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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.

Ito Y, Mason RJ - Respir. Res. (2010)

Bottom Line: IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, National Jewish Health, 1400 Jackson Street, Denver, CO 80206, USA. itoy@njhealth.org

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.

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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.
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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.

Mentions: Human ATII cells were isolated and cultured in vitro for 8 d (2 d adherence, 2 d TGFα and 4 d KGF) with 2 or 20 ng/ml human recombinant IL-13. The protein level of mature SP-C showed significant dose-dependent down-regulation by human recombinant IL-13 (Figure 1A, B). Next we used 20 ng/ml human recombinant IL-13 for a time-course experiment, added it to the cultured cells for the final 2, 4 or 6 d and evaluated the expression of surfactant protein levels on day 8 by immunoblotting (n = 6) (Figure 2A, B). Protein levels of SP-A and mature SP-B were not altered by IL-13, whereas those of mature SP-C and SP-D were greatly down-regulated by 4 or 6 d of treatment with IL-13 (relative increase of mature SP-C: without IL-13 1.0, 2 d IL-13 0.60 ± 0.13, 4 d IL-13 0.35 ± 0.11p = 0.001, 6 d IL-13 0.45 ± 0.19 p = 0.005; SP-D: without IL-13 1.0, 2 d IL-13 0.66 ± 0.12, 4 d IL-13 0.47 ± 0.11 p = 0.002, 6 d IL-13 0.46 ± 0.17 p = 0.003) (Figure 2A, B).


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.

Ito Y, Mason RJ - Respir. Res. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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.
Mentions: Human ATII cells were isolated and cultured in vitro for 8 d (2 d adherence, 2 d TGFα and 4 d KGF) with 2 or 20 ng/ml human recombinant IL-13. The protein level of mature SP-C showed significant dose-dependent down-regulation by human recombinant IL-13 (Figure 1A, B). Next we used 20 ng/ml human recombinant IL-13 for a time-course experiment, added it to the cultured cells for the final 2, 4 or 6 d and evaluated the expression of surfactant protein levels on day 8 by immunoblotting (n = 6) (Figure 2A, B). Protein levels of SP-A and mature SP-B were not altered by IL-13, whereas those of mature SP-C and SP-D were greatly down-regulated by 4 or 6 d of treatment with IL-13 (relative increase of mature SP-C: without IL-13 1.0, 2 d IL-13 0.60 ± 0.13, 4 d IL-13 0.35 ± 0.11p = 0.001, 6 d IL-13 0.45 ± 0.19 p = 0.005; SP-D: without IL-13 1.0, 2 d IL-13 0.66 ± 0.12, 4 d IL-13 0.47 ± 0.11 p = 0.002, 6 d IL-13 0.46 ± 0.17 p = 0.003) (Figure 2A, B).

Bottom Line: IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, National Jewish Health, 1400 Jackson Street, Denver, CO 80206, USA. itoy@njhealth.org

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.

Show MeSH
Related in: MedlinePlus