Role of CCL19/21 and its possible signaling through CXCR3 in development of metallophilic macrophages in the mouse thymus

We have already shown that metallophilic macrophages, which represent an important component in the thymus physiology, are lacking in lymphotoxin-β receptor-deficient mice. However, further molecular requirements for the development and correct tissue positioning of these cells are unknown. To this end, we studied a panel of mice deficient in different chemokine ligand or receptor genes. In contrast to normal mice, which have these cells localized in the thymic cortico-medullary zone (CMZ) as a distinct row positioned between the cortex and medulla, in plt/plt (paucity of lymph node T cells) mice lacking the functional CCL19/CCL21 chemokines, metallophilic macrophages are not present in the thymic tissue. Interestingly, in contrast to the CCL19/21-deficient thymus, metallophilic macrophages are present in the CCR7-deficient thymus. However, these cells are not appropriately located in the CMZ, but are mostly crowded in central parts of thymic medulla. The double staining revealed that these metallophilic macrophages are CCR7-negative and CXCR3-positive. In the CXCL13-deficient thymus the number, morphology and localization of metallophilic macrophages are normal. Thus, our study shows that CCL19/21 and its possible signaling through CXCR3 are required for the development of thymic metallophilic macrophages, whereas the CXCL13–CXCR5 signaling is not necessary.     

Macrophages of the rat thymus after cyclosporin treatment

Young adult Wistar rats received 40 mg/kg of cyclosporin perorally for 21 days. Cyclosporin induced almost total disappearance of thymic medulla, whereas the cortex remained preserved. Although the density of cortical macrophages did not change significantly, their characteristics altered markedly and they became enlarged and rounded. In addition to an increase in acid phosphatase and nonspecific esterase activities, cortical macrophages developed very strong succinic dehydrogenase and chloroacetate esterase activities and a fine, granular, aldehyde fuchsinpositive cytoplasmic content. However, these cytoplasmic granules were PAS-negative and were not sudanophilic. Cortical macrophages retained their normal antigenic properties (which were studied by the use of ED1, ED2 and R-MC 41 monoclonal antibodies). Phagocytic cells in the remaining medullary islands retained their usual characteristics. The changes in cortical macrophages after cyclosporin treatment are discussed, especially in relation to the characteristics of macrophages of the cortico-medullary zone in the normal rat thymus.     

Cytochemical Observations on Malic Dehydrogenase,Lipase, Nonspecific Esterase and Monoamine Oxidase in Chick Liver Cell Cultures Infected with Trichomonas vaginalis*

SYNOPSIS. In trypsin-dispersed chick liver cell cultures malic dehydrogenase activity is localized in granules distributed thruout the cytoplasm of fibroblasts, epithelial cells, and macrophages. A progressive increase of the enzymic activity in all cell culture elements, except for phagocytes whose external or internal surfaces remain in direct contact with the parasites, accompanies infection of the cultures with a relatively pathogenic strain of Trichomonas vaginalis. In such phagocytes most staining for malic dehydrogenase is lost. Epithelial cells and parasite-free macrophages in experimental cultures also have a diffuse cytoplasmic reaction. No lipase activity is present in fibroblasts, but epithelial cells and macrophages in chick liver cell cultures contain numerous reactive granules. A strong diffuse cytoplasmic reaction is found in the epithelial cells and a weaker one in the control phagocytes. In cultures infected with T. vaginalis the enzyme is lost progressively from the epithelium and from those macrophages which have engulfed the parasites or whose external surfaces are invested with the flagellates; however, no significant changes in lipase activity can be found in parasite-free experimental phagocytes. In all cell types found in chick liver cultures, the reaction for nonspecific esterase is localized in cytoplasmic inclusions of varying size, some of which tend to accumulate along the cell membranes and around the nuclei. In addition, a weak diffuse cytoplasmic reaction is seen in the epithelial cells. Most cells in cultures infected with T. vaginalis have a significant increase in esterase activity, the exception being the macrophages which contain parasites within their cytoplasm or those with flagellates applied closely to their external surfaces. Only a few specifically stained granules are retained by such phagocytes. Monoamine oxidase activity is limited to fine granules dispersed in the cytoplasm of fibroblasts, epithelial cells, and macrophages of control cultures. Infection of chick liver cultures with T. vaginalis results in lowered enzyme activity in non-phagocytic cells as well as in macrophages with engulfed flagellates and in those whose external surface are invested with the parasites. The number of reactive inclusions appears to increase in trichomonad-free phagocytes of experimental cultures.     

