Alveolar Epithelial Cells Promote IGF-1 Production by Alveolar Macrophages Through TGF-β to Suppress Endogenous Inflammatory Signals

Abstract

To maintain alveolar gas exchange, the alveolar surface has to limit unnecessary inflammatory responses. This involves crosstalk between alveolar epithelial cells (AECs) and alveolar macrophages (AMs) in response to damaging factors. We recently showed that insulin-like growth factor (IGF)-1 regulates the phagocytosis of AECs. AMs secrete IGF-1 into the bronchoalveolar lavage fluid (BALF) in response to inflammatory stimuli. However, whether AECs regulate the production of IGF-1 by AMs in response to inflammatory signals remains unclear, as well as the role of IGF-1 in controlling the alveolar balance in the crosstalk between AMs and AECs under inflammatory conditions. In this study, we demonstrated that IGF-1 was upregulated in BALF and lung tissues of acute lung injury (ALI) mice, and that the increased IGF-1 was mainly derived from AMs. In vitro experiments showed that the production and secretion of IGF-1 by AMs as well as the expression of TGF-β were increased in LPS-stimulated AEC-conditioned medium (AEC-CM). Pharmacological blocking of TGF-β in AECs and addition of TGF-β neutralizing antibody to AEC-CM suggested that this AEC-derived cytokine mediates the increased production and secretion of IGF-1 from AMs. Blocking TGF-β synthesis or treatment with TGF-β neutralizing antibody attenuated the increase of IGF-1 in BALF in ALI mice. TGF-β induced the production of IGF-1 by AMs through the PI3K/Akt signaling pathway. IGF-1 prevented LPS-induced p38 MAPK activation and the expression of the inflammatory factors MCP-1, TNF-α, and IL-1β in AECs. However, IGF-1 upregulated PPARγ to increase the phagocytosis of apoptotic cells by AECs. Intratracheal instillation of IGF-1 decreased the number of polymorphonuclear neutrophils in BALF of ALI model mice, reduced alveolar congestion and edema, and suppressed inflammatory cell infiltration in lung tissues. These results elucidated a mechanism by which AECs used TGF-β to regulate IGF-1 production from AMs to attenuate endogenous inflammatory signals during alveolar inflammation.

Keywords: AEC; AMs; IGF-1; TGF-β; inflammation.

Figure 1

Increased IGF-1 production in acute LPS lung injury models. (A–D) BALB/c mice were instilled with LPS (4 mg/kg) into the nasal cavity to develop an ALI model. After 24 h of LPS instillation, IGF-1 content was detected in BALF (A) and lung tissue homogenates (B), and the expression of the IGF-1 protein in lung tissues (C) was detected by immunoblotting. **P < 0.01 vs. control group. (D) After ALI model mice were subjected to BAL (ALI + BAL), lung tissues were collected for homogenization, and the IGF-1 content in the homogenate was detected by ELISA. **P < 0.01 vs. the control group, ^##P < 0.01 vs. the ALI group.

Figure 2

IGF-1 is derived from AMs in the ALI model. (A–D) ALI model mice were generated as described in Materials and Methods (Antibodies and Reagents). The 2-CA (10 μg 2-CA in 20 μL PBS) was dripped into the nasal cavity for 12 h before the generation of the ALI model, and AMs were removed from ALI model mice (2-CA + ALI). Mice in the PBS + ALI group were ALI model mice treated with PBS instead of 2-CA. (A) After 24 h of LPS instillation, primary AMs were obtained and counted as described in Materials and Methods (BAL and Acquisition of BALF). **P < 0.01 vs. the ALI group. (B) BALF was obtained as described in Materials and Methods (Establishment of the Acute Lung Injury Model), and the IGF-1 content was detected by ELISA. **P < 0.01 vs. the control group, ^##P < 0.01 vs. the ALI group. (C,D) The expressions of AM band IGF-1 in normal control and ALI model mice were detected by immunoblotting (C) and ELISA (D), respectively. *P < 0.05, **P < 0.01 vs. the control group.

