The effect of interleukin-13 (IL-13) and interferon-γ (IFN-γ) on expression of surfactant proteins in adult human alveolar type II cells in vitro

Abstract

Background: Surfactant proteins are produced predominantly by alveolar type II (ATII) cells, and the expression of these proteins can be altered by cytokines and growth factors. Th1/Th2 cytokine imbalance is suggested to be important in the pathogenesis of several adult lung diseases. Recently, we developed a culture system for maintaining differentiated adult human ATII cells. Therefore, we sought to determine the effects of IL-13 and IFN-γ on the expression of surfactant proteins in adult human ATII cells in vitro. Additional studies were done with rat ATII cells.

Methods: Adult human ATII cells were isolated from deidentified organ donors whose lungs were not suitable for transplantation and donated for medical research. The cells were cultured on a mixture of Matrigel and rat-tail collagen for 8 d with differentiation factors and human recombinant IL-13 or IFN-γ.

Results: IL-13 reduced the mRNA and protein levels of surfactant protein (SP)-C, whereas IFN-γ increased the mRNA level of SP-C and proSP-C protein but not mature SP-C. Neither cytokine changed the mRNA level of SP-B but IFN-γ slightly decreased mature SP-B. IFN-γ reduced the level of the active form of cathepsin H. IL-13 also reduced the mRNA and protein levels of SP-D, whereas IFN-γ increased both mRNA and protein levels of SP-D. IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A.

Conclusions: We demonstrated that IL-13 and IFN-γ altered the expression of surfactant proteins in human adult ATII cells in vitro. IL-13 decreased SP-C and SP-D in human ATII cells, whereas IFN-γ had the opposite effect. The protein levels of mature SP-B were decreased by IFN-γ treatment, likely due to the reduction in active form cathepsin H. Similarly, the active form of cathepsin H was relatively insufficient to fully process proSP-C as IFN-γ increased the mRNA levels for SP-C and proSP-C protein, but there was no increase in mature SP-C. These observations suggest that in disease states with an overexpression of IL-13, there would be some deficiency in mature SP-C and SP-D. In disease states with an excess of IFN-γ or therapy with IFN-γ, these data suggest that there might be incomplete processing of SP-B and SP-C.

Figure 1

IL-13 alters mature SP-C protein level in a dose-dependent manner. Adult human ATII cells were cultured on Matrigel and rat-tail collagen coated inserts in DMEM containing 5% heat inactivated human serum with 2 d of TGFα followed by 4 d of KGF. Panel A shows a representative immunoblot from ATII cells cultured with 2 or 20 ng/ml IL-13 for the final 4 days. Lane 1: day 0 control (freshly isolated ATII cells), Lane 2: empty lane, Lane 3: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF, Lane 4: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF with 4 d 2 ng/ml IL-13, Lane 5: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF with 4 d 20 ng/ml IL-13. Panel B shows the reduction in mature SP-C protein levels after treatment with IL-13 (black) by immunoblotting data normalized to GAPDH (n = 3), which are analyzed by NIH Image. The comparison is to cultures without IL-13. Representative data are shown in Panel A lane 3 to 5. *: p < 0.05 v.s without IL-13.

Figure 2

IL-13 and IFN-γ alter surfactant protein levels. Adult human ATII cells were cultured on Matrigel and rat-tail collagen coated inserts in DMEM containing 5% heat inactivated human serum with 2 d of TGFα followed by 4 d of KGF. Panel A shows representative immunoblot from ATII cells cultured with 20 ng/ml IL-13 for 2, 4 or 6 d or with 100 ng/ml IFN-γ for 4 d. Lane 1: day 0 control, Lane 2: empty lane, Lane 3: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF, Lane 4: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF with 2 d 20 ng/ml IL-13, Lane 5: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF with 4 d 20 ng/ml IL-13, Lane 6: 2 days 10 ng/ml TGFα + 4 d10 ng/ml KGF with 6 d 20 ng/ml IL-13, Lane 7: 2 days 10 ng/ml TGFα + 4 d 10 ng/ml KGF with 4 d 100 ng/ml IFN-γ. Panel B shows surfactant proteins levels from IL-13 (black) time-course treatment immunoblotting data normalized by GAPDH (n = 6), which are analyzed by NIH Image. Only SP-A, mature SP-B, mature SP-C and SP-D data are shown. The comparison is to cultures without IL-13. Representative data are shown in Panel A lane 3 to 6. *: p < 0.05 v.s without IL-13. Panel C shows surfactant proteins levels from IFNγ treatment (gray) immunoblotting data normalized by GAPDH (n = 6), which are analyzed by NIH Image. Representative data are shown in Panel A lane 3 and 7. *: p < 0.05 v.s without IFN-γ.

