Abstract
The multigene family of Ca2+-binding S100 proteins comprises 22 members that
have various important intra- and extracellular roles. The three inflammation-associated
members of this family S100A8, S100A9 and S100A12 (collectively termed
"calgranulins") are constitutive neutrophil and monocyte proteins also expressed by
macrophages within acute and chronic inflammatory lesions, but not in tissue
macrophages. They are expressed in human/murine wounds and by appropriately
activated macrophages, microvascular endothelial cells and keratinocytes in vitro. The
" calgranulins" are implicated in leukocyte activation/deactivation, fatty acid transport,
leukocyte/fibroblast chemotaxis, transmigration and adhesion, embryogenesis, wound
healing, protection against oxidants and antibacterial defence.
Chapter 3 of this thesis explores growth-factor- and cytokine-mediated regulation
and expression of S100A8 and S100A9 in fibroblasts, and demonstrates spatio-temporal
expression of S100A8 in rat dermal wounds. Fibroblasts are stromal resident cells with
important regulatory immune-inflammatory functions in wound healing, tissue
remodelling and fibrosis. Fibroblast migration, proliferation, differentiation and their
synthetic repertoire are modulated by various factors including extracellular matrix
components, growth factors, prostaglandins, reactive oxygen species and cytokines.
Fibroblast growth factor-2 (FGF-2), interleukin-1?(IL-1? and platelet-derived growth
factor (PDGF) are potent fibroblast mitogens; PDGF and transforming growth factor-?
(TGF-? are fibroblast chemoattractants. FGF-2 and IL-1?promote fibroblast
proliferation, whereas TGF-?promotes myofibroblast differentiation and collagen
production. Lipopolysaccharide (LPS), interferon ?(IFN?, tumour-necrosis factor ?
(TNF?, TGF-?and PDGF did not induce the S100A8 gene in fibroblasts whereas FGF-2
(25 ng/ml) maximally induced mRNA 12 hr. after stimulation and this declined over 36
hr. The FGF-2 response was strongly enhanced and prolonged by optimal levels of
heparin (1-10 IU/ml), maximally at 18 hr. post-stimulation. FGF-2/heparin-induced
responses depended on cell-cell contact in vitro. IL-1?(10 U/ml) alone, or in synergy
with FGF-2/heparin strongly induced the gene in 3T3 and primary fibroblasts.
Dexamethasone (10−6 M) enhanced LPS- and FGF-2/-IL-1?induced responses. S100A9
mRNA was not induced by any of these mediators. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblast-like cells by real-time reverse-transcriptase
polymerase chain-reaction. FGF-2-heparin- and IL-1?induced mRNA expression
depended on de-novo protein synthesis and was partially mediated by the mitogenactivated
protein kinase pathway of activation. Preliminary promoter deletion analyses
indicated that FGF-2-responsive elements in the gene promoter were distinct from those
responsive to IL-1? TGF-?(2 ng/ml) significantly suppressed gene induction mediated
by FGF-2 ?heparin/LPS/dexamethasone, but not by IL-1? TGF-?may compromise
mRNA stability. Protein levels in FGF-2-heparin-IL-1?stimulated fibroblasts correlated
well with mRNA levels and expression was mainly cytoplasmic. Immunohistochemistry
indicated S100A8 associated with keratinocytes, neutrophils, macrophage-like cells and
some hair follicles in wounded rat skin. Rat wounds also contained numerous S100A8-
positive fibroblast-like cells 2 and 4 days post-injury; numbers declined by 7 days. Upregulation
of S100A8 by FGF-2/IL-1? down-regulation by TGF-? and time-dependent
expression of S100A8 in wound fibroblasts suggest a role in fibroblast differentiation at
sites of inflammation and repair. Intracellular fibroblast-derived S100A8 may also
regulate intracellular redox equilibrium and antioxidant defence.
Atherosclerosis is a progressive chronic disease with complex aetiology and
pathogenesis. S100A1 and S100B are associated with dendritic cells and lymphocytes in
experimental rodent and human atherosclerotic lesions. Monocytes and macrophages in
plaques of ApoE−/− mice express S100A9 but not S100A8. Myeloperoxidase and HOClmediated
oxidative mechanisms are fundamental in the pathogenesis of atherosclerosis
and S100A8 is exquisitely sensitive to HOCl oxidation which generates sulphinamide
bonds, novel non-reducible cysteine-lysine covalent bonds. Chapter 4 of this thesis
presents novel evidence that, in contrast to the murine ApoE−/− model, the three human
" calgranulins" were expressed in human atherosclerotic plaques, but not in normal
arteries. High levels of S100A8, S100A9 and S100A12 were evident in macrophages and
foam cells. Some neovessels were anti-S100A8-/anti-S100A9-immunoreactive; S100A9
staining was also evident on the extracellular matrix. Patterns of expression of S100A8,
S100A9 and S100A12 were overlapping in serial sections, except that only smooth
muscle cells were S100A12-positive. S100A8 and S100A9 mRNA were also expressed
by macrophages, foam cells and endothelial cells, indicating gene up-regulation rather
than passive protein uptake. Western blotting of plaque extracts revealed monomeric
S100A8, S100A9 and S100A12 and larger complexes. Some were resistant to reduction,
suggesting non-disulfide covalent cross-linking, possibly via sulphinamide bonds. Stable
S100A8-S100A9 complexes were also detected after immunoaffinity purification. In an
in-vitro system, molar ratios of HOCl of >1 generated stable complexes of S100A8 and
S100A9 whereas ~800 and ~100-fold excess HOCl oxidises apolipoprotein B-100 and
BSA, respectively. S100A8 and S100A9 protected low-density lipoprotein (LDL) against
HOCl oxidation in a thiol-independent manner. Because HOCl-oxidised S100s did not
contain epitopes recognised by an antibody used to detect HOCl-oxidised proteins in
plaque, levels of oxidised proteins in plaque are likely to be significantly greater than
described. S100A8 and S100A9 may protect LDL by functioning as HOCl-scavengers.
However, chronic oxidative cross-linking of S100A8 and S100A9 with other proteins and
extracellular matrix components may contribute to plaque pathogenesis.
These studies support key roles for the " calgranulins" in chronic inflammation,
wound healing and atherogenesis possibly by regulating cellular differentiation, activation
and modulation of redox-dependent mechanisms.