Macrophages of the rat thymus after cyclosporin treatment. Histochemical, enzymehistochemical and immunohistochemical study

Young adult Wistar rats received 40 mg/kg of cyclosporin perorally for 21 days. Cyclosporin induced almost total disappearance of thymic medulla, whereas the cortex remained preserved. Although the density of cortical macrophages did not change significantly, their characteristics altered markedly and they became enlarged and rounded. In addition to an increase in acid phosphatase and nonspecific esterase activities, cortical macrophages developed very strong succinic dehydrogenase and chloroacetate esterase activities and a fine, granular, aldehyde fuchsin-positive cytoplasmic content. However, these cytoplasmic granules were PAS-negative and were not sudanophilic. Cortical macrophages retained their normal antigenic properties (which were studied by the use of ED1, ED2 and R-MC 41 monoclonal antibodies). Phagocytic cells in the remaining medullary islands retained their usual characteristics. The changes in cortical macrophages after cyclosporin treatment are discussed, especially in relation to the characteristics of macrophages of the cortico-medullary zone in the normal rat thymus.     

Hydrolytic enzyme activity of rat thymic cells grown in vitro

Summary An electron-microscopic study of the fine-structural localization of acid phosphatase, non-specific esterase and aryl sulphatase activity in cultured rat thymus tissue showed the existance of three different types of cell, two of which conformed to the epithelial reticular cells and the macrophages present in vivo. The origin of the third is unknown. In all three types, and even within the very same cell, the lysosomes differ in activity. This finding argues in favour of the heterogeneity in the lysosomes of thymic cells.     

Ultrastructural and histochemical features of the thymus glands of the adult lungless salamander, Plethodon glutinosus (Caudata: Plethodontidae).

The thymus glands of adult slimy salamanders (Plethodon glutinosus) were examined by light and electron microscopy with the objective of describing the populations of epithelial cells believed to be secretory. The results of various histochemical procedures designed to demonstrate nucleic acids, proteins, lipids, and mucosubstances were evaluated by light microscopy. Each thymus is incompletely subdivided into a variable number of interconnected lobules by trabeculae extending inward from a thin capsule composed of connective tissues. The thymic parenchyma lacks distinct cortical and medullary regions, although developing lymphocytes and plasma cells tend to accumulate in larger numbers in the outermost portions of the glands. Basophils are found regularly in the capsule and trabeculae, but only very rarely within the thymic parenchyma. The epithelial cells of the thymus can be classified into five categories: epithelial reticular cells; three varieties of granulated cells (types I, II, and III), and myoid cells. Epithelial reticular cells form a three-dimensional network which extends throughout all portions of the thymus. Type I and type II granulated cells can be distinguished from one another by various morphological criteria at the ultrastructural level, but only small differences in the composition of their inclusions can be demonstrated histochemically. Both types of granules are composed principally of a proteinaceous material containing an abundance of primary amino and guanidyl groups. In addition, most type I inclusions possess a lipid component that cannot be demonstrated in type II granules. Type III granulated cells possess very small cytoplasmic inclusions resembling those of gastroenteric endocrine cells. Myoid cells contain concentrically arranged myofibrils composed of sarcomeres. In favorably oriented material, small cysts can be identified whose walls are composed of mixtures of type I cells, type II cells, and epithelial reticular cells. Groups of degenerating epithelial cells form lamellated structures corresponding to Hassall's (thymic) corpuscles.     

Ultrastructure of the acinar cells in the submandibular gland of the nine-banded armadillo

There are two discrete lobes comprising the armadillo subman-dibular gland. These two lobes can be defined grossly, histochemically and morphologically with the light and electron microscope. The minor lobe stains more intensely with PAS and AB. When viewed in the electron microscope, the secretory granules of the acinar cells within this lobe appear mucous-like. The granules of the demilune cells are slightly different in appearance. The secretory granules of the acinar cells in the major lobe contain many dense foci embedded in a fibrillar matrix, a substructure not described previously. The demilune cells of this lobe contain secretory granules with a mucous-like structure which is consistent throughout the entire lobe. As in the minor lobe, these demilune cells stain very intensely with PAS and AB.     

