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Netrin-1 regulates the inflammatory response of neutrophils and macrophages, and suppresses ischemic acute kidney injury by inhibiting COX-2-mediated PGE2 production.

Ranganathan PV, Jayakumar C, Mohamed R, Dong Z, Ramesh G - Kidney Int. (2013)

Bottom Line: This was associated with reduced apoptosis, inflammatory cytokine and chemokine expression, and improved kidney function.Moreover, netrin-1 regulates COX-2 expression at the transcriptional level through the regulation of NFκB activation.This could be a potential drug for treating many inflammatory immune disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Vascular Biology Center, Georgia Health Sciences University, Augusta, Georgia 30912, USA.

ABSTRACT
Netrin-1 regulates inflammation but the mechanism by which this occurs is unknown. Here we explore the role of netrin-1 in regulating the production of the prostanoid metabolite PGE2 from neutrophils in in vitro and in vivo disease models. Ischemia reperfusion in wild-type and RAG-1 knockout mice induced severe kidney injury that was associated with a large increase in neutrophil infiltration and COX-2 expression in the infiltrating leukocytes. Administration of netrin-1 suppressed COX-2 expression, PGE2 and thromboxane production, and neutrophil infiltration into the kidney. This was associated with reduced apoptosis, inflammatory cytokine and chemokine expression, and improved kidney function. Treatment with the PGE2 receptor EP4 agonist enhanced neutrophil infiltration and renal injury, which was not inhibited by netrin-1. Consistent with in vivo data, both LPS- and IFNγ-induced inflammatory cytokine production in macrophages and IL-17-induced IFNγ production in neutrophils were suppressed by netrin-1 in vitro by suppression of COX-2 expression. Moreover, netrin-1 regulates COX-2 expression at the transcriptional level through the regulation of NFκB activation. Thus, netrin-1 regulates the inflammatory response of neutrophils and macrophages through suppression of COX-2-mediated PGE2 production. This could be a potential drug for treating many inflammatory immune disorders.

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Flow cytometry analysis of COX-2 expressing immune cells in the kidney. A. Flow cytometry analysis was carried out as described in Materials and Methods. Percentage of COX-2 postive cells shown on each histogram (outside bracket) and percentage of COX-2 positive Gr-1 positive neutrophils, F4/80 positive monocyte/macrophage and CD4 positive T cells were shown inside the bracket on each histogram. B-E. Co-localization of COX-2 and neutrophils in kidney. Immunofluorescence staining of COX-2 and neutrophils in WT mice subjected to IR was carried out as described in Materials and Methods. COX-2 (panel B, green) and neutrophil (panel C, red) staining was seen in the interstitium. Panel D is DAPI nuclear staining. Overlay of panel B, C and D is shown in E. COX-1 and neutrophils were co-localized (yellow staining, arrow). Scale Bar: 50μM.
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Figure 5: Flow cytometry analysis of COX-2 expressing immune cells in the kidney. A. Flow cytometry analysis was carried out as described in Materials and Methods. Percentage of COX-2 postive cells shown on each histogram (outside bracket) and percentage of COX-2 positive Gr-1 positive neutrophils, F4/80 positive monocyte/macrophage and CD4 positive T cells were shown inside the bracket on each histogram. B-E. Co-localization of COX-2 and neutrophils in kidney. Immunofluorescence staining of COX-2 and neutrophils in WT mice subjected to IR was carried out as described in Materials and Methods. COX-2 (panel B, green) and neutrophil (panel C, red) staining was seen in the interstitium. Panel D is DAPI nuclear staining. Overlay of panel B, C and D is shown in E. COX-1 and neutrophils were co-localized (yellow staining, arrow). Scale Bar: 50μM.

