the nitric oxide (NO) synthase inhibitor, N-nitro-l-arginine methyl ester hydrochloride (LNAME, 2 mM; Sigma, St. Louis, MO, USA). Supernatants were collected at 0, 3, 6, and 24 h and assayed for nitrite, as an index of NO production, using the Griess Reagent Kit (Invitrogen, Carlsbad, CA, USA). Measurement of intracellular pH (pHi) THP-1 cells cultured and differentiated with PMA on glass coverslips were loaded with 1 M 2⬘7⬘-bis-(carboxyethyl)-5,6carboxyfluorescein (BCECF)/acetoxymethyl ester (Invitrogen) for 30 min at 37°C. After washing, coverslips were placed in an environmental chamber for live cell imaging and perfused with equilibrated medium at 37°C. BCECF fluorescence was monitored continuously, with an exposure time of 10 s, using a TE2000 inverted fluorescence microscope (Nikon Instruments, Tokyo, Japan) with dual excitation at 500 and 440 nm and emission at 520 nm, as described previously (23). Cells were permeabilized with nigericin to calibrate pHi at the end of each experiment. Determination of cell surface expression of TLR4 and CD14 PMA-differentiated THP-1 cells were exposed to 5 or 20% CO2 for 24 h, then dissociated, blocked in 3% BSA for 30 min, stained with FITC-conjugated anti-TLR4 (Imgenex, San Di2179
ego, CA, USA) or PE-conjugated anti-CD14 (Beckman, Fullerton, CA, USA) antibodies for 30 min, and fixed in 1% paraformaldehyde (PFA). Cell surface expression of TLR4 and CD14 was quantified as the mean fluorescence intensity of the FITC- and PE-positive cells, respectively, using a LSRII flow cytometer (BD Biosciences).
Phagocytosis of polystyrene microspheres by human alveolar macrophages was also assessed by confocal microscopy using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Oberkochen, Germany); these cells were also stained with Alexa Fluor 488-phalloidin (Invitrogen) for filamentous actin (F-actin) and with Hoechst 33342 (Invitrogen) for cell nuclei.
Immunoblotting
Statistics
Whole-cell lysates were prepared in RIPA buffer supplemented with PMSF, sodium orthovanadate, and Complete Protease Inhibitor Cocktail (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Nuclear and cytoplasmic protein fractions were obtained using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). Proteins were separated in 4 –20% gradient gels and transferred to nitrocellulose. Blots were probed with primary antibodies against IB␣, RelA/p65, or -tubulin (all from Santa Cruz Biotechnology); lamin B1 (Zymed Laboratories, San Francisco, CA, USA) or -actin (Abcam, Cambridge, MA, USA); or phosphorylated and total extracellular signal-regulated kinase (ERK), p38 or c-Jun N-terminal kinase (JNK) (all from Cell Signaling Technology, Danvers, MA, USA). Following incubation with appropriate HRP-conjugated secondary antibodies, SuperSignal West Dura Substrate (Thermo Scientific, Rockford, IL, USA) was added, and bands were detected by exposure on X-ray film.
Data are presented as means ⫾ sd. Differences between group means were analyzed by the unpaired Student’s t test or by 1-way ANOVA with the Student-Neuman-Keuls multiple comparison test, as appropriate. Significance was determined at the P ⬍ 0.05 level. Statistical analysis was performed using GraphPad Prism 4 software (GraphPad, San Diego, CA, USA).
Construction of IL-6 promoter-luciferase reporter and luciferase activity assay The mouse IL-6 promoter (⫺2000 to ⫹31) was amplified by PCR using mouse genomic DNA as template and the primers 5⬘-CGGGGTACCACTTAAAAAGCACCTTTTTTAA-3⬘ and 5⬘ATTCTCGAGAGCGGTTTCTGGAATTGACTAT-3⬘. The PCR product was cloned into the Acc65I and XhoI sites of pGL4.10 (Promega, Madison, WI, USA) to create the mouse IL-6 promoter-luciferase reporter, pGL4.10-IL-6/luc. RAW 264.7 cells were transiently transfected with pGL4.10-IL-6/luc (1 g/106 cells) using FuGENE HD (Roche). Cells were cotransfected with the Renilla luciferase reporter plasmid pGL4.74hRLuc/TK (Promega) (50 ng/106 cells) as an internal control to normalize for transfection efficiency. Twenty-four hours after transfection, the cells were stimulated with LPS (1 ng/ml or 100 ng/ml) under normocapnic (5% CO2) or hypercapnic (20% CO2) conditions. After 6 h, cells