Impaired splenic erythropoiesis in phlebotomized mice injected with CL2MDP-liposome: an experimental model for studying the role of stromal macrophages in erythropoiesis.

Erythropoiesis occurs in the presence of erythropoietin (EPO) without macrophages in vitro. In hematopoietic tissues, however, erythroid cells associate closely with stromal macrophages, forming erythroblastic islands via interactions with adhesion molecules. To elucidate the role of macrophages in erythropoiesis, we selectively abrogated stromal macrophages of splenic red pulp of phlebotomized mice by injection with dichloromethylene diphosphonate encapsulated in multilamellar liposomes (CL2MDP-liposome). In the spleen, no erythropoietic activity occurred until 5 days after the treatment. Colony assay revealed that the erythropoiesis was suppressed at the level of CFU-E. The splenic erythropoietic activity gradually developed from day 6 after the treatment, when F4/80+ macrophages began to appear in the red pulp. EPO mRNA was expressed in kidney but not in liver or spleen of phlebotomized mice injected with CL2MDP-liposome, and the serum EPO concentration in these mice was higher than that in phlebotomized mice. These findings suggest that abrogation of stromal macrophages by injection with CL2MDP-liposome impairs the splenic microenvironment for erythropoiesis induced by hypoxic stress, and this may be an excellent experimental model for further characterization of the in vivo role of splenic macrophages in erythropoiesis.

Proliferative response of peripheral blood mononuclear cells and levels of antibody to recombinant protein from Propionibacterium acnes DNA expression library in Japanese patients with sarcoidosis.

BACKGROUND AND AIM OF THE WORK: The causes of sarcoidosis are unknown. Propionibacterium acnes has been isolated from sarcoid lesions, and many genomes of P. acnes or P. granulosum have been detected in all biopsy samples tested from Japanese patients with sarcoidosis. We searched for protein antigens from propionibacteria that caused immune responses in patients with sarcoidosis but not in subjects without sarcoidosis. METHODS: A lambda gt11 genomic DNA expression library of P. acnes was screened with sera from patients with sarcoidosis. Antibodies to a recombinant protein from the insert recovered by the screening were measured in serum and bronchoalveolar lavage (BAL) fluid from patients with or without sarcoidosis by an immunofluorescence-based method. Peripheral blood mononuclear cells from patients with and without sarcoidosis were used to examine the lymphoproliferative response to the protein. RESULTS: Of 180,000 plaques screened, two clones coded for an identical recombinant protein, termed RP35, were recognized by sera. RP35 was the C-terminal region of P. acnes trigger factor. RP35 caused sarcoidosis specific proliferation of the mononuclear cells from 9 (18%) of the 50 patients with sarcoidosis; in a similar way, purified protein derived from Mycobacterium tuberculosis evoked specific responses in 8 (38%) of 21 patients with tuberculosis. Serum levels of IgG and IgA antibodies to RP35 were high in patients with sarcoidosis and other lung diseases. In BAL fluid levels IgG or IgA antibodies were high in 7 (18%) and 15 (39%), respectively, of 38 patients with sarcoidosis, and in 2 (3%) and 2 (3%), respectively, of 63 patients with other lung diseases. CONCLUSIONS: The RP35 protein from P. acnes causes a cellular immune response in some patients with sarcoidosis but not in subjects without sarcoidosis.

Bone remodeling and macrophage differentiation in osteopetrosis (op) mutant mice defective in the production of macrophage colony-stimulating factor.

Mice homozygous for the osteopetrosis (op) mutation are characterized by defective differentiation of osteoclasts, monocytes, and tissue macrophages due to a lack of functional macrophage colony-stimulating factor (M-CSF/CSF-1) activity. In young (4-6 week-old) op/op mice, the bone marrow cavities were filled with spongious bone. In aged (50-72 week-old) op/op mice, the bone marrow cavities were markedly reconstructed and marrow hematopoiesis was expanded. Numbers of osteoclasts and bone marrow macrophages in aged op/op mice were increased but most of the osteoclasts were mononuclear cells and showed poorly developed ruffled borders. Lysosomes of bone marrow macrophages were laden with abundant crystalloid materials in aged op/op mice and aged littermate mice. However, such macrophages were not observed in young op/op mice nor in young littermates. In contrast to the marked increase in numbers of osteoclasts and macrophages in the bone marrow, the number of Kupffer cells in the liver did not increase in aged op/op mice. Kupffer cells in aged op/op mice did not show ultrastructural maturation with aging and contained a few crystalloid structures. M-CSF administration to aged op/op mice induced numerical increases in Kupffer cells and lysosomes in Kupffer cells, disappearance of crystalloid structures in lysosomes of Kupffer cells, and the development of ruffled border in osteoclasts. These findings indicate that M-CSF-independent mechanisms for macrophage and osteoclast development in aged op/op mice are restricted to bone marrow. M-CSF plays important roles in the differentiation of macrophage and osteoclast and the production and function of lysosomes.