Mentions: We hypothesized that netrin-1 regulates neutrophil function and activation by suppressing COX-2-mediated PGE2 production, thereby suppressing inflammatory cytokine and chemokine production and infiltration of neutrophils. To determine this possibility, we examined the expression of COX-1 and COX-2 after reperfusion in WT and RAG-1 knockout mice. COX-1 expression was not altered significantly after reperfusion whereas the expression of COX-2 is significantly up-regulated (Figure 2). Administration of netrin-1 suppressed the expression of COX-2 in both WT and RAG1 knockout animals. Consistent with suppression of COX-2 expression, the ischemia reperfusion-induced increase in PGE2 levels in kidney were suppressed by administration of netrin-1 (Figure 4A). Excretion of PGE2 was also increased in both WT and RAG-1 knockout mice after ischemia reperfusion, which was suppressed by administration of netrin-1 (Figure 4B). To further confirm the netrin-1 effects on COX-2, we measured another COX-2 metabolite, thromboxane B2, which is a degradation product of thromboxane A2. Ischemia reperfusion induced an increase in thromboxane B2 excreation in urine, which was significantly suppressed by administration of netrin-1 (Figure 4C). To determine specific cell type that expressed COX-2, immunolocalization was performed. As shown in Figure 4D, COX-2 expression was seen in the thick ascending limb, distal tubules (also in macula densa) and collecting ducts in sham-operated kidney, but not in proximal tubules. There is some staining in the interstitium, which may be associated with endothelial cells. Twenty four hours after reperfusion, COX-2 expression is highly induced in infiltrating cells. The increase in expression of COX-2 in infiltrating cells is inhibited in netrin-1-treated animals (Figure 4E). The infiltrating cells appear to be neutrophils. To confirm this flow cytometry analysis and co-localization of COX-2 expressing immune cells was performed 24hr after reperfusion. Over 86% of Gr-1 positive neutrophils and 80% of F4/80 positive monocyte/macrophage were positive for COX-2 expression. Very few CD4 T cells were seen in the kidney and 60% of them were positive for COX-2 expression. Consistent with flow cytometric analysis, neutrophils were co-localized for COX-2, confirming that COX-2-expressing infiltrating cells were indeed neutrophils and macrophages (Figure 5).


Netrin-1 regulates the inflammatory response of neutrophils and macrophages, and suppresses ischemic acute kidney injury by inhibiting COX-2-mediated PGE2 production.

Ranganathan PV, Jayakumar C, Mohamed R, Dong Z, Ramesh G - Kidney Int. (2013)

