Sadashige Sakuma

Immunohistochemical Study of Rat Renal Interstitial Fibrosis Induced by Repeated Injection of Cisplatin, with Special Reference to the Kinetics of Macrophages and Myofibroblasts:

Progressive renal interstitial fibrosis occurs following tissue injury, resulting in chronic renal failure. In the fibrogenesis, macrophages are speculated to induce myofibroblasts producing matrix protein. The kinetics of these cells in renal fibrosis induced in rats by repeated injection of cisplatin (CDDP) (2 mg/kg body weight, once weekly) was investigated immunohistochemically. During the 10-wk injection period, epithelial damage of the proximal renal tubules in the corticomedullary junction was seen, followed by cystic dilation of the affected tubules. During the 8-wk recovery period following the seventh injection, the size of the dilated lumina was diminished and atrophic tubules lined by regenerating epithelial cells appeared. Morphometrical analysis revealed that fibrosis began to develop around the dilated renal tubules in the injection period and was more advanced in the following recovery period. Coinciding with development of fibrotic tissues, the number of α-smooth muscle actin-positive myofibroblasts was significantly increased in the areas compared to that of controls. In the injection period, despite a significant increase in myofibroblast number, an elevation of ED-1 (primary antibody)-positive macrophage number was not observed. In the recovery period, however, a significant elevation of macrophage number was noticed in markedly advanced interstitial fibrosis. This suggests that rapid expansion of the macrophage population, probably resulting from release from myelosuppression due to CDDP, might contribute in part to development of myofibroblasts, leading to the augmented fibrosis in the recovery period.

Distribution of Cells Immunopositive for AM-3K, a Novel Monoclonal Antibody Recognizing Human Macrophages, in Normal and Diseased Tissues of Dogs, Cats, Horses, Cattle, Pigs, and Rabbits

The monoclonal antibody AM-3K, which was developed using human pulmonary macrophages as the immunogen, immunocytochemically labels most human macrophages except for blood monocytes and dendritic cell populations. AM-3K also shows cross-reactivity in some animal species. To evaluate the usefulness of AM-3K, the present study investigated the detailed distribution of AM-3K–immunopositive macrophages in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits. Zambonis solution-fixed, paraffinembedded sections were the most available for the immunocytochemistry with AM-3K. In all animal species examined, AM-3K labeled most macrophages in splenic red pulp, lymph node sinuses and thymus, and tissue macrophages in the interstitium of various organs and sites such as the kidneys, lungs, heart, pancreas, intestines, and skin. Alveolar macrophages and perivascular microglial cells were also immunoreactive for AM-3K. Interestingly, Kupffer cells of dogs, cats, and horses were labeled for AM-3K, but those of cattle, pigs, and rabbits were not. Furthermore, in tumor tissues and inflammatory lesions such as liver fibrosis and encephalomalacia that were obtained from dogs, infiltrating macrophages were stained with AM-3K, but not all infiltrating macrophages reacted to AM-3K. In addition, only 30–50% of pulmonary and peritoneal macrophages obtained from cats and dogs were reactive for AM-3K. AM-3K did not react with blood monocytes, dendritic cell populations, and osteoclasts. These observations indicate that AM-3K specifically labels most exudate and tissue macrophages in the animal species examined. However, the expression of antigens recognized by AM-3K on macrophages may be dependent on differential maturation stages or different functions evoked by some conditions. AM-3K immunoreaction products were seen on the cytoplasmic membrane of macrophages by immunoelectron microscopy. AM-3K would be useful for detection of macrophage populations in the animal species examined here.

Immunohistochemical observations on the kinetics of macrophages and myofibroblasts in rat renal interstitial fibrosis induced by cis-diamminedichloroplatinum

It has been speculated elsewhere that growth factors produced by macrophages in response to tissue damage induce a modulation of pre-existing fibroblasts into myofibroblasts, leading to fibrosis. The development of these cells in cis-diamminedichloroplatinum (CDDP)-induced rat renal interstitial fibrosis was observed immunohistochemically. In the cortico-medullary junction, nuclear changes and epithelial necrosis of the proximal renal tubule (mainly the P3 segment) were seen on days 1 and 4 after a single dose (6 mg/kg body weight) of CDDP, and regenerating epithelium appeared on day 7. Gradually developing fibrosis was observed around the affected tubules on days 14 and 28. The increase in fibrosis was confirmed by histometrical analysis. The number of ED-1 (primary antibody) positive macrophages reached a peak in the affected cortico-medullary junction on day 7 and this was accompanied by an increase in muscle actin-positive myofibroblasts. On days 14 and 28, macrophages had declined in number, but the number of muscle actin-positive myofibroblasts in the fibrotic area was still high as compared with control values. Cytoplasmic myofilaments were observed in myofibroblasts by electron microscopy. These findings suggest that the myofibroblasts participate in renal interstitial fibrosis in the rat, and that their appearance may be related to macrophage infiltration in response to tubular injury, at least in the early stages of fibrosis.

