The effect of age and injury on the expression of inducible nitric oxide synthase in facial motor neurons in F344 rats.

Nitric oxide has been implicated in both normal neuronal aging as well as nerve repair events because of its known roles in synaptic plasticity, synaptogenesis and neuroplathologic processes. In this study, we have determined the effect of aging, by comparing brainstem facial motor neurons (FMNs) as well as blood vessels, from adult F344 rats to those in old animals. Inducible nitric oxide synthase (iNOS) expression was determined both by immunohistochemistry using an antibody to iNOS on tissue sections and slot blots. In adult rats, iNOS expression was detectable only in FMNs and not in blood vessels. In old rats, there were robust levels of iNOS protein in blood vessels, while iNOS protein was not detectable in FMNs from old rats. There was also a 12-fold increase in iNOS expression in isolated blood vessels from old rats compared to vessels from adult animals. To determine the effect of injury on iNOS expression, the facial nerve was transected and immunocytochemistry performed as above. After nerve transection in adult rats, iNOS was demonstrable only in blood vessels after 1 day, but by 7 days iNOS protein immunoreactivity was robust in FMNs. In old animals, iNOS protein expression was observed only in FMNs at 1 day, but by 7 days after injury, protein immunoreactivity was localized to the blood vessels. These data suggest that aging and injury differentially affect the expression of iNOS and that the up-regulation of iNOS may be important for the availability of nitric oxide in the aged or injured nervous system.

Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer's disease.

The cerebral microcirculation undergoes specific biochemical changes in Alzheimer's disease. In this study, we have compared the nitric oxide synthase (NOS) activity of brain microvessels isolated from Alzheimer and control brains. L-[3H]-citrulline, the stable co-product generated with nitric oxide (NO) from L-[3H]-arginine, was measured as an indicator of NOS activity. The results indicated a significant increase in NOS activity in microvessels isolated from Alzheimer brains. In addition, using antibodies to both the endothelial and inducible NOS isoforms, we demonstrated a significant increase in enzyme level in Alzheimer-derived vessels. Elevated vascular production of NO, a potentially neurotoxic mediator in the CNS, may contribute to the susceptibility of neurons to injury and cell death in Alzheimer's disease.

Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells.

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7-10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated.

Alternatively spliced pp52 mRNA in nonlymphoid stromal cells.

The 52-kDa phosphoprotein, also reported as lymphocyte-specific gene 1 and WP34, is transcribed as a 1.6-kb mRNA in B lymphocytes, B cell lines, and untransformed T cells. This gene encodes a cytoplasmic and plasma membrane-associated protein that is phosphorylated at a casein kinase II site and reportedly binds calcium. Based on these properties, it has been hypothesized that lymphoid form of the 52-kDa phosphoprotein protein may play a role in lymphocyte signal transduction. We show that alternatively spliced mRNA are expressed from this gene in nonlymphoid cell lines (myocytes, stromal cells, fibroblasts). These cell lines do not express the 1.6-kb lymphoid cell-specific transcript. Instead, mRNA of 2.0 and 2.8 kb are detected in varying abundance. A full-length 2.0-kb cDNA has been cloned and sequenced from the BMS2 stromal cell line by conventional screening and polymerase chain reaction-based methods. This cDNA clone, designated S37, has a single open reading frame encoding a 328 amino acid peptide. The nucleotide sequence of the S37 stromal cell cDNA is identical to that of the lymphocyte derived pp52 cDNA from the 3' poly(A) tail to the codon encoding the amino acid at residue 24. This region of the S37 cDNA clone encodes a protein that is identical to that encoded by the lymphoid pp52 cDNA and includes a casein kinase II phosphorylation site. However, the two clones differ in their 5' nucleotide sequence and their NH3 terminal amino acid sequence. This organization is consistent with alternative exon utilization. These results suggest that tissue-specific control mechanisms are used to generate different forms of lymphoid form of the 52-kDa phosphoprotein mRNA in lymphoid cells versus mesoderm-derived, nonlymphoid cell lineages.

Adipogenesis in a murine bone marrow stromal cell line capable of supporting B lineage lymphocyte growth and proliferation: biochemical and molecular characterization.

