Homocysteine-induced macrophage inflammatory protein-2 production by glomerular mesangial cells is mediated by PI3 Kinase and p38 MAPK

Suresh Shastry, Leighton R James

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Abstract

Background. Homocysteine (Hcy) and inflammatory cytokines have been linked to adverse outcomes in persons with cardiovascular and kidney diseases and recent reports suggest that cytokine-mediated inflammatory infiltrates may be an important contributor to the pathogenesis the aforementioned diseases. Although some reports suggest that Hcy directly influences inflammatory cytokine production, this proposition has not been supported by data from other studies. The objective of the current study was to a) utilize an in vitro cellular model to identify cytokines that may be affected by Hcy and b) examine the role of mitogen activated protein kinase (MAPK) and phosphatidyl inositol 3- (PI3) Kinase in Hcy modulated cytokine production. Methods. Primary rat glomerular mesangial cells (MC, passage 8 to 15), isolated by standard sieving methodology, were exposed to Hcy (15, 50 or 100 M) with L-cysteine (L-Cys; 100 M) serving as a control. An antibody array was used to identify cytokines that were modulated when MCs were exposed to Hcy. Gene expression was assessed by quantitative RT-PCR, while western blotting analysis was used to assess cellular protein levels in the presence and absence of inhibitors of MAPK and PI3 Kinase. Finally, leukocyte adhesion assay was used to examine the effect of Hcy on leukocyte adhesion to glomerular MCs that were maintained in media without, and with, kinase inhibitors. Results. We identified macrophage inflammatory protein 2 (MIP-2) as a key cytokine that manifested increases in both protein and mRNA following exposure of glomerular MC to pathophysiologic Hcy levels (50 M). Further analyses revealed that Hcy-induced MIP-2 was dependent on activation of p38 MAPK and PI3 kinase. MIP-2 enhanced leukocyte adhesion to MC and this MIP-2-enhanced leukocyte adhesion was also dependent on activation of p38 MAPK and PI3K. Finally, we demonstrate that leukocyte adhesion to MC is specifically inhibited by anit-MIP2 antibody. Conclusion. The data suggest that Hcy participates in inflammatory cytokines production by glomerular MC and that Hcy-induced MIP-2 mediates leukocyte adhesion to MC.

Original languageEnglish (US)
Article number27
JournalJournal of Inflammation
Volume6
DOIs
StatePublished - Oct 29 2009
Externally publishedYes

Fingerprint

Chemokine CXCL2
Mesangial Cells
Homocysteine
p38 Mitogen-Activated Protein Kinases
Phosphatidylinositols
Phosphotransferases
Cytokines
Leukocytes
Adhesion
Mitogen-Activated Protein Kinases
Chemical activation
Antibodies
Kidney Diseases
Phosphatidylinositol 3-Kinases
Gene expression
Cysteine
Rats
Assays
Proteins
Cardiovascular Diseases

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry

Cite this

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title = "Homocysteine-induced macrophage inflammatory protein-2 production by glomerular mesangial cells is mediated by PI3 Kinase and p38 MAPK",
abstract = "Background. Homocysteine (Hcy) and inflammatory cytokines have been linked to adverse outcomes in persons with cardiovascular and kidney diseases and recent reports suggest that cytokine-mediated inflammatory infiltrates may be an important contributor to the pathogenesis the aforementioned diseases. Although some reports suggest that Hcy directly influences inflammatory cytokine production, this proposition has not been supported by data from other studies. The objective of the current study was to a) utilize an in vitro cellular model to identify cytokines that may be affected by Hcy and b) examine the role of mitogen activated protein kinase (MAPK) and phosphatidyl inositol 3- (PI3) Kinase in Hcy modulated cytokine production. Methods. Primary rat glomerular mesangial cells (MC, passage 8 to 15), isolated by standard sieving methodology, were exposed to Hcy (15, 50 or 100 M) with L-cysteine (L-Cys; 100 M) serving as a control. An antibody array was used to identify cytokines that were modulated when MCs were exposed to Hcy. Gene expression was assessed by quantitative RT-PCR, while western blotting analysis was used to assess cellular protein levels in the presence and absence of inhibitors of MAPK and PI3 Kinase. Finally, leukocyte adhesion assay was used to examine the effect of Hcy on leukocyte adhesion to glomerular MCs that were maintained in media without, and with, kinase inhibitors. Results. We identified macrophage inflammatory protein 2 (MIP-2) as a key cytokine that manifested increases in both protein and mRNA following exposure of glomerular MC to pathophysiologic Hcy levels (50 M). Further analyses revealed that Hcy-induced MIP-2 was dependent on activation of p38 MAPK and PI3 kinase. MIP-2 enhanced leukocyte adhesion to MC and this MIP-2-enhanced leukocyte adhesion was also dependent on activation of p38 MAPK and PI3K. Finally, we demonstrate that leukocyte adhesion to MC is specifically inhibited by anit-MIP2 antibody. Conclusion. The data suggest that Hcy participates in inflammatory cytokines production by glomerular MC and that Hcy-induced MIP-2 mediates leukocyte adhesion to MC.",
author = "Suresh Shastry and James, {Leighton R}",
year = "2009",
month = "10",
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doi = "10.1186/1476-9255-6-27",
language = "English (US)",
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T1 - Homocysteine-induced macrophage inflammatory protein-2 production by glomerular mesangial cells is mediated by PI3 Kinase and p38 MAPK