Establishment and characterization of cell lines derived from a transplantable rat malignant meningioma: morphological heterogeneity and production of nerve growth factor

Abstract A cell line (KMY-J) was established from a transplantable tumor (MM-KMY) derived from a spontaneous malignant meningioma arising in an aged F344 rat, and three cloned cell lines (KMY-1, KMY-2 and KMY-3) were induced from the parent KMY-J. Morphologically, KMY-J and tumors induced in syngeneic rats by KMY-J showed cell pleomorphism. All neoplastic cells in KMY-J and its tumors were immunoreactive to vimentin; occasional cells reacted to ED1 (rat macrophage/histiocyte-specific antibody) and α-smooth muscle actin (α-SMA), indicating expression of histiocytic or myofibroblastic immunophenotypes of meningioma cells. In contrast, KMY-1, KMY-2 and KMY-3 consisted of a uniform cell population differing from each other. KMY-1-induced tumors were similar histologically to meningeal fibrosarcomas. Dendritic cells seen in KMY-2 cultures gave an appearance of arachnoid trabecular cells. In KMY-3 and its tumors, large round cells and multinucleated giant cells were predominant. Cells of these cloned cell lines also reacted to vimentin, but were negative for ED1 and α-SMA. By the bioassay using PC12 cells and reverse transcription-polymerase chain reaction for nerve growth factor (NGF) mRNA, production of NGF was demonstrated in the parent and cloned cell lines. The present cell lines may prove useful for studying the histological features of meningeal tumors and the bioactive factors produced by meningeal cells.

Immunohistochemical Analysis of Macrophages, Myofibroblasts, and Transforming Growth Factor-β Localization during Rat Renal Interstitial Fibrosis Following Long-Term Unilateral Ureteral Obstruction

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-β (TGF-β, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of α-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-β was found in infiltrating macrophages or myofibroblasts. However, TGF-β was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-β may be renal epithelia. However, the staining intensity for TGF-β on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-β to the advanced stages of UUO-induced renal fibrosis remains to be determined.

Differential Effects of Transforming Growth Factor-β1, a Fibrogenic Factor, on Macrophage-Like Cells (HS-P) and Myofibroblastic Cells (MT-9) In Vitro

Transforming growth factor- β1 (TGF-β1) produced by infiltrating macrophages plays a role in fibrotic disorders through the induction of myofibroblasts. To explore possible mechanisms by which TGF-β1 may act in this context, we investigated effects of TGF-β1 on macrophage-like (HS-P) and myofibroblastic (MT-9) cells, two novel cell lines developed by us. Immunocytochemicall y, the addition of TGF-β1 (0, 0.1, 0.5, and 1.0 ng/ml) dose-dependently suppressed the expressions of antigens recognized by macrophage/histiocyte-specific antibodies (ED1 and ED2) in HS-P cells, whereas the addition concomitantly increased the number of anti-α-smooth muscle actin antibody-positive myofibroblastic cells, suggesting a possible phenotypical modulation of macrophages into myofibroblasts in the fibrotic lesions. By contrast, MT-9 cells did not show such immunophenotypica l changes following TGF-β1 addition. DNA synthesis, measured by tritiated thymidine-incorporation , was inhibited in a dose-dependen tmanner in MT-9 cells by TGF-β1 addition (0, 0.1, 0.2, 0.5, 1.0, 5, and 10 ng/ml), but that in HS-P cells was unchanged. Northern blot analysis revealed that expressions of cell cycle-related early genes, c-jun and c-myc, were increased in HS-P cells after TGF- β1 (1 ng/ml) addition, with c-jun showing peak expression prior to c-myc. By contrast, the peak expressions of c-jun and c-myc were delayed in TGF-β1 (1 ng/ml)-added MT-9 cells, and their levels were less in MT-9 cells than in HS-P cells. Furthermore, TGF-β1 (1 and 10 ng/ml) induced DNA laddering in MT-9 cells, but did not in HS-P cells. Based on these findings, it was speculated that TGF- β1 could have induced G1 arrest in cell cycle and apoptosis in MT-9 cells. The present study showed that there were significant differences in the effects of TGF-β1 between macrophage-like HS-P cells and myofibroblastic MT-9 cells, presumably depending on divergent susceptibilities to TGF- β1 between both cell types. Because such cell types are key cells in the fibrogenesis, HS-P and MT-9 might be useful models for investigating the pathogenesis of fibrosis in vitro.

Characteristics of a rat fibrosarcoma-derived transplantable tumour line (SS) and cultured cell lines (SS-P and SS-A3-1) showing myofibroblastic and histiocytic phenotypes.