Glycogen granules in resting and inflammatory rainbow trout phagocytes--an ultrastructural study

The ultrastructural image of glycogen granules in the cytoplasm of rainbow trout phagocytes in sections stained by the conventional lead or uranyl-lead stains is highly dependent on fixation conditions, the granules being visible only when adequate fixation protocols are used. Morphometry of samples processed for the detection of peroxidase or esterase activities (to specifically label neutrophils and macrophages, respectively), and simultaneously stained for the specific detection of glycogen, showed that inflammatory peritoneal neutrophils were richer in glycogen granules than resting neutrophils. This increase in glycogen content occurs after the migration from the haematopoietic tissues and peripheral blood to the inflamed foci. Glycogen granules could not be found in resting peritoneal macrophages but were found in inflammatory macrophages. The macrophage granules occurred in smaller amounts than in neutrophils, and consisted of granules identical to those of neutrophils together with significantly smaller granules. No evidence for the utilization of glycogen by neutrophils phagocytosing bacteria within the peritoneal cavity was found.     

Identification of two types of melanocyte within the stria vascularis of the mouse inner ear

We have distinguished two types of melanocyte within the intermediate layer of the stria vascularis in the cochlea of normally pigmented mice: light and dark intermediate cells. The light intermediate cells are present in the stria from birth and have the typical appearance of a melanocyte. They are large and dendritic with electron-lucent cytoplasm containing numerous vesicles that show tyrosinase activity, and pigment granules in various stages of development. These granules have the ultrastructural and histochemical characteristics of premelanosomes and melanosomes. The light intermediate cells persist throughout life, but less frequently contain pigment in older animals. The dark intermediate cells, present only in adult mice, vary considerably in number and distribution between animals. Pigment granules, bound within an electron-dense acid phosphatase-rich matrix, form the main component of the dark intermediate cells. The intermediate cells may comprise either two distinct cell populations or different developmental stages of the same cell type; ultrastructural observations suggest the latter. In young mice, light intermediate cells contain the electron-dense matrices, which at later stages of development are found almost exclusively in dark cells. The dark intermediate cells contain few cell organelles other than pigment granules accumulated within lysosomal bodies and they often have pycnotic nuclei. These observations suggest that the dark intermediate cells are a degenerate form of the light intermediate cells. Clusters of melanosomes also occur in the basal cells, and to a much lesser extent in the marginal cells. These cells do not stain after incubation in DOPA, suggesting that they are not capable of melanin synthesis, and therefore probably acquire melanin by donation from adjacent melanocytes. Pigment clusters are also found within the spiral ligament at all stages of development.     

The endocrine cells of the rectum of adult ox.

In order to identify the endocrine cell types in various parts of the Ruminant gut, we have applied ultrastructural, both morphological and cytochemical, techniques, in parallel to the histochemical ones, to study the rectal mucosa of the adult Ox. In these studies we show that: "EC" cells, of the intestinal type, contain predominantly pleiomorphic granules, which are very electron dense and heavily reactive to "Masson" and "Grimelius" methods; "L" cells are recognizable by their numerous granules, which are fairly homogeneous in shape and osmiophilia. They do not react with "Masson" and are weak or negative to Grimelius s reaction. These granules occur near to others that are less dense, unreactive to "Masson", and that contain an argyrophilic matrix, with an eccentric electron dense core, which does not react with silver; "F-like" cells contain granules which are variable in shape, size and osmiophilia. They are unreactive to "Masson" and weak or unreactive to Grimelius silver; "H" cells contain few, small and uniformly osmiophilic granules. These are unreactive to "Masson" and uniformly reactive to "Grimelius". Our data suggest that the morphology, frequency and distribution of the cell types we have identified in the mucosa of the bovine rectum correspond with those reported in large intestine and rectum of Monogastrics, as by other authors described.     

The differentiation of monocytes into macrophages,epithelioid cells,and multinucleated giant cells in subcutaneous granulomas

Summary The peroxidatic (PO) activity of monocytes differentiating into macrophages, epithelioid cells, and multinucleated giant cells in subcutaneous granulomas was investigated with three different media for the demonstration of PO activity. Irrespective of the stage of differentiation, these cells did not show PO activity in the rough endoplasmic reticulum (RER) or nuclear envelope. In addition, it was found that the morphologically characteristic types of granule of the various cells of the monocyte line (the primary granules and secondary granules of monocytes, the macrophage granules, and the epithelioid cell granules), all have distinct cytochemical characteristics.Monocytes lose their primary and secondary granules during differentiation into mature macrophages. Simultaneously, the granules of both types become elongated and the secondary granules lose their halo. In contrast to monocytes, mature macrophages may contain a few microperoxisomes. During the differentiation of macrophages into epithelioid cells or multinucleated giant cells there is an increase in the number of microperoxisomes.     

