Discrete localization of various fatty-acid-binding proteins in various cell populations of mouse retina.

Various fatty acids (FAs) are involved as an energy source in many different functions in the organism. They are also essential ingredients of membranous lipids and act as intracellular signaling molecules. Intracellular fatty-acid-binding proteins (FABPs) comprise a family of soluble lipid-binding proteins with low molecular masses and solubilize long-chain FAs to allow intracellular translocation in the aqueous cytosol. To clarify the functions of FABPs in the retina, which is remarkably rich in polyunsaturated FAs, we have investigated the localization of B (brain type)-, H (heart type)-, E (epidermal type)-, and A (adipocyte type)-FABPs in adult mouse retinae by immunohistochemistry. In order to determine the possible involvement of FABPs in retinal degenerative diseases, we have also examined changes in FABP expression in light-induced photoreceptor cell degeneration (photic injury). The discrete localization of B-, H-, E-, and A-FABP species in various cell populations of the retina has been clarified: B-FABP is mainly localized in the cone photoreceptor cells, H-FABP in some populations of amacrine/bipolar/horizontal interneurons, and E-FABP in ganglion cells, with A-FABP-like immunoreactivity being located in resident microglia of normal retinae. E-FABP has further been localized in invasive macrophages in damaged retinae following photic injury, allowing discrete identification of the resident microglia and invasive macrophages by A- and E-FABP immunoreactivity, respectively.

First disclosure of lipid droplet substructure and myelin translucency in embedment-free section electron microscopy.

In conventional transmission electron microscopy (EM), thinly sectioned specimens embedded in epoxy resin are observed. However, because of a substantial level of electron density of epoxy resin, the possibility cannot be ruled out that bio-structures having electron density similar to that of epoxy resin are not clearly recognized and thus are neglected or misinterpreted in conventional EM. This was the reason to require for embedment-free EM. Embedment-free sections have already been made available reliably by transient embedding in polyethylene glycol (PEG) and subsequent de-embedding through immersion in water, and further by critical-point drying, and this embedment-free EM is thus termed PEG-EM. However, this PEG-EM has not been successful to attract reasonable attention from electron microscopists and instead been misunderstood as a non-reliable method. In this paper, the remarkably enhanced contrast and electron translucency of any observation targets in PEG-EM are clearly demonstrated by comparing with images in conventional EM of adipocytes and neural myelin as examples. These features of PEG-EM, together with faithful correspondence in EM images of any individual substructures between the two methods, confirm the reliability of PEG-EM. Furthermore, the much higher thickness of embedment-free sections together with these features makes the PEG-EM more advantageous than the conventional EM for three-dimensional appreciation of structural elements, which is made by stereo-viewing of sections or by EM tomography. Therefore, the PEG-EM is regarded as an important adjunct to the conventional EM for histological studies and wide application of this method may unravel a new level of histology.

Occurrence of immunoreactivity for adipocyte-type fatty acid binding protein in degenerating granulosa cells in atretic antral follicles of mouse ovary.

The localization of adipocyte-type fatty acid binding protein (A-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Solitary round cells showing the distinct immunoreactivity for A-FABP were detected in 1-6 antral follicles. In sets of two consecutive sections in a mirror alignment on slide glasses which were treated for immunoreactivity for A-FABP and TUNEL reaction separately, cells immunoreactive for A-FABP appeared in the same antral follicles as containing cells exhibiting TUNEL-reaction. In immunoelectron microscopy, A-FABP-immunopositive cells were found to contain highly electron-dense nuclei of round, irregular or crescent shapes together with cytoplasmic remnants without any features of macrophages or cells of extrinsic origin. Therefore the cells were identified as apoptotic granulosa cells. The apoptotic cells immunoreactive for A-FABP were often seen to be enclosed/engulfed in adjacent cells exhibiting normal ultrastructures without containing numerous lysosomes. The present findings suggest that A-FABP is involved in the apoptosis of ovarian granulosa cells, probably through its interaction with peroxisome proliferator activated receptors.

Localization of epidermal-type fatty acid binding protein in macrophages in advanced atretic follicles of adult mice.

The localization of epidermal-type fatty acid binding protein (E-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Numerous macrophages immunopositive for both anti-E-FABP and F4/80 antibodies, together with immunonegative cells, were found in advanced atretic follicles that had eccentric lumens containing deformed ova. While some E-FABP-immunopositive macrophages were spider in shape and appeared singly, others, especially close to the lumen, were round and voluminous and tended to be aggregated. The voluminous macrophages contained phagosomes of various sizes and they were regarded as those actively involved in the phagocytosis of apoptotic granulosa cells. E-FABP-immunopositive macrophages and their processes were often apposed to adjacent immunonegative cells, and some of them lined the lumen containing deformed ova. On the other hand, E-FABP-immunonegative cells in the atretic follicles were classified into two types: the one, a minority, was characterized by small mitochondria containing non-tubular cristae and presumably represented residual granulosa cells, while the other dominant type was characterized by large mitochondria containing tubular cristae and presumably represented theca cells originally surrounding the follicles to be atretic. The present detection of E-FABP-immunopositivity selectively in macrophages of the atretic follicles suggests possible involvement of E-FABP and/or its ligand fatty acids in the process of follicular atresia, and it makes more reliable the identification of the advanced atretic follicles with the antral spaces obliterated, which could provide further details on the histology of the follicular atresia than before.

