In PBMCs, S100A4 was expressed in most CD3 + lymphocytes (Fig. 1a). The analysis showed S100A4 expression in CD4 (Fig. 1b) as well as CD8 positive T-lymphocytes (Fig. 1c). In these cells, S100A4 staining was predominantly cytoplasmic. Also, CD14 + monocytes stained positive for S100A4 (Fig. 1d), displaying a nuclear and cytoplasmic positivity.
In lymph nodes (Fig. 2a), S100A4 staining was observed in areas rich in T cells (Fig. 2c) and in CD68 + histiocytes and dendritic cells (Fig. 2b, d) but not in B cell rich areas (Fig. 2d). In vessels of connective tissue (Fig. 3a, b), S100A4 was highly expressed (nuclear and cytoplasmic) in cells of the subendothelial space, consisting of intimal smooth muscle cells, stellate-shaped pericyte-like cells and in the loose connective tissue of the tunica adventitia. Some positive cells were seen in the tunica media composed of smooth muscle cells, which expressed S100A4 in the cytoplasm. S100A4 was also expressed in the nuclei of most adipocytes of connective tissue (Fig. 3c, d). S100A4 was not detectable in peripheral nerves (Fig. 3c, d), unlike other S-100 antibodies directed against different alpha and beta subtypes(Gonzalez-Martinez et al. 2003). Muscle fibres of skeletal muscle showed staining for S100A4 (Fig. 3e). Less intense and more localised was the S100A4 expression of smooth muscle cells in the urothelial mucosa (Fig. 3f). Also, the urothelium showed cytoplasmic positivity in occasional umbrella cells (Fig. 3f).
In skin (Fig. 4a), S100A4 staining was negative in keratinocytes, but showed strong positive staining in Langerhans cells (Fig. 4b, d). Melanocytes did not express S100A4 (Fig. 4c).
In hypertrophic scar tissue (Fig. 5a), S100A4 showed variable cytoplasmic expression in dermal fibroblasts (Fig. 5d), often stronger in cells co-expressing smooth muscle actin (myofibroblasts; Fig. 5c). S100A4 staining was also seen in CD45 + leukocytes in this tissue (Fig. 5b). Stronger expression of S100A4 was seen in superficial fibromatosis (Fig. 6a, c, d). Also, here S100A4 expression mostly overlapped cells staining positive for smooth muscle actin (Fig. 6b).
Cultured synovial fibroblasts were predominantly negative for S100A4 (Fig. 7a). Smooth muscle actin was rarely expressed by these fibroblasts (Fig. 7b). Accordingly, positive immunohistochemical signal for S100A4 was only clearly detectable in the outer perimeter of 3D cultures of synovial fibroblasts (Fig. 7b), without staining for smooth muscle actin (Fig. 7d). Similarly, fibroblasts of tendon did mostly not or only weakly express S100A4 (Fig. 7e, f).
In the kidney (Fig. 8a, c), S100A4 was expressed in epithelial structures. The strongest and most uniform expression (nuclear and cytoplasmic) was seen in the loop of Henle (Fig. 8b). Also, many cells in the collecting duct expressed S100A4 (Fig. 8b). The distal tubules showed some nuclear and cytoplasmic positivity, more pronounced in the cortex of the kidney (Fig. 8d), with the most homogenous positivity seen in the macula densa (Fig. 8d).
Other investigated organs, including liver, breast, thyroid gland, pancreas, gallbladder, oesophagus, stomach, small intestine, skin and colon, did not express S100A4 in the epithelium (data not shown). The myofibroblast rich stromal cells of the prostate showed a diffuse predominantly moderate cytoplasmic expression of S100A4 (data not shown). The epithelial component in the prostate was negative for S100A4. Only a minority of cells of the uterine myometrium, made up of smooth muscle, showed a weak to moderate cytoplasmic expression of S100A4, the epithelium showed no reactivity for S100A4 (data not shown). The germinal epithelium of testis was negative for S100A4 (data not shown). The hormone secreting cells of the adrenal cortex were negative for S100A4, whereas the surrounding sustentacular cells were positive for S100A4 (nuclear and cytoplasmic).
Astrocytes and oligodendrocytes in brain tissue did not express S100A4, but microglia were positive (data not shown).
In general, the strongest and most consistent nuclear and cytoplasmic S100A4 expression levels were seen in monocytes (Fig. 1d), macrophages and specialised histiocytes, including dendritic cells in lymph nodes (Fig. 2b, d), Langerhans cells of the skin (Fig. 4b, d), Kupffer cells of the liver (data not shown) and microglia of the brain (data not shown).