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Lack of RNase L attenuates macrophage functions.

Yi X, Zeng C, Liu H, Chen X, Zhang P, Yun BS, Jin G, Zhou A - PLoS ONE (2013)

Bottom Line: Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2).Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition.RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF)-β, IL-1β, IL-10, CCL2 and Cox-2.

View Article: PubMed Central - PubMed

Affiliation: Clinical Chemistry Program, Department of Chemistry, Cleveland State University, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Macrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cyclooxygenase-2 (Cox-2) by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN) inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown.

Methodology: Bone marrow-derived macrophages (BMMs) were generated from RNase L(+/+)and (-/-) mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC)-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays.

Conclusions/findings: Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2). Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF)-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions.

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RNase L regulates the expression of Cox-2.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS, 1,000 units/ml of IFN-α, 500 units/ml of IFN-γ, and 100 ng/ml of M-CSF for 14 h. The expression of Cox-2 was determined by Western blot analysis using a monolocnal antibody against mouse Cox-2 (A). RNase L wild type and deficient MEFs were treated with 1 µg/ml of LPS for 14 h. Total RNAs were isolated by using the Trizol Reagent (Invitrogen, CA). The expression of Cox-2, TNF-α, IL-6 and IL-1β was determined by RT-PCR (B). The expression of Cox-2 was measured by real-time PCR (C), which was performed twice in triplicate; the fold change from one of the experiments is present as Mean ±SD, *p<0.05. The production of PGE2 in the media culturing the two types of BMMs treated with LPS as described above was analyzed by ELISA (D). Experiments were performed twice in triplicates. Data are presented as Mean ±SD, *p<0.05.
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pone-0081269-g004: RNase L regulates the expression of Cox-2.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS, 1,000 units/ml of IFN-α, 500 units/ml of IFN-γ, and 100 ng/ml of M-CSF for 14 h. The expression of Cox-2 was determined by Western blot analysis using a monolocnal antibody against mouse Cox-2 (A). RNase L wild type and deficient MEFs were treated with 1 µg/ml of LPS for 14 h. Total RNAs were isolated by using the Trizol Reagent (Invitrogen, CA). The expression of Cox-2, TNF-α, IL-6 and IL-1β was determined by RT-PCR (B). The expression of Cox-2 was measured by real-time PCR (C), which was performed twice in triplicate; the fold change from one of the experiments is present as Mean ±SD, *p<0.05. The production of PGE2 in the media culturing the two types of BMMs treated with LPS as described above was analyzed by ELISA (D). Experiments were performed twice in triplicates. Data are presented as Mean ±SD, *p<0.05.

Mentions: To determine if RNase L is involved in regulating the expression of other proinflammatory genes, we examined the expression of Cox-2, which catalyzes the production of prostaglandins and other eicosanoids, to promote inflammation and is associated with macrophage function [14], [15]. In the experiment, RNase L+/+ and −/− BMMs were treated with LPS and other stimuli, and the expression of Cox-2 was determined by Western blot, RT-PCR and qPCR. As shown in the Figure 4A, the expression of Cox-2 was significantly induced in RNase L +/+ BMMs after the cells were treated with LPS, M-CSF, IFN-α and -γ. However, the induction of Cox-2 was remarkably attenuated in RNase L−/− BMMs, suggesting that RNase L impacts the expression of Cox-2 in the cells induced by LPS. Similar results were also obtained by using mouse embryonic fibroblasts (MEF) after treated with LPS and the samples were analyzed by RT-PCR (Figure 4B). qPCR analysis revealed that the expressing level of Cox-2 in RNase L +/+ BMMs after treatment with LPS was about 3-fold higher than that in RNase L−/− BMMs (Figure 4C). Overall, our results demonstrate that RNase L is involved in the induction of Cox-2 by LPS. To confirm that the increased expression level of Cox-2 results in enhancement of the Cox-2 enzymatic activity in the cells, the production of PGE2 was measured by ELISA. As expected, the production of PGE2 was about 20% higher in the medium of RNase L+/+ BMM in compared to that in the medium of RNase L−/− BMM after the cells were treated with LPS (Figure 4D).


