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Granulomas were induced in rat liver by the injection of glucan, a beta-1,30-polyglucose with an activating effect on macrophages. The livers were fixed by perfusion with glutaraldehyde and processed for cytochemical localization of peroxidase (PO) as well as for scanning and transmission electron microscopy. By light microscopy the granulomas appeared as spherical cell clusters located throughout the liver parenchyma. They exhibited strong PO activity at the periphery with weak to negative reaction at the center. Most granulomas were in direct continuity with adjacent sinusoids. Morphometry revealed that the volume of granulomas increased to about 10 per cent of the total parenchymal volume at 10 days after glucan treatment, decreasing to less than 2% at 90 days. By scanning electron microscopy most cells at the periphery of granulomas were covered with numerous blebs and ruffles, the typical surface features of activated macrophages. By transmission electron microscopy the typical granulomas consisted of mononuclear phagocytes, giant cells, and a few lymphocytes. The phagocytes, as identified by phagocytosis of glucan, exhibited three distinct PO patterns: (1) strong PO activity in the nuclear envelope and endoplasmic reticulum with no activity in cytoplasmic granules; (2) weak or absent reaction in nuclear envelope and endoplasmic reticulum with no PO-positive granules in cytoplasm; and (3) absent reaction in nuclear envelope and endoplasmic reticulum but with a few strongly stained cytoplasmic granules. The first cell type, resembling Kupffer cells, was found mainly at the granuloma periphery. The second cell type, identified ultrastructurally as epithelioid cells, and the third cell type, with PO features of monocytes, were found mainly in the center of granulomas. The multinucleated giant cells which were also present in the center of granulomas exhibited weak to negative PO reaction in nuclear envelope and endoplasmic reticulum and contained large numbers of glucan particles. These observations suggest that most cells in the center of the granulomas, including the giant cells, are derived from blood monocytes. The mononuclear phagocytes at the periphery, on the other hand, seem to be for the most part former Kupffer cells.

The mononuclear phagocytes (Kupffer cells) in the normal rat liver can be distinguished from the endothelial cells on the basis of their endogenous peroxidase activity in the endoplasmic reticulum and their exclusive ability to phagocytose large (0.81 mum.) latex particles. Using these cellular markers we have investigated the effects of an estrogen upon the mitotic activity and the ultrastructure of individual types of littoral cells in the rat liver. Adult female rats received a single 10-mg. injection of diethylstilbestrol, and at daily intervals up to 6 days their livers were fixed by perfusion and processed for localization of peroxidase. Mitotic figures were rare in untreated control animals, but dividing littoral cells with both positive and negative peroxidase reaction could be identified. The exclusive localization of injected latex particles in dividing peroxidase-positive cells indicated that peroxidase reaction identified the Kupffer cells not only in the interphase but also during the mitotic division. In estrogen-treated animals there was a sharp rise in the mitotic activity of littoral cells; the activity reached its peak on the 3rd day and returned to normal levels on the 6th day after the initial injection. A breakdown of the dividing cells on the basis of their peroxidase reactivity revealed that nearly the entire population of dividing cells consisted of peroxidase-negative endothelial cells. In addition, numerous hyperactive Kupffer cells containing large phagolysosomes with phagocytosed peripheral blood cells and latex particles were seen. Intermediate cell-types with cytochemical features between Kupffer cells and endothelial cells or between monocytes and Kupffer cells were not encountered. Because of the limited phagocytic capacity of hepatic endothelial cells, our observations would provide morphologic evidence in support of previous physiologic studies, indicating that estrogen treatment has little or no effect upon the particle clearing function of the reticuloendothelial system in rats. The rare but clear demonstration of dividing Kupffer cells in liver sinusoids would indicate that these cells are capable of self-replication in situ. Finally, our observations suggest that estrogens may play an important role in the pathophysiology of endothelial cells.