AU - Shastry, Suresh

AU - James, Leighton R

PY - 2009/10/29

Y1 - 2009/10/29

N2 - Background. Homocysteine (Hcy) and inflammatory cytokines have been linked to adverse outcomes in persons with cardiovascular and kidney diseases and recent reports suggest that cytokine-mediated inflammatory infiltrates may be an important contributor to the pathogenesis the aforementioned diseases. Although some reports suggest that Hcy directly influences inflammatory cytokine production, this proposition has not been supported by data from other studies. The objective of the current study was to a) utilize an in vitro cellular model to identify cytokines that may be affected by Hcy and b) examine the role of mitogen activated protein kinase (MAPK) and phosphatidyl inositol 3- (PI3) Kinase in Hcy modulated cytokine production. Methods. Primary rat glomerular mesangial cells (MC, passage 8 to 15), isolated by standard sieving methodology, were exposed to Hcy (15, 50 or 100 M) with L-cysteine (L-Cys; 100 M) serving as a control. An antibody array was used to identify cytokines that were modulated when MCs were exposed to Hcy. Gene expression was assessed by quantitative RT-PCR, while western blotting analysis was used to assess cellular protein levels in the presence and absence of inhibitors of MAPK and PI3 Kinase. Finally, leukocyte adhesion assay was used to examine the effect of Hcy on leukocyte adhesion to glomerular MCs that were maintained in media without, and with, kinase inhibitors. Results. We identified macrophage inflammatory protein 2 (MIP-2) as a key cytokine that manifested increases in both protein and mRNA following exposure of glomerular MC to pathophysiologic Hcy levels (50 M). Further analyses revealed that Hcy-induced MIP-2 was dependent on activation of p38 MAPK and PI3 kinase. MIP-2 enhanced leukocyte adhesion to MC and this MIP-2-enhanced leukocyte adhesion was also dependent on activation of p38 MAPK and PI3K. Finally, we demonstrate that leukocyte adhesion to MC is specifically inhibited by anit-MIP2 antibody. Conclusion. The data suggest that Hcy participates in inflammatory cytokines production by glomerular MC and that Hcy-induced MIP-2 mediates leukocyte adhesion to MC.

AB - Background. Homocysteine (Hcy) and inflammatory cytokines have been linked to adverse outcomes in persons with cardiovascular and kidney diseases and recent reports suggest that cytokine-mediated inflammatory infiltrates may be an important contributor to the pathogenesis the aforementioned diseases. Although some reports suggest that Hcy directly influences inflammatory cytokine production, this proposition has not been supported by data from other studies. The objective of the current study was to a) utilize an in vitro cellular model to identify cytokines that may be affected by Hcy and b) examine the role of mitogen activated protein kinase (MAPK) and phosphatidyl inositol 3- (PI3) Kinase in Hcy modulated cytokine production. Methods. Primary rat glomerular mesangial cells (MC, passage 8 to 15), isolated by standard sieving methodology, were exposed to Hcy (15, 50 or 100 M) with L-cysteine (L-Cys; 100 M) serving as a control. An antibody array was used to identify cytokines that were modulated when MCs were exposed to Hcy. Gene expression was assessed by quantitative RT-PCR, while western blotting analysis was used to assess cellular protein levels in the presence and absence of inhibitors of MAPK and PI3 Kinase. Finally, leukocyte adhesion assay was used to examine the effect of Hcy on leukocyte adhesion to glomerular MCs that were maintained in media without, and with, kinase inhibitors. Results. We identified macrophage inflammatory protein 2 (MIP-2) as a key cytokine that manifested increases in both protein and mRNA following exposure of glomerular MC to pathophysiologic Hcy levels (50 M). Further analyses revealed that Hcy-induced MIP-2 was dependent on activation of p38 MAPK and PI3 kinase. MIP-2 enhanced leukocyte adhesion to MC and this MIP-2-enhanced leukocyte adhesion was also dependent on activation of p38 MAPK and PI3K. Finally, we demonstrate that leukocyte adhesion to MC is specifically inhibited by anit-MIP2 antibody. Conclusion. The data suggest that Hcy participates in inflammatory cytokines production by glomerular MC and that Hcy-induced MIP-2 mediates leukocyte adhesion to MC.

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