Figure 3

IL-13 and IFN-γ alter surfactant protein mRNA levels. Adult human ATII cells were cultured on Matrigel and rat-tail collagen coated inserts in DMEM containing 5% heat inactivated human serum with 2 d TGFα followed by 4 d KGF. Panel A shows mRNA data from ATII cells cultured with 20 ng/ml IL-13 (black) for 2, 4 or 6 d. mRNA levels for surfactant proteins were measured by quantitative real-time PCR and normalized to the constitutive probe 36B4 (n = 6). *: p < 0.05 v.s without IL-13. Panel B shows mRNA data from ATII cells cultured with 100 ng/ml IFN-γ (gray) for 4 d. mRNA levels for surfactant proteins were normalized to the constitutive probe 36B4 by quantitative real-time PCR (n = 6). *: p < 0.05 v.s without IFN-γ

Figure 4

IFN-γ increases whereas IL-13 decreases proSP-C. Adult human ATII cells were cultured on Matrigel and rat-tail collagen coated inserts in DMEM containing 5% heat inactivated human serum for 2 d with10 ng/ml TGFα followed by 4 d 10 ng/ml KGF with or without 4 d 20 ng/ml IL-13 or 100 ng/ml IFN-γ. Panel A shows protein levels by immunoblotting for four different proSP-C bands normalized by GAPDH from 6 different donors cells stimulated by 4 d 20 ng/ml IL-13 or 100 ng/ml IFN-γ, which are analyzed by NIH Image. Representative protein levels by immunoblotting are shown on Figure 1 Panel B lane 3, 5 and 7. White bar: without IL-13 and IFN-γ, black bar: with 20 ng/ml IL-13 for 4 d, grey bar: with 100ng/ml IFN-γ for 4 d. *: p < 0.05 v.s without IL-13 or IFN-γ. Panel B shows immunocytochemistry for proSP-C. Top picture: without IL-13 or IFN-γ, middle one: with IL-13, bottom one: with IFN-γ. green: proSP-C, blue: DAPI. These three pictures were taken at the same exposure times.

Figure 5

IFN-γ reduces cathepsin H but not ABCA3. Adult human ATII cells cultured on Matrigel and rat-tail collagen in DMEM containing 5% heat inactivated human serum with 2 d TGFα followed by 4 d KGF with 20 ng/ml IL-13 or with 100 ng/ml IFN-γ for the final 4 days. Panel A shows representative active form of cathepsin H protein levels by immunoblotting and protein levels by immunoblotting for the active form cathepsin H normalized by GAPDH from 6 different donors, which are analyzed by NIH Image. Lane 1: day 0 control, Lane 2: empty, Lane 3: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF, Lane 4: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF with 4 d 20 ng/ml IL-13, Lane 5: 2 d 10 ng/ml TGFα + 4 d 10 ng/ml KGF with 4 d 100 ng/ml IFN-γ. White bar: without IL-13 and IFN-γ, black bar: with 20 ng/ml IL-13 for 4 d, gray bar: with 100 ng/ml IFN-γ for 4 d. *: p < 0.05 v.s. without IL-13 and IFN-γ. Panel B shows representative ABCA3 protein levels by immunoblotting and protein levels by immunoblotting for ABCA3 normalized by GAPDH from 6 different donors, which are analyzed by NIH Image. Lane order is same as in Panel A. White bar: without IL-13 and IFN-γ, black bar: with 20 ng/ml IL-13 for 4 d, gray bar: with 100 ng/ml IFN-γ for 4 d. *: p < 0.05 v.s. without IL-13 and IFN-γ.