Pathogenesis of Lactobacillus Casei Cell Wall Extract-Induced Cardiovasculitis
Keiji Suzuki, Kyoumi Nakasato, Shoko Okitsu-Negishi* and Kazuya Yoshino*
Division of Histopathology, Department of Laboratory Sciences, Gunma University School of Health Sciences, 3-39-15 Showamach, Maebashi, Gunma 371-8514 and *Department of Pediatrics, Teikyo University School of Medicine, Tokyo 173-8605
Received for publication April 3, 1998 and in revised form October 31, 1998
In this experiment, coronary angitis with fibrinoid necrosis, aortitis, endocarditis of the aortic and pulmonary valves, and pericarditis were induced by a single intraperitoneal injection of the water soluble cell wall extract from Lactobacillus casei (LCWE). Inflammatory cell infiltration of primarily monocytes and neutrophils was found throughout the aortae, the endocardium, the coronary arteries, and the pericardium. Other organs showed no such inflammatory changes.
Immunohistochemically, overexpression of VCAM-1, ICAM-1, MCP-1, IL-1$\alpha$, IL-6, and TNF-$\alpha$ of endothelial cells of the aorta, coronary artery, capillaries and endocardium, and pericardium was observed after intraperitoneal injection of LCWE. These adhesion molecules and cytokines showed the strongest expression 24 and 48 hr after intraperitoneal injection of LCWE. The expression of ICAM-1, MCP-1, IL-1$\alpha$, IL-6, and TNF-$\alpha$ in endothelial cells and in the pericardium decreased 1 week after LCWE injection. VCAM-1 expression of endothelial cells disappeared 1 week after the injection. In conclusion, LCWE-induced overexpression of cytokines and adhesion molecules in endothelial cells of the aorta, coronary artery, capillaries and endocardium, and pericardium may contribute to cardiovasculitis of the mice.
Ubiquitous Expression of bcl-2 Protein in Mitotic Nuclei and Chromosomes
Akinobu Nakano, Shigeru Morikawa and Takayuki Harada
Department of Pathology, Shimane Medical University, Izumo 693-8501
Received for publication August 10, 1998 and in revised form November 24, 1998
We investigated the expression of bcl-2 oncoprotein in human cells by immunocytochemistry using an anti-bcl-2 antibody. Expression of bcl-2 was detected in and around mitotic nuclei and chromosomes from prophase to early telophase in all types of long-term cultured cell lines irrespective of the level of its expression in the cytoplasm. Western blot analysis showed a discrete but distinct protein band, which corresponded to the 26 kDa $\alpha$-form of bcl-2, in the lysate of cells showing only mitotic bcl-2. No other immunoreactions, including those corresponding to the $\beta$-form, were detected. RT-PCR combined with nested PCR showed bcl-2 mRNA in the same cell lines and in situ hybridization confirmed its presence in the cytoplasm of almost all interphase cells. The appearance of nuclear bcl-2 was not due to aberrant genetic regulation induced by an artificial in vitro environment. It was also not unique to neoplastic cells as nuclear and chromosomal bcl-2 was detected in freshly prepared specimens from B-cell lymphomas, myelodysplastic syndrome, and primary cultures of normal fibroblasts during mitosis. On the other hand, the level of cytoplasmic bcl-2 varied from one cell line to another; 8 carcinoma lines did not express cytoplasmic bcl-2, whereas many B cell lines expressed high levels of the protein. Bcl-2 appeared to accumulate in mitotic nuclei, because immunocytochemical reaction product was always stronger in mitotic nuclei than in the cytoplasm when the expression of bcl-2 was induced in peripheral blood lymphocytes by ConA stimulation. Our results indicate that bcl-2 protein is ubiquitously expressed in all cells around mitotic nuclei or chromosomes, whereas its distribution in the cytoplasm seems to reflect differences in genetic regulation based on cell lineage, differentiation stage or neoplastic state.
