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

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      Journal of Inflammation (London, England)

      BioMed Central

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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 μM) with L-cysteine (L-Cys; 100 μM) 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 μM). 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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          Most cited references 38

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          Single step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction. Anal

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            Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream.

            The recent flood of reports using real-time Q-PCR testifies to the transformation of this technology from an experimental tool into the scientific mainstream. Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers. The range of applications of real-time Q-PCR is immense and has been fueled in part by the proliferation of lower-cost instrumentation and reagents. Successful application of real-time Q-PCR is not trivial. However, this review will help guide the reader through the variables that can limit the usefulness of this technology. Careful consideration of the assay design, template preparation, and analytical methods are essential for accurate gene quantification.
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              Chemokines.

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                Author and article information

                Journal
                J Inflamm (Lond)
                Journal of Inflammation (London, England)
                BioMed Central
                1476-9255
                2009
                26 September 2009
                : 6
                : 27
                Affiliations
                [1 ]Department of Medicine, University of Texas Southwestern Center, Dallas, TX, USA
                Article
                1476-9255-6-27
                10.1186/1476-9255-6-27
                2764696
                19781090
                Copyright © 2009 Shastry and James; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Research

                Immunology

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