Ciliary neurotrophic factor (CNTF) plus soluble CNTF receptor alpha increases cyclooxygenase-2 expression, PGE2 release and interferon-gamma-induced CD40 in murine microglia.
BACKGROUND
Ciliary neurotrophic factor (CNTF) has been regarded as a potent trophic factor for motor neurons. However, recent studies have shown that CNTF exerts effects on glial cells as well as neurons. For instance, CNTF stimulates astrocytes to secrete FGF-2 and rat microglia to secrete glial cell line-derived neurotrophic factor (GDNF), which suggest that CNTF exerts effects on astrocytes and microglia to promote motor neuron survival indirectly. As CNTF is structurally related to IL-6, which can stimulate immune functions of microglia, we hypothesized that CNTF might exert similar effects.


METHODS
We performed 2-D and 1-D proteomic experiments with western blotting and flow cytometry to examine effects of CNTF on primary microglia derived from neonatal mouse brains.


RESULTS
We show that murine microglia express CNTF receptor alpha (CNTFRalpha), which can be induced by interferon-gamma (IFNgamma). Whereas IL-6 activated STAT-3 and ERK phosphorylation, CNTF did not activate these pathways, nor did CNTF increase p38 MAP kinase phosphorylation. Using 2-D western blot analysis, we demonstrate that CNTF induced the dephosphorylation of a set of proteins and phosphorylation of a different set. Two proteins that were phosphorylated upon CNTF treatment were the LYN substrate-1 and beta-tubulin 5. CNTF weakly stimulated microglia, whereas a stronger response was obtained by adding exogenous soluble CNTFRalpha (sCNTFRalpha) as has been observed for IL-6. When used in combination, CNTF and sCNTFRalpha collaborated with IFNgamma to increase microglial surface expression of CD40 and this effect was quite pronounced when the microglia were differentiated towards dendritic-like cells. CNTF/sCNTFRalpha complex, however, failed to increase MHC class II expression beyond that induced by IFNgamma. The combination of CNTF and sCNTFRalpha, but not CNTF alone, enhanced microglial Cox-2 protein expression and PGE2 secretion (although CNTF was 30 times less potent than LPS). Surprisingly, Cox-2 production was enhanced 2-fold, rather than being inhibited, upon addition of a gp130 blocking antibody.


CONCLUSIONS
Our studies indicate that CNTF can activate microglia and dendritic-like microglia similar to IL-6; however, unlike IL-6, CNTF does not stimulate the expected signaling pathways in microglia, nor does it appear to require gp130.
Lin
Hsiao-Wen
HW
Department of Neurology and Neurosciences, UMDNJ-New Jersey Medical School Cancer Center, Newark, NJ, USA. hwlin2002@hotmail.com
Jain
Mohit Raja
MR
Li
Hong
H
Levison
Steven W
SW
D000818:Animals; D019013:CD40 Antigens / Q000378:metabolism*; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000378:metabolism*; D053739:Ciliary Neurotrophic Factor Receptor alpha Subunit / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D050822:Cytokine Receptor gp130 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005802:Genes, MHC Class II / Q000502:physiology; D007371:Interferon-gamma / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D050796:STAT3 Transcription Factor / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D019013:CD40 Antigens; D020934:Ciliary Neurotrophic Factor; D053739:Ciliary Neurotrophic Factor Receptor alpha Subunit; C494200:Il6st protein, mouse; D015850:Interleukin-6; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D014404:Tubulin; D050822:Cytokine Receptor gp130; D007371:Interferon-gamma; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone