Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage

Abstract

Elevated blood and tissue CO(2), 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 P(CO2) 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)-kappaB-dependent 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-beta, which do not require NF-kappaB. Notably, hypercapnia did not affect LPS-induced degradation of IkappaB alpha, 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 P(CO2) 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.

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, P_CO2 36 mmHg) or hypercapnia (20% CO₂, P_CO2 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.

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, P_CO2 36 mmHg) or hypercapnia (20% CO₂, P_CO2 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.

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, P_CO2 36 mmHg) or hypercapnia at 9% CO₂ (P_CO2 64 mmHg), 12.5% CO₂ (P_CO2 88 mmHg), or 20% CO₂ (P_CO2 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.

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 (pH_e 7.26), 12.5% CO₂-preequilibrated RPMI 1640 containing 25 mM NaOH (pH_e 7.46), or 5% CO₂-preequilibrated RPMI 1640 containing 15 mM HCl (pH_e 7.29), as indicated. pH_i 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.

Figure 5.

Hypercapnia inhibits IL-6 expression triggered by multiple TLRs but does not block LPS induction of IL-10 or IFN-β. 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-β mRNAs by qPCR (n=3).

Figure 6.

Hypercapnia does not affect LPS-stimulated IκBα 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 IκBα. B) Alternatively, cytoplasmic and nuclear fractions were prepared and immunoblotted for RelA/p65, with β-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.

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 μg 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 μg/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).

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-μm sections from the middle of a Z stack spanning the height of the cell monolayer.