Abstract
Cluster of differentiation (CD)-40 is a cell surface receptor belonging to the tumor necrosis factor receptor family that plays a critical role in the regulation of immune responses. We have previously shown that the cytokine interferon (IFN)-gamma induces CD40 expression in microglia. Herein, we have elucidated the molecular mechanisms underlying IFN-gamma induction of CD40 gene expression in microglia/macrophages. IFN-gamma up-regulates CD40 expression at the transcriptional level, and this regulation involves the STAT-1alpha transcription factor. Microglia from STAT-1alpha-deficient mice were refractive to IFN-gamma induction of CD40 expression, illustrating the importance of STAT-1alpha in this response. Functional analysis of the CD40 promoter indicates that two gamma activated sequence elements as well as two Ets elements are involved in IFN-gamma induction of CD40 promoter activity. STAT-1alpha binds to the gamma activated sequence elements, whereas PU.1 and/or Spi-B bind to the Ets elements. The expression of PU.1 and Spi-B, in conjuction with STAT-1alpha activation, correlates with IFN-gamma inducibility of CD40 expression. Collectively, our data demonstrate the involvement of STAT-1alpha, PU.1, and Spi-B in IFN-gamma induction of CD40 gene expression in cells of the macrophage lineage.
Highlights
CD401 is a 50-kDa type I phosphoprotein member of the tumor necrosis factor-receptor superfamily
IFN-␥-induced CD40 Gene Expression Is Mediated at the Transcriptional Level and Requires STAT-1␣—We have shown previously that IFN-␥ is the most potent inducer of CD40 expression in microglia, the resident macrophage of the brain [17]
Because microglia and macrophages are derived from the same lineage [32], we wished to compare the extent of IFN-␥ induction of CD40 expression in macrophages with that of microglia
Summary
Recombinant Proteins and Reagents—Recombinant murine IFN-␥ was purchased from Genzyme (Boston, MA) and recombinant human IFN-␥ was from Biogen (Cambridge, MA). The murine cells (RAW264.7, EOC13, primary microglia, A20, and NIH 3T3) were scraped, incubated with 100 l of 2.4G2 hybridoma supernatant (which contains rat anti-mouse Fc␥R antibody) supplemented with 10% normal mouse serum for 30 min at 4 °C, washed, incubated with 10 g/ml of anti-CD40 antibody for 30 min at 4 °C, washed, incubated with 10 g/ml of biotinylated anti-rat IgG2a, washed, incubated with 10 g/ml of phosphatidylethanolamine-conjugated strepavidin, washed, and were fixed in a final volume of 200 l of 1% paraformaldehyde. EMSA was performed with 5 g of nuclear extract in a total volume of 15 l of binding buffer (50 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 4% glycerol, 0.5 mM dithiothreitol, 4 mM Tris-Cl (pH 7.5), 1 g of polydeoxyinosinic-deoxycytidyl acid, and 20,000 cpm 32P-labeled oligonucleotide probe), and incubated on ice for 15 min. Values for CD40 expression were normalized to GAPDH levels for each experimental condition
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