were harvested, lysed, and analyzed using the Dual-Luciferase® Reporter Assay System (Promega). Phagocytosis assay Differentiated THP-1 macrophages or human alveolar macrophages were cultured in 5 or 20% CO2 overnight. Red fluorescent 1.0 m polystyrene microspheres (FluoSpheres; Invitrogen) or Alexa Fluor 488-conjugated heat-killed Staphylococcus aureus (S. aureus BioParticles; Invitrogen) were opsonized with 50% human AB serum (Mediatech, Manassas, VA, USA) for 30 min at 37°C. Opsonized microspheres or heatkilled S. aureus were incubated with macrophages in normocapnia or hypercapnia for 1 h at 37°C. Noninternalized beads or bacteria were quenched using 0.25 mg/ml trypan blue. Cells were washed with cold PBS and fixed in 1% PFA. Phagocytosis of polystyrene microspheres was quantified by flow cytometry. For each condition, the phagocytic index was determined by multiplying the percentage of cells containing beads by the mean fluorescence of all cells containing beads. Phagocytosis of polystyrene microspheres and heat-killed bacteria was also assessed microscopically in adherent THP-1 cells using a Nikon TE200 inverted fluorescence microscope. 2180
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RESULTS Hypercapnia inhibits TNF and IL-6 expression in macrophages stimulated with LPS To examine the effect of hypercapnia on macrophage innate immune responses, we first utilized THP-1 cells differentiated by exposure for 48 h to a low concentration of PMA. THP-1 cells differentiated in this manner acquire a mature macrophage phenotype, with highlevel expression of CD14 and TLR4, and robust induction of inflammatory cytokines on subsequent stimulation with LPS (24, 25). As expected, in differentiated THP-1 cells cultured under normocapnic conditions (5% CO2, PCO2 36 mmHg), LPS markedly up-regulated expression of TNF and IL-6 mRNA (Fig. 1A, B) and protein (Fig. 1C, D). However, when THP-1 macrophages were stimulated with LPS under hypercapnic conditions (20% CO2, PCO2 140 mmHg), induction of TNF and IL-6 mRNA (Fig. 1A, B) and protein (Fig. 1C, D) was significantly attenuated. Suppression of TNF and IL-6 by hypercapnia did not result from reduced cell viability, because exposure of THP-1 cells to 10 or 20% CO2, with or without LPS, caused no measurable cytotoxicity, even after 24 h (Supplemental Table 1). Similarly, 20% CO2 inhibited LPS-induced TNF and IL-6 synthesis in primary human (Fig. 2A, B) and mouse (Fig. 2C, D) alveolar macrophages, and IL-6 mRNA expression in murine RAW 264.7 macrophages (Supplemental Fig. 1B). We next determined the effect of lower levels of hypercapnia on LPS-induced cytokine expression. As shown in Fig. 3A, hypercapnia reduced IL-6 mRNA levels in a concentration-dependent manner. In particular, the highly significant inhibition of IL-6 expression at 9% CO2 (PCO2 64 mmHg) and 12.5% CO2 (PCO2 88 mmHg), well within the range of PCO2 commonly seen in patients with severe COPD and other lung diseases (6, 26), indicates that hypercapnia suppresses cytokine expression at CO2 levels that are clinically relevant. As noted, cells were exposed to hypercapnia in the above experiments in an atmosphere that contained 21% O2. Nevertheless, to assess whether elevated CO2 might act via a pathway intersecting with the hypoxia response, we determined the effect of hypercapnia on
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Figure 1. Hypercapnia inhibits LPS-induced TNF and IL-6 mRNA and protein expression in THP-1 macrophages. PMA-differentiated THP-1 macrophages were cultured under normocapnic conditions, then stimulated with LPS (1 ng/ml), or exposed to vehicle alone, in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (20% CO2, PCO2 140 mmHg). For cells stimulated in hypercapnia, LPS or vehicle was added in culture medium that had been preequilibrated overnight in 20% CO2/21% O2/69% N2. A, B) Cells were harvested at 3 h for RNA isolation. C, D) Supernatants were collected at 4, 6, and 8 h for analysis of cytokine secretion. TNF and IL-6 mRNAs were analyzed by qPCR, and cytokine levels in supernatants were determined by CBA (n⫽3 for all). *P ⬍ 0.01, †P ⬍ 0.001 vs. 5% CO2.