The haematopoietic supraneural organ of adult, sexually immature river lampreys (Lampetra fluviatilis [L.] Gray) with particular reference to azurophil leucocytes.

The haematopoietic tissue in the supraneural organ of the freshwater river lamprey (Lampetra fluviatilis L. Gray) was studied in sexually immature animals. Besides erythro- and granulopoietic elements, macrophages, reticular cells, fibroblasts and glycogen-rich fat cells were seen. Developing granulocytes of the lamprey contain one type of azurophil granules originating from small cytoplasmic (Golgi) vesicles. The lamprey's azurophil granulocytes seem to be homologous with those of fishes. However, the granulocytes of fishes, studied thus far, show granules with only one type of inclusion, whereas in lamprey the granulocyte inclusions are variable in size and shape. Thus, lamprey granulocytes are, in this respect, reminiscent of similar cells of higher vertebrates. The PAS and alkaline phosphatase reactions, common markers of vertebrate neutrophil leucocytes, are very weak in the haematopoietic tissue granulocytes of the lamprey, and intense in the blood cells of the same animal. Lamprey granulocytes, similarly to the granulocytes of Chondrostei and Elasmobranchiata, do not stain with peroxidase, naphthol-AS-D-chloroacetate esterase and sudan black B. The haematopoietic tissue contains a relatively high number of degenerated granulocytes.     

Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells

Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.     

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.     

An excursion through the ultrastructural world of quick-frozen pancreatic islets

In this article we have presented a philosophical and historical perspective on quick freezing, freeze-drying, freeze-substitution, and immunocytochemical localization of pancreatic islet hormones. A compilation of our findings indicates that quick-freezing does not produce any gross distortion of islet tissue; the amount of usable islet tissue for ultrastructural analysis is approximately 13 micron deep from the frozen edge; three different cell types can be identified in quick-frozen tissue based on general morphological characteristics; freeze-substitution with tetrahydrofuran produces a unique ultrastructural appearance in which ribosomes are particularly striking; with the use of protein A-gold, insulin and glucagon can be localized immunocytochemically on silver-gray (50-nm-thick) sections treated with 1% ovalbumin at room temperature overnight; secretory granules of quick-frozen alpha and beta cells may exist in either a swollen or condensed state; swollen beta cell secretory granules contain a filamentous material that demonstrates immunogold labeling for insulin; insulin and glucagon can be localized within the cisternae of endoplasmic reticulum; our methods provide not only discrete immunocytochemical localization of hormone, but also well-preserved cellular compartments; energy electron loss spectroscopy (EELS) has shown that quantifiable nitrogen maps can be used as an index of hormone packaging in secretory granules; and the sectioning properties of secretory granules at the ultramicrotome change when islet tissue is unosmicated and sectioned on glycerol.     

Analysis of hemopoietic lineage of accessory cells in the developing thymus of Xenopus laevis

The developmental history of accessory cells in the thymus was studied by grafting hemopoietic stem cells into cytogenetically distinct frog embryos (diploid-2N or triploid-3N) before the establishment of circulation and overt differentiation and colonization of the thymus. The DNA content of cortical thymocytes and circulating erythrocytes was quantified by staining with propidium iodide and measuring the amount of red fluorescence emitted by individual nuclei with the use of flow cytometry. Accessory cells from thymic medulla were separated by incubating for 2 hr on glass slides. For comparison, the developmental history of peritoneal macrophages was examined as representative, myeloid-derived phagocytic cells. DNA content of adherent cells was quantified by staining with the DNA-specific Feulgen reaction and measuring light absorption of individual nuclei by microdensitometry. Thymic accessory cells were subdivided into phagocytic and nonphagocytic phenotypes on the basis of latex bead ingestion. Phagocytic cells in the thymus were usually nonspecific esterase positive and phenotypically resembled peritoneal macrophages. Nonphagocytic cells from the thymus were usually esterase negative and had a dendritic morphology characterized by branched cytoplasmic extensions. Nonphagocytic cells were positive for cytoplasmic RNA based on staining with methyl green-pyronin Y. Phagocytic cells from both the thymus and the peritoneal cavity had no levels of cytoplasmic RNA detectable by this method. Analysis of the embryonic derivation of thymic accessory cells, based on the proportion of cells carrying the cytogenetic marker, demonstrated that thymic lymphocytes and thymic accessory cells were a concordant pair of cells, distinct from myeloid-derived erythrocytes and possibly macrophages. These experiments provide circumstantial evidence suggesting thymocytes and thymic accessory cells could arise from a bipotential precursor that diverges into these separate lineages after colonization of the epithelial thymic rudiment during early development.     

