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TNF-alpha AND IL-1 POTENTIATE AXON PROLIFERATION FROM MYENTERIC NEURONS IN CO-CULTURES
P-Y Gougeon, S Lourenssen, MG Blennerhassett
GIDRU, Department of Medicine, Queen’s University, Kingston, Ontario
BACKGROUND: Inflammatory bowel disease (IBD) causes smooth muscle hyperplasia and hypertrophy, which may underlie resulting changes in organ contractility. Inflammation also leads to significant neuronal loss, but is followed by a dramatic increase in axon numbers.
HYPOTHESIS: The intestine responds to inflammatory factors by inducing axonal proliferation to compensate for neuronal loss.
AIMS: Identify inflammation factors that stimulate axonal growth and examine their influence on the interplay between neurons, glia, smooth muscle cells, and the extracellular matrix. TNF-alpha and IL-1 are two pro-inflammatory cytokines associated with IBD and the rat TNBS model of colitis, suggesting they are potential targets.
METHODS and RESULTS: The effect of daily addition of TNF-alpha (50 ng/ml) or IL-1 (25 ng/ml) to a rat intestinal smooth muscle and myenteric plexus (SMMP) in vitro co-culture model was assessed by immunocytochemistry for the pan-neuronal marker HuD and the axonal marker SNAP-25. This revealed that TNF-alpha (44.8±11.9%) and IL-1 (31.5±9.6%) potentiated axonal growth by myenteric neurons. In addition, the media from the cytokine-treated co-cultures contained an increase in MMP-9 while MMP-2 levels were unaffected, as determined via zymogram. Employing a reductionist approach, we found that cytokine-treated pure cell lines of smooth muscle cells from either the circular or longitudinal muscle layers of the rat colon secreted MMP-9, while identically treated neonatal or adult glial cell lines secreted no detectable MMP-9 by zymogram. To evaluate the role of MMP-9 in TNF-alpha and IL-1-mediated axonal growth, SMMP co-cultures were treated with a MMP-9 inhibitor (5 nM, Calbiochem). The inhibitor had no effect on DMEM control co-cultures, but reduced TNF-alpha (50 ng/ml) and IL-1 (25 ng/ml)-induced axonal proliferation by 56.6±1.3% and 55.2±16.5% respectively.
CONCLUSION: These novel findings suggest that TNF-alpha and IL-1 stimulate myenteric axon growth at least in part through the induction of MMP-9 release from smooth muscle cells. The capacity of these cytokines to increase MMP-9 levels in the deeper layers of the SMMP may be highly beneficial to the muscle wall by helping axonal re-growth after inflammation and therefore preventing extensive smooth muscle hyperplasia and deficiencies in contractility.
Funded by GIDRU CIHR training grant, CCFC and NSERC