Lack of RNase L attenuates macrophage functions.

Yi X, Zeng C, Liu H, Chen X, Zhang P, Yun BS, Jin G, Zhou A - PLoS ONE (2013)

RNase L regulates the expression of Cox-2.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS, 1,000 units/ml of IFN-α, 500 units/ml of IFN-γ, and 100 ng/ml of M-CSF for 14 h. The expression of Cox-2 was determined by Western blot analysis using a monolocnal antibody against mouse Cox-2 (A). RNase L wild type and deficient MEFs were treated with 1 µg/ml of LPS for 14 h. Total RNAs were isolated by using the Trizol Reagent (Invitrogen, CA). The expression of Cox-2, TNF-α, IL-6 and IL-1β was determined by RT-PCR (B). The expression of Cox-2 was measured by real-time PCR (C), which was performed twice in triplicate; the fold change from one of the experiments is present as Mean ±SD, *p<0.05. The production of PGE2 in the media culturing the two types of BMMs treated with LPS as described above was analyzed by ELISA (D). Experiments were performed twice in triplicates. Data are presented as Mean ±SD, *p<0.05.
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pone-0081269-g004: RNase L regulates the expression of Cox-2.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS, 1,000 units/ml of IFN-α, 500 units/ml of IFN-γ, and 100 ng/ml of M-CSF for 14 h. The expression of Cox-2 was determined by Western blot analysis using a monolocnal antibody against mouse Cox-2 (A). RNase L wild type and deficient MEFs were treated with 1 µg/ml of LPS for 14 h. Total RNAs were isolated by using the Trizol Reagent (Invitrogen, CA). The expression of Cox-2, TNF-α, IL-6 and IL-1β was determined by RT-PCR (B). The expression of Cox-2 was measured by real-time PCR (C), which was performed twice in triplicate; the fold change from one of the experiments is present as Mean ±SD, *p<0.05. The production of PGE2 in the media culturing the two types of BMMs treated with LPS as described above was analyzed by ELISA (D). Experiments were performed twice in triplicates. Data are presented as Mean ±SD, *p<0.05.
Mentions: To determine if RNase L is involved in regulating the expression of other proinflammatory genes, we examined the expression of Cox-2, which catalyzes the production of prostaglandins and other eicosanoids, to promote inflammation and is associated with macrophage function [14], [15]. In the experiment, RNase L+/+ and −/− BMMs were treated with LPS and other stimuli, and the expression of Cox-2 was determined by Western blot, RT-PCR and qPCR. As shown in the Figure 4A, the expression of Cox-2 was significantly induced in RNase L +/+ BMMs after the cells were treated with LPS, M-CSF, IFN-α and -γ. However, the induction of Cox-2 was remarkably attenuated in RNase L−/− BMMs, suggesting that RNase L impacts the expression of Cox-2 in the cells induced by LPS. Similar results were also obtained by using mouse embryonic fibroblasts (MEF) after treated with LPS and the samples were analyzed by RT-PCR (Figure 4B). qPCR analysis revealed that the expressing level of Cox-2 in RNase L +/+ BMMs after treatment with LPS was about 3-fold higher than that in RNase L−/− BMMs (Figure 4C). Overall, our results demonstrate that RNase L is involved in the induction of Cox-2 by LPS. To confirm that the increased expression level of Cox-2 results in enhancement of the Cox-2 enzymatic activity in the cells, the production of PGE2 was measured by ELISA. As expected, the production of PGE2 was about 20% higher in the medium of RNase L+/+ BMM in compared to that in the medium of RNase L−/− BMM after the cells were treated with LPS (Figure 4D).

Bottom Line: Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2).Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition.RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF)-β, IL-1β, IL-10, CCL2 and Cox-2.

View Article: PubMed Central - PubMed

Affiliation: Clinical Chemistry Program, Department of Chemistry, Cleveland State University, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Macrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cyclooxygenase-2 (Cox-2) by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN) inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown.

Methodology: Bone marrow-derived macrophages (BMMs) were generated from RNase L(+/+)and (-/-) mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC)-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays.

Conclusions/findings: Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2). Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF)-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions.

Show MeSH
Related in: MedlinePlus