Figure 3

IGF-1 production is enhanced in AMs by LPS-stimulated AEC-CM. (A) AECs (1 ×10⁶ cells/well) were inoculated into a 6-well plate and allowed to adhere before treatment with different doses of LPS (1, 10, 50, and 100 ng/mL) for 4 h and collection of AEC-CM. Primary AMs were extracted as described in Materials and Methods and cultured (1 ×10⁶ cells/well). After the cells adhered to the wall, AEC-CM was added for 2 h. After several washes, AMs were resuspended in RPMI-1640 complete medium and cultured for an additional 12 h. AM-CM was collected and the IGF-1 content was detected by ELISA. Immunoblotting was used to detect IGF-1 protein expression in AMs. **P < 0.01 vs. the 0 ng/mL group. (B) Primary AMs (1 ×10⁶ cells/well) were inoculated into a 6-well plate and stimulated with LPS (10 ng/mL) or AEC-CM (10 ng/mL, LPS stimulation group) for 12 h. The expression of AM IGF-1 was detected by immunoblotting, and the content of IGF-1 in AM-CM was detected by ELISA. *P < 0.05, **P < 0.01 vs. the control group.

Figure 4

Identification of factors produced by LPS-treated AECs that induce IGF-1 secretion by AMs. (A) AECs (2 ×10⁵ cells/well) were inoculated into a 24-well plate and stimulated with LPS (10 ng/mL) for 2, 8, and 24 h. The supernatants were analyzed by ELISA for IL-6, GM-CSF, IL-1β, MCP- 1, TNF-α, and TGF-β content. **P < 0.01 vs. the 2 h group. (B) Primary AMs (1 ×10⁶ cells/well) were inoculated into 6-well plates and stimulated h with different concentrations (1, 10, 100, and 1,000 ng/mL) of IL-1β, TNF-α, MCP-1, and TGF-β for 24 h. The IGF-1 content in AM-CM was measured by ELISA. *P < 0.05 and **P < 0.01 vs. the 0 ng/mL group. (C) Primary AMs (1 ×10⁶ cells/well) were inoculated into 6-well plates and stimulated with TGF-β (100 ng/mL) for 24 h. The expression level of IGF-1 in AMs was detected by western blotting. **P < 0.01 vs. the control group.

Figure 5

TGF-β is necessary for IGF-1 production in AMs induced by LPS-stimulated AEC-CM. (A–C) AECs (1 ×10⁶ cells/well) were inoculated into a 6-well plate, stimulated with LPS (10 ng/mL) for 4 h, and AEC-CM was collected. AEC-CM was incubated for 30 min at 4°C with anti-MCP-1 antibody (MCP-Ab) or anti-TGF-β antibody (TGF-β Ab) or isotype control antibody (Iso Ab). Primary AMs were stimulated with the treated AEC-CM, washed, and resuspended in RPMI-1640 complete medium for 12 h. The content of IGF-1 in AM-CM was detected by ELISA (A), and the expression of IGF-1 in AM was detected by western blotting (B). In addition, AECs were treated with pirfenidone (50 ng/μL) for 1 h before stimulation with LPS (10 ng/mL), and the AEC-CM was collected and added to AMs for 2 h. After washing and resuspending AMs in culture medium for 12 h, the IGF-1 content in AM-CM was detected by ELISA (C). **P < 0.01 vs. the control group, ^##P < 0.01 vs. the AEC-CM group.

Figure 6

IGF-1 elevation in the ALI model is TGF-β dependent. (A,B) ALI model mice were generated as described in Materials and Methods (Antibodies and Reagents). At 24 h before LPS instillation, pirfenidone (400 μg/kg) (A) or TGF-β antibody (400 μg/kg) (B) was instilled into the nasal cavity once every 12 h. At 24 h after LPS instillation, the content of TGF-β in the BALF of each group of mice was measured by ELISA. **P < 0.01 vs. the control group, ^##P < 0.01 vs. the ALI group.

Figure 7

TGF-β induces IGF-1 production in AMs via the PI3K/Akt signaling pathway. (A) Primary AMs (1 ×10⁶ cells/well) were inoculated into 6-well plates and stimulated with different concentration of TGF-β (0, 1, 10, and 100 ng/mL) for 24 h. The expression of p-Akt in AMs was detected by western blotting. **P < 0.01 vs. the 0 ng/ml group. (B,C) Primary AMs (1 ×10⁶ cells/well) were inoculated into a 6-well plate and stimulated with Wortmannin (1 mM) for 2 h, followed by TGF-β (100 ng/mL) treatment for 24 h. IGF-1 protein expression in AMs was detected by immunoblotting, and the IGF-1 content in AM-CM was detected by ELISA. **P < 0.01 vs. the control group, ^##P < 0.01 vs. the TGF-β group.