An Interlaboratory Validation Study of the Improved Transformation Assay Employing Balb/c 3T3 Cells: Results of a Collaborative Study on the Two-stage Cell Transformation Assay by the Non-genotoxic Carcinogen Study Group.

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its interlaboratory reproducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin-transferrin-ethanolamine-sodium selenite and 2% fetal bovine serum (FBS) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1µg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In the presence of 0.2µg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells.

Expression of scavenger receptor class A and CD14 in lipopolysaccharide-induced lung injury.

CD14 and macrophage scavenger receptor class A type I and II (MSR-A) are receptors for lipopolysaccharide (LPS). In this study, the expressions of both receptors in the lung after administration of LPS in aerosol to mice with a nebulizer were observed. Bronchiolar epithelial cells and alveolar macrophages immediately incorporated LPS and expressed CD14. CD14-positive neutrophils then appeared in the alveolar space followed by the appearance of MSR-A-expressing cells in the vascular lumen, pulmonary interstitium, and alveolar space. Numbers of apoptotic cells increased after 1 day, and MSR-A-expressing macrophages actively incorporated apoptotic bodies. Daily administration of macrophage colony stimulating factor (M-CSF) to the mice resulted in increased levels of MSR-A expression and reduced levels of CD14 as well as several cytokine expressions, leading to shortening of the inflammatory process. The numbers of apoptotic cells were reduced in M-CSF injected mice. These findings imply that CD14 acts as an immediate expressing receptor for LPS and MSR-A exerts a protective function by scavenging LPS and apoptotic cells in LPS-induced lung injury.

The role of Kupffer cells and regulation of neutrophil migration into the liver by macrophage inflammatory protein-2 in primary listeriosis in mice.

Depletion of mouse Kupffer cells and splenic macrophages following intravenous administration of liposome-entrapped clodronate severely reduced host resistance to primary infection with Listeria monocytogenes. Infection of clodronate-treated mice with a sublethal dose of L. monocytogenes resulted in death of the mice within 3 days. The macrophage depletion resulted in marked increases in bacterial growth in the liver and spleen, but not in other tissues. The proliferation of L. monocytogenes was observed in a large number of hepatocytes that underwent apoptosis. Infiltration of neutrophils in the liver and rapid formation of microabscesses were observed in the control mice after L. monocytogenes infection. However, there was less accumulation of neutrophils in the liver of Kupffer cell-depleted mice than in the control mice. Expression of macrophage inflammatory protein-2 (MIP-2) was enhanced in the livers of both the control and Kupffer cell-depleted mice after L. monocytogenes infection. MIP-2 was also induced in a murine hepatocyte cell line following L. monocytogenes infection. The administration of neutralizing anti-interleukin-8 receptor homolog antibody severely abrogated neutrophil infiltration into the Listeria-infected mouse liver. Anti-MIP-2 antibody moderately reduced neutrophil infiltration and microabscess formation in the liver. These findings indicate that Kupffer cells protect hepatocytes from L. monocytogenes infection and the resultant apoptosis. Moreover, MIP-2 and its related molecules produced by the infected hepatocytes regulate neutrophil infiltration and microabscess formation in primary listeriosis.

Complete remission of multiple hepatocellular carcinomas associated with hepatitis C virus-related, decompensated liver cirrhosis by oral administration of enteric-coated tegafur/uracil.

We report a case of complete remission of multiple hepatocellular carcinomas after oral administration of enteric-coated tegafur/uracil. A 77-yr-old woman was diagnosed as having recurrent hepatocellular carcinoma associated with decompensated liver cirrhosis. We administered enteric-coated tegafur/uracil to this patient. After 1 month of oral administration, there was a decrease in tumor markers. An image analysis showed disappearance of hepatocellular carcinoma. No recurrence of the hepatocellular carcinoma was recognized for 18 months up to the time of the patient's death, which was due to massive bleeding from a hemorrhagic rectal ulcer. At autopsy, the tumor lesion had necrotized. Oral administration of enteric-coated granules containing tegafur/uracil may provide an effective treatment for hepatocellular carcinoma.

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice.

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.