Abstract A transplantable tumour line (SS) was established in syngeneic rats from a spontaneous fibrosarcoma that had arisen in the submandibular salivary gland of a 24-month-old male F344 rat. A cell line (SS-P) was induced from SS, and a cloned cell line (SS-A3-1) was isolated from SS-P. The primary tumour consisted of oval to spindle-shaped cells arranged in bundles with abundant collagen fibres; ultrastructurally, neoplastic cells exhibited fusiform morphology with prominent rough endoplasmic reticulum. SS tumours showed marked interlacing fascicle and herring-bone growth patterns. SS-P and SS-A3-1 were simmilar morphologically to each other, consisting of oval, spindle or polygonal cells and occasional multinucleated giant cells. Tumours induced by SS-P and SS-A3-1 were histologically similar to SS tumours. Immunohistochemically, all cells in the primary tumour, SS tumours and tumours induced both by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures gave a positive reaction to vimentin. Interestingly, neoplastic cells reacting to ED1 (rat macrophage/histiocyte-specific antibody) and α-smooth muscle actin (α-SMA) appeared in SS tumours and tumours induced by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures. Cells with histiocytic fine structures and myofibroblastic cells with cytoplasmic actin-like microfilaments were also observed by electron microscopy. The present rat fibrosarcoma-derived transplantable tumour line (SS) and cell lines (SS-P and SS-A3-1) might express myofibroblastic and histiocytic phenotypes, probably depending on the surrounding conditions. These cell lines may prove useful for studying the mechanisms of phenotypic plasticity in neoplastic fibroblasts.

Gicerin, a cell adhesion molecule, participates in the histogenesis of retina

Gicerin is a novel cell adhesion molecule that belongs to the immunoglobulin superfamily. Gicerin protein adheres to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family, and also exhibits homophilic adhesion. Heterophilic adhesion of gicerin to NOF is thought to play an active role in neurite outgrowth of developing retinal cells in vitro. In this study, we examined the adhesion activity of gicerin during the retinal development of Japanese quail using an antibody directed against gicerin, to elucidate the biological importance of gicerin in retinal histogenesis. Immunohistochemical and Western blot analysis showed that gicerin was highly expressed in the developing retina but suppressed in the mature retina. The aggregation of neural retinal cells from 5-day embryonic quail retina was significantly inhibited when incubated with a polyclonal antibody to gicerin, suggesting that gicerin protein participates in the adhesion of neural retinal cells of the developing retina. Furthermore, histogenesis of retina both in the organ cultures and in ovo embryos was severely disrupted by incubation with a gicerin antibody. These findings provide evidence that gicerin plays an important role in retinal histogenesis.

Morphological characteristics of a transplantable histiocytic sarcoma (HS-J) in F344 rats and appearance of renal tubular hyaline droplets in HS-J-bearing rats

A transplantable tumour (HS-J) was established from a spontaneous histiocytic sarcoma found in a 24-month-old male F344 rat. Serial transplantations (seven generations) were made in syngeneic male and female rats by means of intraperitoneal or subcutaneous implants, with a 100% take rate. Rats given HS-J implants developed large nodules locally, with metastasis to distant organs. HS-J tumours consisted mainly of round to oval cells with abundant cytoplasm, arranged in a compact sheet. Enzyme- and immuno-histochemical examination showed that neoplastic cells reacted with ED1 (rat monocyte/macrophage-specific antibody), lysozyme, alpha 1-antitrypsin and lysosomal enzymes (acid phosphatase and non-specific esterase), indicating derivation from cells of the monocyte/macrophage lineage. The majority of neoplastic cells were negative for ED2 (rat tissue macrophage-specific antibody). Abnormal accumulations of hyaline droplets in the proximal renal tubular epithelial cells were seen in HS-J-bearing rats. The droplets were faintly immunopositive for lysozyme, but negative for alpha-2u globulin and albumin. It was considered that excessive production of the protein by tumour cells might lead to subsequent overload in renal tubules. HS-J may prove beneficial for studying the biological behaviour of monocyte/macrophage-derived tumours in the rat.

Genetics of susceptibility to radiation-induced apoptosis in colon: two loci on Chromosomes 9 and 16

Apoptosis, a mechanism for removal of genetically damaged cells and for maintenance of desired size of cell populations, has been implicated in tumor development. Previously, we defined polymorphic loci for susceptibility to apoptosis of thymocytes Rapop1, Rapop2, and Rapop3 on mouse Chromosomes 16, 9, and 3, respectively, using recombinant congenic CcS/Dem strains, each of which contains a random set of 12.5% STS/A genome in the genetic background of BALB/cHeA. The STS/A alleles at these loci confer lower susceptibility to radiation-induced apoptosis of thymocytes than the BALB/cHeA. In the present study, we tested susceptibility of colon crypt cells to radiationinduced apoptosis. In contrast to apoptosis in thymus, the STS/A mice were more susceptible to apoptosis in colon than the BALB/ cHeA. Among the CcS/Dem strains, CcS-4, CcS-7, and CcS-16 were more susceptible to apoptosis in colon than the BALB/cHeA; in thymus, the CcS-7 mice are less susceptible, and the CcS-4 and CcS-16 are not different from the BALB/cHeA. Thus, individual CcS/Dem strains showed different apoptosis susceptibility in the two organs. Analysis of (CcS-7 × BALB/cHeA)F2 hybrids revealed linkage of susceptibility to radiation-induced apoptosis of colon crypt cells to two loci on Chrs 9 and 16, to which Rapop2 and Rapop1 are mapped. The STS/A allele at the locus on chromosome 9 results in high susceptibility to apoptosis of colon crypt cells in mice homozygous for the BALB/cHeA allele at the locus on Chr 16. Although these two loci may be identical to Rapop1 and Rapop2, they affect apoptosis in colon in a way different from that in thymus.