Figure 8

IGF-1 prevents LPS-induced p38 MAPK activation and MCP-1, TNF-α, and IL-1β production in AECs. (A–C) AECs (1 ×10⁶ cells/well) were inoculated into a 6-well plate and pre-stimulated with IGF-1 (50 ng/mL) for 1 h, followed by LPS (10 ng/mL) for a total of 24 h. (A) The expression of p-p38 MAPK in AECs was detected by immunoblotting. (B) The mRNA expression levels of the cytokines TNF-α, IL-1β, and MCP-1 in AECs was detected by qRT-PCR. (C) The cytokines TNF-α, IL-1β, and MCP-1 in AEC-CM were detected by ELISA. **P < 0.01 vs. the control group, ^##P < 0.01 vs. the LPS group.

Figure 9

IGF-1 promotes apoptotic cell phagocytosis by AECs through PPARγ. (A,B) AECs (1 ×10⁵ cells/well) were inoculated into a 6-well plate, stimulated with different concentrations of IGF-1 (0, 10, 20, 40, and 60 ng/mL) for 12 h, and co-cultured with FITC-labeled apoptotic cells for a total of 4 h. Phagocytosis of apoptotic cells was detected by flow cytometry (A) and fluorescence microscopy (B). **P < 0.01 vs. the 0 ng/mL group. (C) AECs (1 ×10⁶ cells/well) were inoculated into a 6-well plate, stimulated with IGF-1 (60 ng/mL) for 24 h, and qRT-PCR was used to detect PPARγ, LxRA, and LxRB mRNA expression in AECs. **P < 0.01 vs. the control group. (D) AECs (1 ×10⁵ cells/well) were inoculated into a 6-well plate and treated with IGF-1 at different concentrations (0, 10, 20, 40, and 60 ng/mL) for 24 h. The protein expression of PPARγ was detected by western blotting. **P < 0.01 vs. the 0 ng/ml group. (E) AECs were stimulated with IGF-1 (60 ng/mL) for 6, 12, and 24 h, and the protein expression of PPARγ in AECs was detected by western blotting. **P < 0.01 vs. the 0 h group. (F) AECs (1 ×10⁵ cells/well) were inoculated into a 6-well plate, transfected with PPARγ siRNA or scrambled (Sc) siRNA for 36 h, and stimulated with IGF-1 (60 ng/mL) for 12 h. FITC-labeled apoptotic cells were added to the culture for 4 h. AEC phagocytosis of apoptotic cells was detected by flow cytometry. **P < 0.01 vs. the control group, ^##P < 0.01 vs. the IGF-1 group.

Figure 10

IGF-1 reduces airway inflammation in ALI mice. (A–D) ALI model mice were prepared as described in Materials and Methods (Antibodies and Reagents). At 24 h before LPS nasal drops, BALB/c mice received nasal drops of IGF-1 (0.8 mg/kg) once every 12 h. At 24 h after LPS treatment, lung tissues and BALF were collected from mice. (A) The lung tissues of each group were stained with HE, and the pathological damage was observed under a microscope. (B) The BALF of each group of mice was centrifuged, cell pellets were smeared and stained with Wright's staining solution, and the number of neutrophils was counted. (C) Wet-dry weight ratios of lung tissues in each group of mice were calculated as described in Materials and Methods (Western Blot Analysis). (D) The total protein content of BALF in each group of mice was measured according to Materials and Methods (Wet-Dry Weight Ratio of Lung Tissue). **P < 0.01 vs. the control group, ^##P < 0.01 vs. the ALI group.

Figure 11

Schematic of the two-way communication between AMs and AECs in LPS-induced airway inflammation. LPS induced the production of inflammatory factors such as TNF-α, MCP-1, IL-1β by AECs through p38 MAPK signaling. AECs also produced TGF-β in response to LPS stimulation. TGF-β induced IGF-1 production by AMs through the PI3K/Akt signaling pathway. IGF-1 prevented LPS-induced p38 MAPK activation and MCP-1, TNF-α, and IL-1 β production in AECs, and it also promoted AEC phagocytosis of apoptotic cells through PPARγ.