Differential localization of brain-type and epidermal-type fatty acid binding proteins in the adrenal gland of mice.

In immuno-light and -electron microscopy, brain-type fatty acid binding protein (B-FABP) is localized in the sustentacular cells enclosing the chromaffin cells in the adrenal medulla. This represents another new feature commonly shared by the sustentacular cells and ganglionic satellite cells, the latter of which has already been reported to localize this molecule, and suggests a common feature in lipid metabolism shared by the two cells enclosing peripheral neurons and paraneurons. On the other hand, epidermal-type fatty acid binding protein (E-FABP) is localized in two discrete cells in the adrenal gland: the one is a subpopulation of intra-adrenal macrophages which are intensely immunoreactive for F4/80, a marker of macrophages, and are rich in pleomorphic lysosomes. Because of their direct apposition to adjacent cortical endocrine cells and medullary chromaffin cells, the macrophages may be involved not only in phagocytosis of degenerating adrenal cells but also in exertion of some yet unknown effects on the endocrine function of the cortical and medullary cells via humoral factors such as cytokines which have recently been known to be secreted by macrophages. The other is a population of cells having scanty perikaryal cytoplasm poor in organneles and several thinny extended processes in the cortex and exhibiting weak immunoreactivity for E-FABP. The possible natures of these cells immunoreactive for E-FABP are discussed in view of a subpopulation of endothelial cells or the dendritic cells of antigen-presenting property.

Specific localization of epidermal-type fatty acid binding protein in dendritic cells of splenic white pulp.

Dendritic cells in the splenic white pulp of mice were intensely immunoreactive for epidermal-type fatty acid binding protein (E-FABP). This specific immunostaining revealed a clear difference in morphology between the dendritic cells in the periarterial lymphoid sheath (PALS) and follicular dendritic cells in the follicles in terms of cell sizes and process branching. No immunoreactivity was detected in dendritic cells in the marginal zones and the red pulp, although endothelial cells of almost all capillaries in the red pulp were immunoreactive for E-FABP. After peritoneal injection of lipopolysaccharide, the immunoreactive cells in PALS progressively enlarged and became rounded in shape with a peak in size at 24 h postinjection and they eventually resumed the dendritic form at 48 h postinjection. Within each of the enlarged immunoreactive cell perikarya were included small immunonegative apoptotic cells, presumptive lymphocytes. Taken together, E-FABP is useful as a marker for dendritic cells in the splenic white pulp, and may be involved through combination with fatty acids in antigen presentation and retention as well as in cytokine production.

Localization of brain-type fatty acid-binding protein in Kupffer cells of mice and its transient decrease in response to lipopolysaccharide.

Brain-type fatty acid-binding protein (B-FABP) was localized in Kupffer cells of liver of postnatal day 10 (P10) and older mice in immunolight and electron microscopy as well as by in situ hybridization histochemistry. The immunoreaction products were localized in the cytoplasmic matrix but not within the nucleus. After peritoneal injection of lipopolysaccharide (LPS), the immunoreaction for B-FABP decreased markedly in Kupffer cells at 1 h postinjection and thereafter gradually recovered to the preinjection level by 24 h postinjection, although no decrease in the mRNA expression was detected in Northern blotting throughout the course after the injection. The specific localization of B-FABP, but not the other FABPs, in Kupffer cells, and its rapid decrease after LPS injection suggest the intimate involvement of B-FABP in Kupffer cells in the inflammatory reaction, probably through mediation of n-3 polyunsaturated fatty acids, which are strong binders of B-FABP.

Localization of epidermal-type fatty acid binding protein in the thymic epithelial cells of mice.

The immunoreactivity for epidermal-type fatty acid binding protein of epidermis type (E-FABP) was selectively localized in the epithelial cells of both cortex and medulla of mouse thymus. The cortical epithelial cytoreticulum was clearly visible with the intense immunoreactivity and the immunoreactive cytoreticulum extended intricately throughout the thymic cortex to enclose thymocytes. In the thymic medulla, the immunoreactivity was variable in intensity among the epithelial cells and there was a tendency that epithelial cells containing more numerous tonofilament bundles were less immunoreactive. Considering the possibility that FABPs function as intracellular carriers for unsaturated long chain fatty acids, the present finding suggests that E-FABP in the thymic epithelial cells, especially the cortical ones because of their extensive location, are intimately involved in the metabolic processes of fatty acids including production of bioactive substances, such as prostaglandin and leukotriene, which are known to exert some regulation of thymic immune responses.