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A3 was generated as an antibody recognizing somatic stem cells in rat tissues. We investigated the distribution of A3-positive cells in developing rat hair follicles by immunolabeling. A3-positive cells began to be seen in the hair germ and peg in fetuses and neonates; the positive cells were epithelial cells above basal cells. Furthermore, A3-positive cells were seen in the outer root sheath adjacent to the bulge in mature hair follicles. Double immunofluorescence revealed that these A3- positive epithelial cells reacted to E-cadherin (for all epithelial elements) but not to CK15 (for basal cells/epithelial stem cells) or to nestin (for stem cells), indicating that A3-positive epithelial cells are suprabasal cells in the developing epidermic hair follicle. Additionally, spindle-shaped mesenchymal cells surrounding the hair peg and mature hair follicle reacted to A3; in double immunofluorescence, the A3-positive cells were located outside collagen type IV-positive glassy membrane, and reacted to vimentin (for mesenchmal cells), Thy-1 (for immature mesenchymal cells), CD34 (for stem cells) and nestin, but not to α- smooth muscle actin (for myofibroblasts); the positive cells were regarded as immature mesenchymal cells with stem cell nature in the connective tissue sheath of developing hair follicles. A3-positive epithelial and mesenchymal cells did not show proliferating activity. Collectively, it is considered that A3-positive cells seen in developing rat hair follicles may be quiescent postprogenitor cells with the potential to differentiate into either highly-differentiated epithelial or mesenchymal cells. A3 would become a useful antibody to know the kinetics of rat hair follicle-constituting cells.

Macrophages play important roles in host defense and homeostasis. In contrast to adulthood, far less is known about macrophage populations in fetuses and neonates. Macrophages were evaluated in the developing rat skin at different anatomical sites (head, anterior dorsal, posterior dorsal, and abdomen) of F344 rats obtained on gestational days 18 and 20, on neonatal days 1-21, and at adult weeks 5-15. The numbers of macrophages in the epidermis, dermis or perifollicular areas that were positive for ED1 (exudative macrophages with activated phagocytosis), ED2 (resident macrophages), and OX6 (antigen-presenting cells) were evaluated. There were no differences in macrophage numbers among the anatomical sites. In the epidermis, only OX6 cells were seen, with gradually increased numbers in neonates and adults. In the dermis, many ED1 cells were already seen in fetuses, and the number peaked on neonatal day 4, and remained at that level until adulthood. By contrast, ED2 and OX6 cells began to be seen after birth and their numbers continued to increase until adulthood; ED2 cells were distributed diffusely in the dermis, whereas ED1 and OX6 cells were present exclusively in the upper dermis. In perifollicular areas, ED1, ED2 and OX6 cells began to be seen after birth, and their numbers gradually increased until adulthood. Some macrophages in dermal and perifollicular areas gave double-positive reactions to ED1+ED2+, ED1+OX6+ or OX6+ED2+. Increased mRNA levels of colony stimulating factor-1 and monocyte chemoattractant protein-1 appeared to correspond to the emergence of rat macrophages. Skin macrophages were shown to be heterogeneous in distribution and function; the information from this study should be very useful for future investigations of experimentally induced rat skin lesions.

Macrophages play important roles in host defense mechanisms. In the brain, besides microglial cells, meningeal macrophages are present. However, the pathobiological characteristics of meningeal macrophages in rats remain to be investigated. In normal meninx, immunohistochemically, macrophages reacting to CD163 (macrophage scavenger receptor) and major histocompatibility complex (MHC) class II-expressing cells (involving activated macrophages or dendritic cells) were sporadically seen without age-dependent changes. Injection of lipoplysaccharide (LPS) (5 μg; Escherichia coli) into the cerebrum increased the number of anti-CD68-positive macrophages (with greater phagocytic activity) in the meninx, with a peak at 12 h during observation period until 48 h; MHC class IIexpressing cells showed a gradual increase in number from 3 h after injection; however, anti-CD163-positive macrophages did not show significant change. In in vitro studies, LPS (0, 0.02, 0.05, 0.5, 5, 50 and 100 μg/ml) was added to KMY-1 or KMY-2 cells, both of which had been established from a rat malignant meningioma. KMY-1 originally reacted to CD163, but LPS addition at 0.5 μg/ml and greater concentrations decreased the anti- CD163-positive cell number and instead increased the anti-CD68-positive cell number. LPS-treated KMY-2 increased the anti-CD163-positive cell number at 0.05 and 0.5 μg/ml. By RT-PCR methods, LPS (0, 0.5, 5, 50, and 100 μg/ml)-treated KMY-1 and KMY-2 showed an increase in mRNA of monocyte chemoattractant protein- 1 (MCP-1, a chemokine), and LPS-treated KMY-2 increased mRNA of nerve growth factor (NGF, an immunological effecter). Collectively, under LPS treatment, macrophages with heterogeneous functions appear in rat meninx; rat meninx-forming cells may be involved in pathogenesis of meningeal inflammation by expressing different immunophenotypes and by producing regulatory proinflammatory factors such as MCP-1 and NGF.