Flow cytometry analysis of COX-2 expressing immune cells in the kidney. A. Flow cytometry analysis was carried out as described in Materials and Methods. Percentage of COX-2 postive cells shown on each histogram (outside bracket) and percentage of COX-2 positive Gr-1 positive neutrophils, F4/80 positive monocyte/macrophage and CD4 positive T cells were shown inside the bracket on each histogram. B-E. Co-localization of COX-2 and neutrophils in kidney. Immunofluorescence staining of COX-2 and neutrophils in WT mice subjected to IR was carried out as described in Materials and Methods. COX-2 (panel B, green) and neutrophil (panel C, red) staining was seen in the interstitium. Panel D is DAPI nuclear staining. Overlay of panel B, C and D is shown in E. COX-1 and neutrophils were co-localized (yellow staining, arrow). Scale Bar: 50μM.
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Figure 5: Flow cytometry analysis of COX-2 expressing immune cells in the kidney. A. Flow cytometry analysis was carried out as described in Materials and Methods. Percentage of COX-2 postive cells shown on each histogram (outside bracket) and percentage of COX-2 positive Gr-1 positive neutrophils, F4/80 positive monocyte/macrophage and CD4 positive T cells were shown inside the bracket on each histogram. B-E. Co-localization of COX-2 and neutrophils in kidney. Immunofluorescence staining of COX-2 and neutrophils in WT mice subjected to IR was carried out as described in Materials and Methods. COX-2 (panel B, green) and neutrophil (panel C, red) staining was seen in the interstitium. Panel D is DAPI nuclear staining. Overlay of panel B, C and D is shown in E. COX-1 and neutrophils were co-localized (yellow staining, arrow). Scale Bar: 50μM.
Mentions: We hypothesized that netrin-1 regulates neutrophil function and activation by suppressing COX-2-mediated PGE2 production, thereby suppressing inflammatory cytokine and chemokine production and infiltration of neutrophils. To determine this possibility, we examined the expression of COX-1 and COX-2 after reperfusion in WT and RAG-1 knockout mice. COX-1 expression was not altered significantly after reperfusion whereas the expression of COX-2 is significantly up-regulated (Figure 2). Administration of netrin-1 suppressed the expression of COX-2 in both WT and RAG1 knockout animals. Consistent with suppression of COX-2 expression, the ischemia reperfusion-induced increase in PGE2 levels in kidney were suppressed by administration of netrin-1 (Figure 4A). Excretion of PGE2 was also increased in both WT and RAG-1 knockout mice after ischemia reperfusion, which was suppressed by administration of netrin-1 (Figure 4B). To further confirm the netrin-1 effects on COX-2, we measured another COX-2 metabolite, thromboxane B2, which is a degradation product of thromboxane A2. Ischemia reperfusion induced an increase in thromboxane B2 excreation in urine, which was significantly suppressed by administration of netrin-1 (Figure 4C). To determine specific cell type that expressed COX-2, immunolocalization was performed. As shown in Figure 4D, COX-2 expression was seen in the thick ascending limb, distal tubules (also in macula densa) and collecting ducts in sham-operated kidney, but not in proximal tubules. There is some staining in the interstitium, which may be associated with endothelial cells. Twenty four hours after reperfusion, COX-2 expression is highly induced in infiltrating cells. The increase in expression of COX-2 in infiltrating cells is inhibited in netrin-1-treated animals (Figure 4E). The infiltrating cells appear to be neutrophils. To confirm this flow cytometry analysis and co-localization of COX-2 expressing immune cells was performed 24hr after reperfusion. Over 86% of Gr-1 positive neutrophils and 80% of F4/80 positive monocyte/macrophage were positive for COX-2 expression. Very few CD4 T cells were seen in the kidney and 60% of them were positive for COX-2 expression. Consistent with flow cytometric analysis, neutrophils were co-localized for COX-2, confirming that COX-2-expressing infiltrating cells were indeed neutrophils and macrophages (Figure 5).

Bottom Line: This was associated with reduced apoptosis, inflammatory cytokine and chemokine expression, and improved kidney function.Moreover, netrin-1 regulates COX-2 expression at the transcriptional level through the regulation of NFκB activation.This could be a potential drug for treating many inflammatory immune disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Vascular Biology Center, Georgia Health Sciences University, Augusta, Georgia 30912, USA.

ABSTRACT
Netrin-1 regulates inflammation but the mechanism by which this occurs is unknown. Here we explore the role of netrin-1 in regulating the production of the prostanoid metabolite PGE2 from neutrophils in in vitro and in vivo disease models. Ischemia reperfusion in wild-type and RAG-1 knockout mice induced severe kidney injury that was associated with a large increase in neutrophil infiltration and COX-2 expression in the infiltrating leukocytes. Administration of netrin-1 suppressed COX-2 expression, PGE2 and thromboxane production, and neutrophil infiltration into the kidney. This was associated with reduced apoptosis, inflammatory cytokine and chemokine expression, and improved kidney function. Treatment with the PGE2 receptor EP4 agonist enhanced neutrophil infiltration and renal injury, which was not inhibited by netrin-1. Consistent with in vivo data, both LPS- and IFNγ-induced inflammatory cytokine production in macrophages and IL-17-induced IFNγ production in neutrophils were suppressed by netrin-1 in vitro by suppression of COX-2 expression. Moreover, netrin-1 regulates COX-2 expression at the transcriptional level through the regulation of NFκB activation. Thus, netrin-1 regulates the inflammatory response of neutrophils and macrophages through suppression of COX-2-mediated PGE2 production. This could be a potential drug for treating many inflammatory immune disorders.

Show MeSH
Related in: MedlinePlus