expression of mRNA for vascular endothelial growth factor (VEGF), a prototypical hypoxia-responsive gene (27). We found that hypercapnia had no effect on VEGF mRNA levels (Supplemental Fig. 2A). Likewise, hypercapnia did not increase mRNA for the heat shock protein, Hsp70 (Supplemental Fig. 2B). These results indicate that inhibition of cytokine expression by elevated CO2 involves a pathway distinct from those involved in responses to hypoxia and heat stress. In the experiments described so far, macrophages exposed to hypercapnia were cultured under normocapnic conditions until just prior to stimulation, at which point culture medium that had been preequilibrated in hypercapnia at the indicated CO2 level was placed on the cells, LPS was added, and the culture plates were immediately placed in an incubator chamber containing the same concentration of CO2. To
determine whether exposure of the cells to hypercapnia prior to LPS stimulation might result in different effects on cytokine expression than simple acute exposure to elevated CO2, we incubated cells under hypercapnic conditions for various times prior to addition of LPS. Figure 3B shows that preexposure of THP-1 cells to 20% CO2 for up to 24 h resulted in no greater inhibition of IL-6 secretion than when cells were stimulated with LPS immediately after addition of medium preequilibrated with 20% CO2. Having thus established that hypercapnia’s effect on IL-6 expression was rapid in onset, we next tested the reversibility of this inhibition. Figure 3C shows that when THP-1 cells were incubated in 20% CO2 for 18 h, then stimulated with LPS in 5% CO2, inhibition of IL-6 mRNA expression by hypercapnia was completely lost. The reversibility of hypercapnia’s effect confirms that elevated CO2 does
Figure 2. Hypercapnia inhibits LPS-induced TNF and IL-6 protein expression in human and mouse alveolar macrophages. Human and mouse alveolar macrophages were cultured under normocapnic conditions, then stimulated with LPS (1 ng/ml) in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (20% CO2, PCO2 140 mmHg). For macrophages stimulated in hypercapnia, LPS or vehicle was added in culture medium that had been preequilibrated overnight in 20% CO2/21% O2/69% N2. Supernatants were collected at 6 h, and TNF and IL-6 levels were determined by CBA. A, B) Human alveolar macrophages (n⫽3). C, D) Mouse alveolar macrophages (n⫽3). *P ⬍ 0.001, †P ⬍ 0.05, ‡P ⬍ 0.01 vs. 5% CO2. CO2 INHIBITS MACROPHAGE INNATE IMMUNE RESPONSES
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Figure 3. Inhibition of LPS-induced IL-6 secretion by hypercapnia is concentration dependent, rapid, and reversible. A) PMA-differentiated THP-1 macrophages were cultured under normocapnic conditions, then stimulated with LPS (1 ng/ml), or exposed to vehicle alone, in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia at 9% CO2 (PCO2 64 mmHg), 12.5% CO2 (PCO2 88 mmHg), or 20% CO2 (PCO2 140 mmHg). For cells stimulated in hypercapnia, LPS or vehicle was added in culture medium that had been preequilibrated at the indicated CO2 concentration. At 3 h after LPS stimulation, cells were harvested for isolation of RNA, which was subsequently analyzed by qPCR (n⫽3). B) PMA-differentiated THP-1 macrophages were cultured in 5% CO2 (open bar) or for 0, 1, 4, or 24 h in 20% CO2 (solid bars), and then stimulated with LPS (1 ng/ml) in normocapnia or hypercapnia, respectively. For cells stimulated in hypercapnia, LPS was added in culture medium that had been preequilibrated in 20% CO2. At 6 h after LPS stimulation, supernatants were collected for determination of IL-6 by CBA (n⫽2). C) PMA-differentiated THP-1 cells were incubated for 18 h in 5% CO2 (open and shaded bars) or 20% CO2 (solid bar), then stimulated with LPS (1 ng/ml). LPS was added in medium preequilibrated with 5% CO2 (open and solid bars) or 20% CO2 (shaded bar), after which cultures were incubated at the corresponding CO2 concentration, as indicated. At 3 h after LPS stimulation, cells were harvested for isolation of RNA, which was subsequently analyzed by qPCR (n⫽3). *P ⬍ 0.001 vs. 5% CO2; †P ⬍ 0.001 vs. other treatments.
not inhibit IL-6 by altering cell viability. Further, the rapid onset and reversibility of IL-6 inhibition suggest that hypercapnia may act by triggering reversible posttranslational modifications of key proteins that regulate LPS-mediated cytokine responses. Hypercapnia inhibits IL-6 expression independently of extracellular and intracellular acidosis Because CO2/HCO3⫺ is the major buffer system in RPMI 1640 (as in extracellular fluids in vivo), exposure of cells to increased PCO2 is accompanied by reduced pH in the culture medium. We therefore asked whether the effect of hypercapnia on IL-6 expression could be attributed to extracellular acidosis. Figure 4A shows that addition of 25 mM NaOH to the culture medium [to prevent the fall in extracellular pH (pHe) otherwise associated with exposure to 12.5% CO2] provided no protection against the hypercapnia-induced decrease in LPS-induced IL-6 mRNA expression. (These experiments utilized 12.5% CO2 because excessive amounts of NaOH, or other buffers not tolerated by the cells, would be required to buffer the acidosis associated with higher levels of CO2.) This finding indicates that hypercapnia inhibits cytokine mRNA expression by a mechanism independent of extracellular acidosis. On the other hand, acidification of culture medium with increasing concentrations of HCl also caused a progressive decrease in LPS-stimulated IL-6 mRNA expression (Fig. 4B), raising the possibility that hypercapnia could inhibit IL-6 expression by a second mechanism dependent on extracellular acidosis as well. To gain further insight into the role of reduced pH in mediating the effect of hypercapnia on IL-6, we 2182
