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Identification of inflammatory mediators by screening for glucocorticoid-attenuated response genes
1Department of Pediatrics, UCLA School of Medicine 90095-1752, USA.
Abstract:
We describe an approach for identifying novel inflammatory mediators, based on screening for immediate early/primary response genes whose induction by an inflammatory stimulus is attenuated by glucocorticoids. This procedure can be applied to a wide range of cell types and tissues, using a variety of inducers. In an initial test of this idea, we identified cDNAs for 12 LPS-induced, glucocorticoid-attenuated response genes (GARGs) by differential hybridization screening of a lambda phage cDNA library from murine 3T3 fibroblasts. Seven of the GARGs were known genes, including the chemokines JE/MCP-1, fic/MARC/MCP-3, and crg2/IP-10. One of the novel cDNAs was a new C-X-C chemokine that we designated LIX, for LPS-induced C-X-C chemokine. Because the 12 GARG cDNAs were identified in a single screening of only 15,000 phage, and four were found as single isolates, these results suggest that there are many GARGs not yet described. Furthermore, six of the seven known GARGs encode proteins that modulate intercellular communication. These results support our hypothesis that GARGs predominantly encode products that function in paracrine cell communication. Here we provide an overview of the GARG strategy and the differential hybridization procedures used in our initial screening. A variety of other methods for identifying differentially expressed genes may be used in future searches for novel GARGs.
Insights
Researchers identified novel inflammatory mediators called glucocorticoid-attenuated response genes (GARGs) by screening for genes affected by inflammation and steroids. This method helps discover new molecules involved in cell communication.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Inflammatory responses involve complex gene expression changes.
- Glucocorticoids are potent anti-inflammatory agents that modulate gene expression.
- Identifying novel inflammatory mediators is crucial for understanding disease pathogenesis.
Purpose of the Study:
- To develop and apply a screening strategy for identifying novel inflammatory mediators.
- To characterize genes whose expression is induced by inflammatory stimuli and suppressed by glucocorticoids.
- To investigate the role of these genes in intercellular communication.
Main Methods:
- Screening for immediate early/primary response genes.
- Utilizing differential hybridization to screen a cDNA library from murine 3T3 fibroblasts.
- Inducing inflammatory responses with lipopolysaccharide (LPS) and assessing gene induction.
- Evaluating the effect of glucocorticoids on gene induction.
Main Results:
- Identified 12 lipopolysaccharide-induced, glucocorticoid-attenuated response genes (GARGs).
- Included known chemokines (JE/MCP-1, fic/MARC/MCP-3, crg2/IP-10) and a novel C-X-C chemokine (LIX).
- Seven of the 12 GARGs encode proteins involved in intercellular communication, supporting the paracrine communication hypothesis.
Conclusions:
- The GARG screening strategy is effective for identifying novel inflammatory mediators.
- GARGs predominantly encode proteins involved in paracrine cell communication.
- Further research using this strategy can uncover numerous undescribed GARGs.