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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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Related in: MedlinePlus

RNase L regulates the expression of cytokines and chemokines.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS for 14 h. The secretory level of certain cytokines and chemokines in the media was measured by using an ELISA kit for each of the analyzers. Experiments were performed two times in triplicates. Data are presented as Mean ±SD. *p<0.0001, **p<0.05.
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pone-0081269-g003: RNase L regulates the expression of cytokines and chemokines.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS for 14 h. The secretory level of certain cytokines and chemokines in the media was measured by using an ELISA kit for each of the analyzers. Experiments were performed two times in triplicates. Data are presented as Mean ±SD. *p<0.0001, **p<0.05.

Mentions: It has been well known that macrophages are able to secret a large number of cytokines, chemokines and growth factors under stimulation, which in turn regulate their own functions and promote adaptive immunity [26]. Since our results showed that RNase L was involved in macrophage migration and endocytosis, we were intrigued to determine if RNase L regulates the production of these factors contributing to the function of macrophages. RNase L +/+ and −/− BMMs were treated with LPS and the secreted products of TNF-α, CCL-2, IL-1β, IL-6, IL-10 and TGF-β, which are believed to be associated with cell migration and macrophage functions, in the media were analyzed by using ELISA. As shown in Figure 3, RNase L is necessary for the efficient induction of CCL-2 and IL-10, and lack of RNase L reduced 63% and 78% of the secretory level of CCL-2 and IL-10, respectively, under stimulation with LPS. However, RNase L only has a modest effect on the production of TNF-α and TGF-β. Although the basal level of IL-6 in RNase L+/+ BMMs is significantly higher (25%) than that in RNase L−/− BMMs, RNase L seemed not to contribute to the induction of IL-6 at all in the cells by LPS. Interestingly, RNase L apparently inhibited the production of IL-1β and M-CSF after the cells were treated with LPS. The secretory level of IL-1β and M-CSF was 2.3- and 1.3-folds higher respectively in the medium of BMMs deficient RNase L than that in the medium of wild type BMMs. To confirm the results, we generated an RNase L deficient Raw 264.7 cell line, a mouse macrophage cell line, in which RNase L was knocked down by using RNase L hairpin RNA (ShRNA) lentiviral particles (Figure S1). RNase L knocked down Raw 264.7 cells were treated with LPS and the level of these factors described above in the media was determined by ELISA. As expected, the results were similar to that obtained in BMMs (Figure S2).


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 cytokines and chemokines.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS for 14 h. The secretory level of certain cytokines and chemokines in the media was measured by using an ELISA kit for each of the analyzers. Experiments were performed two times in triplicates. Data are presented as Mean ±SD. *p<0.0001, **p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852499&req=5

pone-0081269-g003: RNase L regulates the expression of cytokines and chemokines.RNase L deficient and wild type BMMs were treated with 1 µg/ml of LPS for 14 h. The secretory level of certain cytokines and chemokines in the media was measured by using an ELISA kit for each of the analyzers. Experiments were performed two times in triplicates. Data are presented as Mean ±SD. *p<0.0001, **p<0.05.
Mentions: It has been well known that macrophages are able to secret a large number of cytokines, chemokines and growth factors under stimulation, which in turn regulate their own functions and promote adaptive immunity [26]. Since our results showed that RNase L was involved in macrophage migration and endocytosis, we were intrigued to determine if RNase L regulates the production of these factors contributing to the function of macrophages. RNase L +/+ and −/− BMMs were treated with LPS and the secreted products of TNF-α, CCL-2, IL-1β, IL-6, IL-10 and TGF-β, which are believed to be associated with cell migration and macrophage functions, in the media were analyzed by using ELISA. As shown in Figure 3, RNase L is necessary for the efficient induction of CCL-2 and IL-10, and lack of RNase L reduced 63% and 78% of the secretory level of CCL-2 and IL-10, respectively, under stimulation with LPS. However, RNase L only has a modest effect on the production of TNF-α and TGF-β. Although the basal level of IL-6 in RNase L+/+ BMMs is significantly higher (25%) than that in RNase L−/− BMMs, RNase L seemed not to contribute to the induction of IL-6 at all in the cells by LPS. Interestingly, RNase L apparently inhibited the production of IL-1β and M-CSF after the cells were treated with LPS. The secretory level of IL-1β and M-CSF was 2.3- and 1.3-folds higher respectively in the medium of BMMs deficient RNase L than that in the medium of wild type BMMs. To confirm the results, we generated an RNase L deficient Raw 264.7 cell line, a mouse macrophage cell line, in which RNase L was knocked down by using RNase L hairpin RNA (ShRNA) lentiviral particles (Figure S1). RNase L knocked down Raw 264.7 cells were treated with LPS and the level of these factors described above in the media was determined by ELISA. As expected, the results were similar to that obtained in BMMs (Figure S2).

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