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determined the effects of elevated CO2, and of acidification of cell culture medium with HCl, on pHi in THP-1 cells loaded with the pH-sensitive dye, BCECF. As shown in Fig. 4C, switching cells from normocapnic to hypercapnic medium without buffering (12.5% CO2, pHe 7.26) caused pHi to decrease from ⬃7.2 to ⬃7.0 within 2 min, after which it began to gradually increase. When cells were switched from normocapnic to hypercapnic medium buffered with NaOH (12.5% CO2, pHe 7.46), again pHi decreased to ⬃7.0, but in this case, it increased more quickly, returning fully to baseline (⬃7.2) within 15 min. It should be noted that the unbuffered and buffered hypercapnia conditions used here to assess changes in pHi are identical to the hypercapnia conditions that resulted in ⬎50% reductions in IL-6 expression in Fig. 4A. Notably, in contrast to both hypercapnia conditions, switching from normocapnic medium at normal pH (5% CO2, pHe 7.48) to normocapnic medium acidified with HCl (5% CO2, pHe 7.29) resulted in a much greater decline in pHi to ⬃6.5, which persisted with little change for the duration of the experiment. These results are similar to the effects of hypercapnia and normocapnic acidosis on pHi we recently reported in alveolar epithelial cells (23, 28). Of note, the hypercapnia-induced decrease in pHi from 7.2 to 7.0 seen in THP-1 cells represents a 60% increase in [H⫹], which was transient, whereas the decrease in pHi from 7.2 to 6.5 associated with normocapnic acidosis represents a 500% increase in [H⫹], without attenuation over time. The small degree and transient nature of the reductions in pHi caused by hypercapnia, in contrast to the much larger and sustained fall in pHi resulting from normocapnic acidosis,
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Figure 4. Hypercapnia inhibits IL-6 expression independently of extracellular and intracellular acidosis. A, B) PMA-differentiated THP-1 macrophages were stimulated with LPS (1 ng/ml) in normocapnia (5% CO2) or hypercapnia (12.5 or 20% CO2) in RPMI 1640 without additions, or with added NaCl, NaOH, or HCl at the indicated concentrations. At 3 h after LPS stimulation, cells were harvested for isolation of RNA, which was subsequently analyzed by qPCR (n⫽3). C) PMA-differentiated THP-1 macrophages adhered to glass coverslips were loaded with BCECF, washed, and perfused with RPMI 1640 containing 5% CO2. At time 0, perfusion medium was changed to 12.5% CO2-preequilibrated RPMI 1640 without additions (pHe 7.26), 12.5% CO2-preequilibrated RPMI 1640 containing 25 mM NaOH (pHe 7.46), or 5% CO2-preequilibrated RPMI 1640 containing 15 mM HCl (pHe 7.29), as indicated. pHi was monitored continuously by fluorescence microscopy. Data are shown for a single cell under each condition and are representative of measurements on 10 cells in each of 4 –7 experiments/condition. *P ⬍ 0.001 vs. 5% CO2; †P ⬍ 0.001 vs. 5% CO2 ⫹ 25 mM NaCl; ‡P ⬍ 0.001 vs. 5% CO2 ⫹ 20 mM HCl; §P ⬍ 0.001 vs. 5% CO2 and 5% CO2 ⫹ 20 mM NaCl.
suggest that hypercapnic suppression of IL-6 does not result from intracellular acidosis. This conclusion is reinforced by the fact that preexposure of THP-1 cells to hypercapnia for up to 24 h, by which time pHi should long since have recovered to baseline, inhibited LPSinduced IL-6 synthesis to an identical degree as acute exposure to hypercapnia initiated at the same time as LPS stimulation (Fig. 3B). On the other hand, the finding that normocapnic acidosis caused a much greater and persistent decline in pHi than did hypercapnia suggests that acidification of culture medium with HCl inhibits IL-6 expression by a mechanism related to intracellular acidosis and distinct from that mediating the effect of elevated CO2. Hypercapnia inhibits IL-6 expression mediated by activation of multiple TLRs but does not affect LPS-induced IL-10 or IFN- We next sought to define the locus of hypercapnia’s inhibitory effect on LPS-stimulated cytokine expresCO2 INHIBITS MACROPHAGE INNATE IMMUNE RESPONSES
sion. As assessed by flow cytometry, hypercapnia did not affect surface expression of TLR4 or CD14 on differentiated THP-1 cells (Fig. 5A). At the same time, 20% CO2 inhibited IL-6 synthesis induced not only by LPS, but also by ligands for TLR1/2, TLR5, and TLR6/2 (Fig. 5B). Thus, IL-6 inhibition by hypercapnia was not specific to LPS activation of TLR4. In addition, the fact that LPS-induced IL-6 and TNF expression requires canonical NF-B signaling (29, 30), and that hypercapnia decreased IL-6 expression triggered by ligands for multiple TLRs that all signal through a common pathway leading to activation of NF-B (31), suggested that elevated CO2 might interfere with cytokine expression by inhibiting one or more steps in the TLR-NF-B pathway. To gain insight into whether NF-B might be a key target of elevated CO2, we examined the effects of hypercapnia on expression of IL-10 and IFN-, cytokines stimulated by LPS via pathways independent of canonical NF-B signaling (32, 33). Notably, hypercapnia had no inhibitory effect on LPS-induced expression of either IL-10 (Fig. 5C) or IFN- (Fig. 5D). This 2183
Figure 5. Hypercapnia inhibits IL-6 expression triggered by multiple TLRs but does not block LPS induction of IL-10 or IFN-. A) PMA-differentiated THP-1 macrophages were exposed to 5 or 20% CO2 for 18 h, stained with FITC-conjugated anti-TLR4 and PE-conjugated anti-CD14 antibodies, and TLR4 and CD14 cell surface expression were determined by flow cytometry. B) THP-1 macrophages were stimulated with ligands for TLRs in 5 or 20% CO2, and 6 h later supernatants were collected and analyzed for IL-6 by CBA (n⫽2). *P ⬍ 0.05 vs. 5% CO2. C, D) THP-1 macrophages (2⫻106 cells/well) were stimulated with LPS (1 ng/ml) in 5 or 20% CO2, and 3 h later cells were harvested for isolation of RNA, which was subsequently analyzed for IL-10 and IFN- mRNAs by qPCR (n⫽3).