Multiple injections of glucan, a beta-1,3-polyglucose with a potent stimulating effect on the mono-nuclear phagocytes, induced hemopoiesis in adult rat liver. Hemopoietic islands were present in the connective tissue of the portal triads and in the perisinusoidal spaces. The use of peroxidase cytochemistry facilitated the detection of such islands at the light microscopic level and the identification of their cellular components at the ultrastructural level. The hemopoietic colonies consisted of erythroid and myeloid series. The erythropoietic cells comprised erythroblasts, normoblasts, and reticulocytes, all of which contained peroxidase reaction product diffusely in their cytoplasm. The granulopoietic series consisted of promyelocytes (peroxidase activity in nuclear envelope, endoplasmic reticulum, Golgi apparatus, and granules) and myelocytes (peroxidase only in granules). The hemopoietic islands were always associated with macrophages which exhibited peroxidase activity in NE and endoplasmic reticulum but not in the Golgi apparatus. This association between hemopoietic cells and "glucan-activated" macrophages shows the central role of macrophages in differentiation of hemopoietic stem cells and suggests that the macrophage-activating potency of glucan and its effect on hepatic hemopoiesis are interrelated.

The effects of a new piperazine derivative BM 15766, which inhibits the biosynthesis of cholesterol at the 7-dehydrocholesterol-delta 7-reductase step, upon the ultrastructure of rat liver and the serum lipids, have been investigated. Treated animals showed a marked reduction in total sterol content in serum with simultaneous reduction of triglycerides. The catalase activity in liver homogenates was unchanged, carnitine acetyltransferase increased only slightly, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase was augmented by a factor of 2. In sections stained with alkaline 3,3'-diaminobenzidine for catalase, distinct proliferation of peroxisomes (PO) in perivenous regions of the hepatic lobules was noted in rats of both sexes. In male animals many PO showed loop-like extensions and invaginations of their limiting membranes into the matrix. Such alterations were seen less frequently in female animals; instead, females exhibited in the same regions of the hepatic lobules, large aggregates of PO, smooth endoplasmic reticulum, and mitochondria with longitudinal cristae. Close contacts of PO and fenestrated segments of smooth endoplasmic reticulum were noted in both sexes. These observations demonstrate the marked adaptive response of rat hepatocyte organelles to severe hypocholesterolemia induced by BM 15766. The alterations of PO may reflect attempts to increase their surface membrane, which plays a crucial role in the exchange of substrates between the cytoplasm and the peroxisomal matrix. Moreover, the close association of PO and smooth endoplasmic reticulum could facilitate the shuttle of lipid intermediates between these two intracellular compartments involved in the biosynthesis of complex lipids.

The cytochemical localization of catalase and peroxidase in various sinusoidal cells of rat liver, i.e., in Kupffer cells, endothelial cells, and fat-storing cells has been investigated. The alkaline 3,3'-diaminobenzidine technique reveals distinct "catalase-positive particles" in all three cell types. The particles are round to oval-shaped, measuring 0.1 to 0.3 mum. in diameter. The diaminobenzidine reaction product is distributed uniformly over their matrix, often obscuring the distinct limiting membrane. In fat-storing cells the particles appear in close proximity of lipid droplets. No evidence of fusion of the limiting membrane of the particles with that of phagolysosomes containing latex particles was observed. The "catalase-positive particles" appeared often in close proximity of endoplasmic reticulum, but by examining consecutive serial sections we could not find any convincing evidence of direct continuity between the two organelles. In addition to catalase there is an endogenous peroxidase in the endoplasmic reticulum and nuclear envelope of Kupffer cells. Whereas peroxidase is sensitive to aldehyde fixation and has its optimal pH in the neutral range, the staining for catalase requires prior fixation with glutaraldehyde and isoptimal at pH 10.5. By using proper fixation and incubation conditions the two enzymes have been visualized selectively, and it is demonstrated that they occupy two distinct intracellular compartments within the Kupffer cells: the catalase in the matrix of particles and the peroxidase in the endoplasmic reticulum. The possible functional role of catalase in various sinusoidal cells is briefly discussed.