This thesis addresses the hypothesis that the sonic hedgehog (Shh) signalling
pathway is up regulated in response to cellular injury and that this signal acts as
a potential pro-fibrogenic signal in pulmonary inflammation. In vitro studies
utilised human (A549) and mouse (CMT) type II like epithelial cell lines in an
analysis of epithelial response to injury. An immediate up regulation and release
of soluble Shh in response to hydrogen peroxide exposure was identified in
mouse epithelium, utilising both RT-PCR and a novel Shh ELISA developed
here. Subsequent up-regulation and release of GM-CSF was shown to be Shh
independent. Human epithelial cells demonstrated a similar release of Shh,
suggesting a common pathway in both species. In contrast GM-CSF was not up
regulated in human cells, but IL-8 up regulation did occur. The relevance of
these studies to in vivo signalling was ascertained through use of the FITC
instillation mouse model of inflammatory fibrosis.Previous work by our group has illustrated epithelial up-regulation of Shh in
areas of inflammatory fibrotic disease in the FITC model, and in biopsies from
patients with idiopathic pulmonary fibrosis. Transoral intratracheal delivery of
FITC on two occasions six weeks apart reproduced previous histological
findings made in the lab using a surgical intratracheal FITC instillation.
However, use of improved immunohistochemical (IHC) techniques would
suggest that, unlike human disease, Shh expression in the FITC mouse model
is not solely restricted to epithelium in areas of inflammation or fibrosis. To
determine the Shh responder cell populations in human disease and determine
the validity of the IHC findings in the FITC model, a laser micro-dissection
technique was developed. Viable mRNA for sequences including the Shh
receptor patched (Ptc), and downstream signalling component smoothened
(Smo) have been recovered from archival formalin fixed human biopsy material,
sufficient for RT-PCR and agarose gel electrophoresis visualisation. mRNA
analysis of material derived from fibrotic areas of a single standard section of
archival human lung biopsy has now been performed.Observations correlating levels of FITC specific lgG1 antibody and disease
severity in FITC treated animals suggest an immuno-modulatory role for T
lymphocyte dependent, Th2 type antibodies in disease progression. In support
of this iBALT has also been identified in diseased lung and T-lymphocyte
depletion results in disease amelioration.Taken together these findings suggest that Shh is an indicator of acute cellular
injury, but that the principal component of FITC induced fibrotic disease
progression is immune mediated and that this does not depend on Shh up
regulation