confirmed that hypercapnia alters gene expression in a specific, rather than a generalized, manner and was consistent with the possibility that hypercapnia decreases LPS-induced TNF and IL-6 expression by inhibiting activation of NF-B. Hypercapnia does not block degradation of IB␣, nuclear translocation of RelA/p65, or activation of MAP kinases Next, we specifically assessed whether hypercapnia interferes with activation of NF-B in stimulated macrophages. First, we examined the effect of hypercapnia on proteasomal degradation of IB␣, which is triggered by IB kinase (IKK)-mediated phosphorylation of IB␣ following LPS stimulation (34). As shown in Fig. 6A, under normocapnic conditions, LPS triggered the expected degradation of IB␣, followed by recovery of expression within 60 min, and hypercapnia had no effect on this process. Accompanying degradation of IB␣, RelA/p65 translocated in time-dependent fashion from the cytosol to the nucleus, which was also unaffected by hypercapnia (Fig. 6B). Thus, hypercapnia does not block the signaling steps downstream of TLRs leading to activation of IKK, IB␣ degradation and nuclear accumulation of RelA/p65. The critical targets me2184
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diating hypercapnic inhibition of TNF and IL-6 must therefore be downstream of these steps in NF-B activation, or part of another regulatory pathway. Besides NF-B, mitogen-activated protein (MAP) kinases have been shown to play a role in activating TNF and IL-6 gene transcription (35–37). Therefore, we examined the effects of hypercapnia on activation of ERK, p38, and JNK in THP-1 macrophages stimulated with LPS. Figure 6C shows that LPS caused timedependent phosphorylation of ERK, p38, and JNK, in each case peaking at 20 min, and that there was no difference in the degree or timing of MAP kinase activation in hypercapnic vs. normocapnic culture conditions. Thus, hypercapnia does not inhibit induction of TNF and IL-6 gene expression by interfering with LPS-induced MAP kinase activation. It was previously reported that culture of macrophages in 5% CO2, as compared to air (0% CO2), enhanced LPS-stimulated NO production and increased tyrosine nitration of surfactant proteins (38). We therefore considered the possibility that hypercapnia might increase NO production, leading to nitration and inactivation of NF-B or other proteins required for TNF or IL-6 gene expression. To test this, we measured nitrite, as an index of NO production, in murine RAW 264.7 macrophages following stimulation with LPS in 5 and
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Figure 6. Hypercapnia does not affect LPS-stimulated IB␣ degradation, RelA/p65 nuclear translocation or MAP kinase activation. PMA-differentiated THP-1 macrophages were stimulated with LPS (100 ng/ml) in 5 or 20% CO2, after which cells were harvested at the indicated times. A) Whole-cell lysates were analyzed by immunoblot for total cellular IB␣. B) Alternatively, cytoplasmic and nuclear fractions were prepared and immunoblotted for RelA/p65, with -tubulin and Lamin B1 as cytoplasmic and nuclear loading controls, respectively. C) Whole-cell lysates were analyzed by immunoblot for phosphorylated and total ERK, p38, and JNK. Immunoblots are representative of 3 experiments with similar results.