Recent advances in long-term bone marrow (BM) culture techniques have allowed investigators to dissect cellular components responsible for lympho hematopoiesis. Consequently, a number of "stromal" cell clones have been developed which are capable of supporting B lineage lymphocyte growth and proliferation in vitro by direct cell-cell interactions and the release of cytokines. While much work has focused on the support function of these cells, questions remain regarding their own differentiation potential. We have examined adipogenesis in the cloned BM stromal cell, BMS2. The presence of hydrocortisone, methylisobutylxanthine, or 30% fetal calf serum each accelerated adipocyte differentiation. This process was accompanied by the accumulation of triglycerides and cholesterol esters along with the induction of adipocyte-specific enzymes. Likewise, the steady-state level of mRNA transcripts increased for genes related to lipid metabolism. However, the pattern of mRNA expression in BMS2 adipocytes differed from that of a well-established, pre-adipocyte cell line, 3T3-L1, with respect to the following genes: glycerol phosphate dehydrogenase, CAAT/enhancer binding protein and angiotensinogen. Adipocyte BMS2 cells retailed the ability to support stromal cell-dependent B lineage lymphocytes in methylcellulose assays. The adipocytes continued to express macrophage-colony-stimulating factor mRNA constitutively and interleukin 6 mRNA in an inducible manner, similar to the BMS2 pre-adipocytes. Together, these data document a close developmental relationship between a specialized fibroblasts and adipocytes in the BM and suggest that adipocyte stromal cells may play an active role in lympho-hematopoiesis.

Response of bone marrow stromal cells to adipogenic antagonists.

Adipocytes constitute a major part of the bone marrow stroma in vivo and may play an active role in lymphohematopoiesis. Earlier studies had shown that the bone marrow stromal cell clone BMS2 was capable of adipocyte differentiation in vitro, in addition to its well-defined ability to support B lymphopoiesis. We now demonstrate that the process of adipogenesis in this functional bone marrow stromal cell clone can be inhibited by the cytokines interleukin-1 alpha, tumor necrosis factor, and transforming growth factor beta. Exposure of preadipocyte BMS2 cells to these agents blocked the induction of adipocyte differentiation as assessed by morphologic criteria and analysis of the neutral lipid content. Both interleukin-1 alpha and tumor necrosis factor elicited a rapid transient elevation in the steady-state mRNA levels of c-fos, c-jun, and JE. When added to differentiated adipocytes, the three cytokines continued to act as adipogenic antagonists. This was indicated by concentration- and time-dependent decreases in the activity of an adipocyte-specific enzyme, lipoprotein lipase. These changes in enzyme activity correlated directly with a decrease in steady-state levels of lipoprotein lipase mRNA. Another RNA marker of adipocyte differentiation (adipsin) was less influenced by the adipogenic antagonists. This may reflect the longer half-life of this mRNA transcript compared with those of lipoprotein lipase. Our results dramatically demonstrate that the differentiation state of bone marrow stromal cells can be modulated by exogenous factors in vitro. It is also the first report that transformation growth factor beta regulates the activity of lipoprotein lipase. These data suggest potential physiologic actions for these cytokines in vivo within the overall context of lymphohematopoiesis.

Characterization of murine bone marrow and spleen-derived stromal cells: analysis of leukocyte marker and growth factor mRNA transcript levels.

Stromal cells are believed to regulate lympho-hematopoiesis through direct cell-cell interactions and the release of growth factors. Many questions remain, however, about their lineage derivation and functional heterogeneity. We previously prepared a panel of stromal cell lines from murine spleen and bone marrow and characterized them based on their ability to support lymphocyte growth in long-term cultures. These cells are now compared with respect to their expression of various immunoglobulin superfamily and cytokine genes by Northern blot analysis. These results indicate that although stromal cells appear to be mesodermal in origin, they are not closely related developmentally to the hematopoietic progenitor cells they support. The potential production of at least six cytokines was demonstrated. All clones constitutively expressed mRNA for macrophage colony stimulating factor, interleukin-6, transforming growth factor beta and neuroleukin. The most potent lymphocyte supporting clones also made interleukin 7 constitutively. Previous findings had suggested that these clones responded to exogenous stimuli and this has now been demonstrated in terms of induced expression of IL-6 and G/M-CSF mRNA. Interleukin 6 mRNA levels were markedly upregulated by exposure of cells to LPS, TNF, IL-1, IL-6, IL-7, and EGF. G/M-CSF mRNA levels were "superinduced" by the combination of LPS and cycloheximide, a protein synthesis inhibitor. These responses are similar to ones documented by investigators working with endothelial cells and fibroblasts. Together, these data suggest that stromal cells are a multifunctional component of the lymphopoietic microenvironment and may be active participants in a complex, cytokine-mediated regulatory network.