Relationship between structure and T-lymphocyte maturation in human thymomas. Enzyme histochemical and immunohistological studies

Twenty-six human thymomas were studied in an attempt to correlate their morphological appearance with the type and degree of T-lymphocyte maturation, as determined by acid alpha-naphthyl-acetate esterase (ANAE) activity and immunological analysis. Four normal human thymuses were used for purposes of comparison. Two morphological patterns were identified in the thymomas. The distinction was based largely on similarities between the neoplastic epithelial cells and normal cortical and medullary epithelial cells, and on the relative proportions of epithelial cells and lymphocytes. By these criteria "medullary" and "cortical" patterns were identified. In several thymomas both patterns were present in the same tumor ("mixed-type pattern"), producing alternating dark cortical-like areas and lighter foci of medullary differentiation. A good correlation was found between the two patterns and the phenotype of the T-associated lymphoid component. ANAE activity, which was completely lacking in normal cortical thymocytes, was almost absent in the phenotypically immature T-cells of cortical-type thymomas. By contrast, in the medullary-type thymomas, T-cells showed immunological features in common with medullary thymocytes. This was characterized by strong ANAE activity in the majority of cells with a staining pattern corresponding to that of peripheral T-lymphocytes. In addition, most of the proliferating epithelial cells in medullary-type thymomas stained strongly with anti-cytokeratin and anti-epidermal-type keratin antisera. In the mixed-type thymomas the epithelial cell morphology and the immunohistochemical and enzymic features of the T-cells were found to be closely related to the respective cortical--or medullary-like areas. It was concluded that the various characteristics of normal thymic cortex and medulla studied are also present in thymomas. In particular, in medullar-type thymomas the presence of many of the features of normal thymic medulla, such as a squamous cell component, macrophages and interdigitating reticulum cells, may constitute a microenvironment which operates actively in T-cell education. This may account for the functional activities, characteristic of peripheral T-lymphocytes, which T-lymphocytes attain in these thymomas.     

Preliminary observations on the ultrastructure of the human laryngeal glands: the secreting cells

The secretory portions of human laryngeal glands consist of seromucous and mucous cells. Seromucous cells contain granules either homogeneously dense or endowed with a bipartite structure, consisting of a clear peripheral halo and a central dense portion. In any case, however, they exhibit a morphological appearance different from that observed in seromucous granules of salivary glands. Mucous cells vary considerably according to the functional state.     

The distribution of some hydrolytic enzymes in the cells of the digestive gland of certain lamellibranchs and gastropods

Five hydrolytic enzymes (acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and non-specific esterase) have been studied histochemically in the cells of the digestive gland of Mytilus edulis, Helix aspersa , and certain other lamellibranchs and gastropods. All the enzymes studied have basically similar distributions.
In the digestive cells, the enzymes occur in cytoplasmic granules which are believed to be primary lysosomes; in vacuoles which contain phagocytosed food material; and in vacuoles containing lipofuscin granules, which are the residues of digestive activity.
In the basiphil cells of M. edulis , most of the enzymes are localized in a few cytoplasmic granules; non-specific esterase, however, is found throughout the cytoplasm. In the calcium cells of H. aspersa and the other pulmonate gastropods studied, the enzymes are either in cytoplasmic granules, or distributed diffusely throughout the cytoplasm. Acid phosphatase is also found in the calcium spherules, especially in H. aspersa.
In the excretory cells of H. aspersa and the other pulmonates studied, the enzymes are found in granules in the cytoplasm, and in the lipofuscin granules which lie in the vacuoles of these cells.