20% CO2. As expected, LPS induced a large increase in nitrite production under normocapnic conditions, but this was not affected by hypercapnia (Supplemental Fig. 1A). In addition, the increase in nitrite production was only detectable 24 h after LPS stimulation, but not at 3 or 6 h, times at which hypercapnia decreased expression of TNF and IL-6 mRNA and protein, respectively (Figs. 1–3 and Supplemental Fig. 1B). Furthermore, the NO synthase inhibitor L-NAME did not block the hypercapnia-induced decrease in IL-6 mRNA expression (Supplemental Fig. 1B), even though it eliminated LPS-induced NO production completely (Supplemental Fig. 1C). Taken together, these results indicate that NO does not mediate the inhibitory effect of hypercapnia on macrophage TNF and IL-6 expression. Hypercapnia decreases IL-6 promoter-driven luciferase activity and does not affect IL-6 mRNA stability The finding that hypercapnia did not affect activation of either NF-B or MAP kinases led to the question whether elevated CO2 reduced TNF and IL-6 expression by inhibiting gene transcription or by a posttranscriptional mechanism. To address this, we constructed a mouse IL-6 promoter-luciferase reporter plasmid, pGL4.10-IL-6/luc, which we used to transiently transfect RAW 264.7 macrophages. RAW 264.7 macrophages were used because hypercapnia inhibited IL-6 expression in these cells (Supplemental Fig. 1B), as in THP-1 cells and human and mouse alveolar macrophages, but unlike THP-1 cells and primary macrophages, RAW 264.7 cells are easy to transfect with high efficiency. As shown in Fig. 7A, LPS dose-dependently CO2 INHIBITS MACROPHAGE INNATE IMMUNE RESPONSES
stimulated IL-6 promoter-driven luciferase activity in cells transfected with pGL4.10-IL-6/luc, and this activity was significantly inhibited by culture in 20% as compared to 5% CO2. This finding indicates that hypercapnia inhibits gene transcription driven by the IL-6 promoter. To determine whether hypercapnia might also inhibit IL-6 expression by a post-transcriptional mechanism affecting mRNA stability, we stimulated differentiated THP-1 macrophages with LPS in normocapnia, added actinomycin D (Act D), and assessed IL-6 mRNA levels in the presence of 5 or 20% CO2 over time. Figure 7B shows that IL-6 mRNA levels declined at identical rates in 5 and 20% CO2, indicating that hypercapnia does not alter IL-6 mRNA degradation. The lack of an effect of elevated CO2 on IL-6 mRNA half-life is consistent with the finding that hypercapnia did not alter activation of p38 or ERK, which are known to influence TNF and IL-6 expression by regulating the stability of their respective mRNAs (39, 40). Hypercapnia inhibits phagocytosis Phagocytosis is a fundamental macrophage function that is critical for host defense against microbial pathogens. TLR4- and TLR2-dependent signaling is essential for phagocytosis of bacteria and phagosome maturation in macrophages (41). Furthermore, it has recently been shown that activation of NF-B is required for bacterial phagocytosis in a murine macrophage cell line (42). Because our data indicate that hypercapnia inhibits transcription of NF-B-regulated genes (although apparently not by inhibiting NF-B itself), we examined whether hypercapnia also interfered with phagocytosis. Indeed, compared to normocapnia, 20% CO2 caused 2185
Figure 7. Hypercapnia inhibits IL-6 promoter-driven luciferase activity but does not alter IL-6 mRNA degradation. A) RAW 264.7 cells were transfected with pGL4.10-IL-6/luc and pGL4.74-hRLuc/TK (1 g and 50 ng/106 cells/well, respectively) and 24 h later stimulated with LPS (1 or 100 ng/ml) or medium alone in 5 or 20% CO2. After 6 h, cells were harvested and assayed for firefly luciferase and Renilla luciferase activities. Normalized firefly luciferase activity for each condition is represented as fold-change relative to unstimulated normocapnic cells (n⫽3). *P ⬍ 0.05 vs. 5% CO2. B) PMA-differentiated THP-1 macrophages were stimulated with LPS (1 ng/ml) in 5% CO2 for 3 h, at which point Act D (5 g/ml) was added, and the cells were either maintained in 5% CO2 or switched to 20% CO2. Cells were harvested at the indicated times for isolation of RNA, which was subsequently analyzed by qPCR. Amount of IL-6 mRNA remaining at each time is expressed as a percentage of the quantity of IL-6 mRNA present at time 0, when Act D was added (n⫽3).
⬎40% reduction in ingestion of opsonized polystyrene beads (Fig. 8A, B) and similarly reduced uptake of heat-killed S. aureus (Fig. 8C) in THP-1 cells. Hypercapnia also inhibited phagocytosis of opsonized polystyrene beads by human alveolar macrophages, shown in Fig. 8D (5% CO2: 13.8⫾2.2 beads/cell, n⫽42 cells; 20% CO2: 7.0⫾0.5 beads/cell, n⫽52 cells; P⬍0.01). Because phagocytosis requires remodeling of the actin cytoskeleton (43), we also stained these cells with Alexa Fluor 488-phalloidin to visualize F-actin. Confocal imaging shows that following phagocytosis, F-actin appeared in a similar predominantly cortical distribution in both 5 and 20% CO2, without obvious differences in staining intensity between the 2 conditions (Fig. 8D). This similarity notwithstanding, the reduction of phagocytosis by elevated CO2 in both differentiated THP-1 cells and primary human alveolar macrophages indicates that, besides cytokine synthesis, hypercapnia inhibits another key macrophage function required for host defense.