Immunohistochemical Localization of Lipoprotein Lipase and Apolipoprotein E in Human Atherosclerotic Lesions
Masahiro Araki, Jianglin Fan and Teruo Watanabe
Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575
Received for publication September 26, 1998 and in revised form November 19, 1998
Lipoprotein lipase (LPL) and apolipoprotein E (apo E) are both involved in the metabolism of triglyceride-rich lipoproteins. In the arterial wall, these two proteins may be associated with foam cell formation during atherogenesis, yet their localization and cellular sources in the arterial intima are not fully defined. To evaluate the potential roles of these two proteins in various stages of human atherosclerosis, we studied the distribution of LPL and apo E in diffuse intimal thickening, fatty streaks and fibrous plaques of aorta and coronary artery using specific antibodies against human LPL and apo E. In aorta with diffuse intimal thickening, LPL was found in both intimal and medial smooth muscle cells whereas apo E was present only in the intima and was mainly associated with the surrounding extracellular matrix. In fatty streaks and fibrous plaques, LPL and apo E were widely distributed, and were either cell-associated or bound to the extracellular matrix. Double immunohistochemical staining using LPL/apo E and macrophage-specific antibodies revealed that LPL or apo E was colocalized with macrophage-derived foamy and nonfoamy cells. Furthermore, LPL and apo E were variably associated with the extracellular matrix in these areas. As the lesions advanced, LPL was diffusely stained in macrophages and smooth muscle cells in the proximity of the lipid core and fibrous cap, whereas apo E was associated with macrophages around the lipid core. These immunohistochemical findings suggest that LPL can be produced by a variety of cellular sources in the arterial wall, including smooth muscle cells and macrophages, while apo E is mainly derived from macrophages. We speculate that LPL secretion and apo E secretion from macrophages in atherosclerotic lesions are regulated differently and their respective functions in foam cell formation during atherogenesis merit further investigation.
Key words: Macrophages, Foam cells, Apolipoprotein E, Lipoprotein lipase, Atherosclerosis
Age-Related Changes in the Immunohistochemical Localization of Inducible Nitric Oxide Synthase (iNOS) and Nuclear Factor-kappa B (NF-$\kappa$B) in the Mouse Cochlea
1 Department of Anatomy, Aichi Medical University, Nagakute-cho, Yazako, Aichi-gun, Aichi 480-1103 and 2 Department of Otorhinolaryngology, Aichi Medical University, Nagakute-cho, Yazako, Aichi-gun, Aichi 480-1103
Received for publication October 2, 1998 and in revised form November 12, 1998
Localization of iNOS and NF-$\kappa$B in the cochlear tissues of ddy-strain mice was studied immunohistochemically with reference to ageing. Severe structural disorders with a decrease in the number of cells were found, especially in the organ of Corti and in the spiral ganglion of older mice. Throughout all age-spans studied, iNOS-like immunoreactivity was found in the cells of Hensen, Claudius and the outer spiral sulcus epithelium, and it increased dramatically with age. A very small number of cells in the stroma of the spiral ligament and spiral limbus of younger mice up to 12 months of age showed weak iNOS immunoreactivity, while the spiral ganglion cells of the younger mice were almost negative. In mice after 18 months of age, however, strong iNOS immunoreactive cells remarkably increased in the spiral ligament and spiral limbus. Moreover, spiral ganglion neurons that survived from age-related cell death also showed strong reactivity. The immunohistochemical localization of NF-$\kappa$B and its change with age were nearly the same as those of iNOS. The possible roles of iNOS and NF-$\kappa$B in the changes of mouse cochlear tissues with advancing age were discussed.
1 Second Department of Pathology, 2 Department of Orthopaedic Surgery, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017
Received for publication November 19, 1998 and in revised form December 15, 1998
We investigated the expression of osteonectin and osteopontin during fracture healing of mouse tibiae by in situ hybridization (ISH) and the effects of fibrin(ogen) on the expression of bone matrix proteins by Northern blot analysis. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibiae. On days 2, 4, 7 and 14 after the operation, the fractured bones were excised, fixed with 4% paraformaldehyde (PFA) and decalcified with 20% EDTA to prepare 5-$\mu$m sections. Digoxigenin (DIG) labeled single-stranded DNA probes generated by uni-directional polymerase chain reaction (PCR) were used for ISH.