DISCUSSION In this report we have documented that hypercapnia inhibits LPS-stimulated macrophage TNF and IL-6 mRNA and protein expression in a rapid, reversible, and concentration-dependent manner, at levels of PCO2 in the same range as seen in patients with acute and chronic lung disease. This inhibition occurred without an effect on macrophage viability, without induction of hypoxia- or heat shock-responsive genes, and indepen2186
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dently of extracellular and intracellular acidosis and of NO signaling. Moreover, hypercapnia inhibited LPSstimulated TNF and IL-6 expression without affecting LPS induction of IL-10 or IFN-, indicating that the effects of elevated CO2 on cytokine expression are selective. This selectivity implies that hypercapnia acts through mechanisms specific to regulation of TNF and IL-6 and not shared by IL-10 or IFN-. NF-B is a key activator of TNF and IL-6 gene transcription (29, 30) that is not required for expression of IL-10 or IFN- (32, 33). Whether hypercapnia might exert its effects on cytokine expression by blocking activation of NF-B was therefore an obvious question. However, we found that hypercapnia had no effect on LPS-induced degradation of IB␣ or nuclear translocation of RelA/p65, indicating that NF-B was not the target of elevated CO2. Hypercapnia also did not block activation of ERK, p38, or JNK MAP kinases, which have been shown to increase TNF and IL-6 transcription through activation of AP-1 (35–37). Despite the fact that hypercapnia did not impair activation of NF-B or the MAP kinases, it did inhibit IL-6 promoter-driven luciferase activity. This finding, in conjunction with the observation that elevated CO2 did not alter the stability of IL-6 mRNA, indicates that hypercapnia inhibits IL-6 gene transcription. The critical targets that mediate hypercapnic inhibition of IL-6 expression may include other transcription factors known to activate IL-6 gene transcription, such as cyclic AMP response element or CCAAT-enhancer binding protein family members (44, 45), as well as transcriptional coactivators or repressors. Alternatively, there may exist a separate CO2 response
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Figure 8. Hypercapnia impairs phagocytosis of polystyrene microspheres and heat-killed S. aureus. PMA-differentiated THP-1 macrophages or human alveolar macrophages were incubated in 5 or 20% CO2, and serum-opsonized red fluorescent FluoSpheres (200 microspheres/cell) or Alexa Fluor 488-conjugated heat-killed S. aureus (50 bacteria/cell) were added. A) After 1 h, THP-1 macrophages incubated with fluorescent microspheres were harvested, and phagocytosis was quantitated by flow cytometry (n⫽4). *P ⬍ 0.05 vs. 5% CO2. B, C) Alternatively, adherent THP-1 macrophages were analyzed by standard fluorescence microscopy to visualize cellular uptake of microspheres (red; B) or labeled bacteria (green; C). D) At 1 h after addition of fluorescent microspheres, adherent human alveolar macrophages were permeabilized and stained with Alexa Fluor 488-phalloidin to visualize F-actin (green) and with Hoechst 33342 for nuclei (blue), and then analyzed by confocal microscopy; the images are 0.26-m sections from the middle of a Z stack spanning the height of the cell monolayer.
factor that, on binding CO2, specifically regulates transcription of IL-6 and other genes. Our findings are consistent with previous reports that hypercapnia inhibited secretion of TNF by mouse peritoneal (46) and rat alveolar (47) macrophages. We have extended these observations by showing that elevated CO2 also inhibits expression of IL-6, but not IL-10 or IFN-; that inhibition of IL-6 expression is rapid, reversible, and independent of extracellular or intracellular acidosis and NO signaling; that hypercapnia does not inhibit activation of NF-B or MAP kinases; and that elevated CO2 inhibits IL-6 promoter activity and does not alter IL-6 mRNA stability. In another study, Takeshita et al. (48) reported that hypercapnia inhibited ICAM-1 and IL-8 expression triggered by LPS in human pulmonary artery endothelial cells. In contrast to our observations, these authors found that hypercapnia inhibited LPS-induced activation of NFB. Thus, hypercapnia appears to affect pulmonary artery endothelial cells differently than macrophages. That the effects of elevated CO2 may differ depending on cell type is further demonstrated by a recent report showing that long-term exposure of malignant colon and lung epithelial cell lines to hypercapnia (without agonist stimulation) actually activated NF-B and increased inflammatory cytokine synthesis (49). TNF and IL-6 are innate immune effectors that play CO2 INHIBITS MACROPHAGE INNATE IMMUNE RESPONSES
important roles in host defense. Both cytokines enhance neutrophil and macrophage phagocytosis and killing of gram-positive and gram-negative bacteria, in part by increasing adhesion molecule expression and oxygen radical generation (11, 12). In murine models of pneumonia caused by Klebsiella, E. coli, or Pseudomonas aeruginosa, bacterial growth was increased and survival decreased by administration of a TNF-receptor blocking antibody (14) and in TNF gene-deficient animals (16). Conversely, administration of recombinant TNF (13) or gene therapy to augment endogenous TNF expression (15) enhanced bacterial clearance and improved pneumonia survival. Mice deficient in IL-6 also demonstrated impaired bacterial clearance and increased mortality in pneumonia because of Chlamydia trachomatis (17). TNF and IL-6 are also important in fighting infections due to fungal organisms (18 –20) and tuberculosis (21). Finally, IL-6 is required for differentiation of helper T cells that produce IL-17 (22), which plays a key role in orchestrating newly recognized cytokine networks critical for lung host defense (50, 51). The foregoing body of data strongly suggests that reduced synthesis of TNF and IL-6 could account, at least in part, for the high rates of lung infection and mortality reported in patients with hypercapnia (3–5, 7, 8). Phagocytosis is another macrophage innate immune 2187
function critical for killing of microbial pathogens (52) we found to be inhibited by hypercapnia. The fact that TNF and IL-6 both enhance phagocytosis (11, 12) raises the possibility that impaired synthesis of one or both of these cytokines could underlie the phagocytic defect caused by hypercapnia. However, we have been unable to overcome inhibition of phagocytosis by adding recombinant TNF and IL-6 to macrophages cultured in hypercapnia (data not shown), suggesting the involvement of other mechanisms. Hyperoxia (95% O2) has been shown to interfere with phagocytosis in association with a large increase in F-actin content in macrophages (53). Hypercapnia did not cause a similar increase in F-actin in our study. However, we assessed F-actin only at a single time point (1 h) after addition of particles to the cells. Further studies will be required to determine hypercapnia interferes with phagocytosis by altering the dynamics of actin or other cytoskeletal elements (54), which might be detectable at earlier times after initiation of the phagocytic process. The potential clinical importance of hypercapniainduced suppression of innate immunity is supported by a recent study showing that hypercapnic acidosis decreased bacterial clearance and worsened physiological parameters of lung injury in a model of pneumonia due to E. coli in rats (55). On the other hand, in 2 additional recent reports from the same investigators (56, 57), hypercapnic acidosis actually attenuated lung injury associated with E. coli pneumonia in the rat model. The reason for discrepant results among these studies is unclear, but may have to do with use of different rat strains, varying infection and hypercapnia exposure protocols, and inability to separate the effects of hypercapnia and acidosis with the methods utilized. These discrepancies highlight the need for additional studies to more fully elucidate the effects of hypercapnia on host defense in animal models and in the clinical setting. Besides their roles in host defense, TNF and IL-6 are well known for their ability to promote inflammation and their association with tissue injury. This suggests that inhibition of these cytokines by hypercapnia would have antiinflammatory effects that might be beneficial in certain circumstances. Indeed, hypercapnia has been shown to protect against LPS-induced lung injury in rats (58) and, in some (59, 60) but not all (61) studies, to ameliorate ventilator-induced lung injury in rabbits. These results and the rat pneumonia studies described above indicate that although hypercapnia may be harmful in the setting of infection, it can attenuate lung injury due to sterile inflammatory insults, such as LPS or high-tidal-volume mechanical ventilation, at least under certain conditions. Intriguingly, we recently found that hypercapnia also impacts the innate immune response in Drosophila melanogaster (62). We have shown that hypercapnia inhibits the expression of multiple antimicrobial peptides (AMPs), key innate immune effectors in the fruit fly. Transcription of the affected AMPs is regulated by pathways homologous to the canonical NF-B pathway 2188
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in vertebrates. Interestingly, hypercapnic inhibition of AMP expression in Drosophila was reversible and independent of acidosis, NO signaling, and induction of hypoxia or heat shock genes, all features shared with the CO2 effect on macrophage IL-6 expression in the current study. Elevated CO2 also inhibited agonistinduced AMP expression without blocking cleavage of the Drosophila IB-like domain, Relish, similar to our finding that hypercapnia did not prevent degradation of IB␣ or NF-B activation triggered by LPS in macrophages. Additionally, hypercapnia blocked activity of the promoter for the AMP, Diptericin, analogous to its effect on IL-6 promoter activity. Furthermore, we found that hypercapnia increased the mortality of flies infected with a variety of pathogenic bacteria. Together, our current findings in human and murine macrophages and our data in Drosophila indicate that hypercapnia suppresses innate immune responses in widely divergent species. Moreover, the similarity of our data in the mammalian and Drosophila systems suggests the striking possibility that the mechanisms underlying hypercapnia’s effects may be evolutionarily conserved. Recent studies have documented the presence of pathways for sensing and responding to CO2 in Drosophila (63, 64), Caenorhabditis elegans (65– 67), and mammals (23, 28). Although specific CO2-sensitive receptors have been identified in gustatory neurons in Drosophila (63, 64), the molecular mechanisms for CO2 sensing in non-neuronal cells remain to be identified. Nevertheless, our observations on hypercapnic suppression of innate immune responses in human and mouse macrophages, along with our findings in Drosophila, clearly indicate that cells of the immune system are critical targets whose function can be altered by elevated CO2 in important ways. Future studies are warranted to further elucidate the molecular mechanisms by which hypercapnia suppresses innate immunity, whether these mechanisms are fundamentally conserved, and their clinical significance in COPD, ARDS, and other lung diseases. The authors thank Y. Gruenbaum, R. M. Pope, A. Kato, P. J. Bryce, and S. C. Shin for helpful discussions, and Aisha Nair for technical assistance. This work was supported by grants from the National Institutes of Health (HL068546 and HL078860 to R.P.S., HL085534 to J.I.S., and HL072891 to P.H.S.S.), the American Heart Association (grant-in-aid 0855686G to G.J.B.), and the Department of Veterans Affairs (Merit Review to P.H.S.S.).
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Received for publication June 17, 2009. Accepted for publication January 21, 2010.
WANG ET AL.