Immunohistochemistry revealed fibrin(ogen) at the site of fracture hematoma 2-4 days after fracture and subsequent accumulation of mesenchymal cells. On days 4-14 after the fracture, osteonectin signals were predominantly expressed in proliferating chondrocytes at the endochondral ossification site and in osteoblasts of the marginal woven bone. Osteopontin was expressed on osteoblasts lining the surface of the marginal woven bone at the mid-late phase of fracture healing. In vitro, Northern blot analysis showed that treatment of the mesenchymal cells, C3H10T1/2, with fibrin(ogen) enhanced the steady-state level of osteonectin and osteopontin gene expression. These data suggested that fibrin(ogen) in the hematoma may be important for inducing bone matrix genes in immature mesenchymal cells at the early phase of fracture repair.
Key words: Bone matrix protein, In situ hybridization, Polymerase chain reaction, Fracture healing
1 First Department of Internal Medicine and 2 Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611
Received for publication November 20, 1998 and in revised form December 1, 1998
Histochemical distribution of sugar chains in the substantia gelatinosa of the dorsal horn in the rat spinal cord, where there are mainly small-diameter sensory neurons, was studied with labeled lectins used as a probe. Cryostat sections were prepared from the pre-fixed thoracic spinal cord. For light microscopy, the sections were stained with the biotinylated lectins followed by avidin-Cy3, and observed under a confocal laser scanning microscope. For electron microscopy, the sections were treated with horseradish peroxidase (HRP)-labeled lectins.
Among more than 20 different kinds of lectins tested, GS I-B4, Griffonia simplicifolia I-B4, was shown light-microscopically to selectively bind to sites in nerve fibers of the substantia gelatinosa, and also to those on the surface of unmyelinated nerve fibers and Schwann cells in the dorsal root. Electron microscopy revealed that GS I-B4 bound to the axolemma of the unmyelinated nerve fibers and microglial cell membranes in the substantia gelatinosa, and also to the axolemma of the unmyelinated nerve fiber and to the surface layer of the myelinated nerve fiber in the dorsal root, i.e., to the Schwann cell membrane. On the other hand, GS I-B4 binding was negative in the oligodendroglia except for the trans side of the Golgi complex.
The present results indicate that the carbohydrate structure containing Gal $\alpha$1-3Gal sequence and related sugar structures, which has a strong affinity for GS I-B4, is distributed in the axolemma of the unmyelinated nerve fibers, in the microglial cell membranes in the substantia gelatinosa, and in the Schwann cell membranes in the dorsal root. A marked difference between the oligodendroglial cell membrane and the Schwann cell membrane was found in terms of the distribution of Gal$\alpha$1-3Gal-type sugar structures. Differences in these myelin-forming cells may have some relevance to their functions as well as to demyelinating disease processes selectively confined to the central or the peripheral nervous system.
Rapid Communication Parallel Distribution of Bcl-2 and Bax in Hypertrophied Chondrocytes of the Mandibular Condylar Cartilage from the Senescence-accelerated Mouse (SAMP3)
Mina Kawasaki, Yoshifumi Tajima, Jun Ohno, Kaoru Kusama and Satoshi Yokose*
Department of Oral Pathology and *Department of Operative Dentistry, Meikai University School of Dentistry, Sakado, Saitama 350-0283
Received for publication October 26, 1998 and in revised form December 16, 1998
To demonstrate the distribution of apoptotic cell death in the temporomandibular joint of SAMP3 mice, showing degenerative joint disease resulting in overt deformation of the mandibular condyle, immunohistochemical localization of Bcl-2 and Bax in the condylar cartilage of the animals was examined. Histological changes revealed an apparent deformity of the condyles at around 6 months of age, and thereafter showed severe degeneration in accordance with advancing age until 13 months. The apoptotic changes by the Bcl-2 and Bax expressions were observed in the hypertrophied chondrocytes abutting on the endochondral ossification site, as well as in the synovial membrane, throughout all ages examined. The expression of both of these molecules remained to localize in these site and increased with advancing age, in which proceeding matrix degradation and chondroid bone formation took place. In addition, the nidus of metaplastic chondrocytes that protruded into the joint space of the aged animals also demonstrated positive immunoreactivities. The radical production and parallel distribution of Bcl-2 and Bax in the hypertrophied chondrocytes in the affected condyles might indicate that some hypertrophic cells may undergo apoptosis, and others, survive, to regulate the cartilage degradation occurring in the mandibular condyles of SAMP3 mice.