Mimicking phosphorylation at Ser-48 strongly reduces surface expression of human macrophage scavenger receptor class A: implications on cell motility.

The role of human macrophage scavenger receptor A1 (SRA1) in the development of atherosclerotic lesions is still scarcely defined. Substituting serine 48 in human SRA1 by an aspartate demonstrated that (1) surface expression of the mutated receptor was 13-fold decreased; (2) the amount of cell-associated Texas red-labeled acetylated low density lipoprotein (LDL) in mutant receptor-expressing cells was almost three-fold reduced; (3) the migration of mutant receptor-transfected cells towards surfaces coated with oxidized LDL decreased by almost 60% compared to cells that were transfected with the wild type receptor. Phosphorylation of the cytoplasmic part of SRA1 may help to modulate the residence time of macrophages in atherosclerotic lesions.

Tenascin-R associates extracellularly with parvalbumin immunoreactive neurones but is synthesised by another neuronal population in the adult rat cerebral cortex.

The molecular components surrounding a neurone serve as recognition cues for the nerve terminals and glial processes that contact them and the constellations formed by these inputs will therefore be determined by the blend of adhesive and repulsive components therein. Using immunohistochemical methods, we observed that the large extracellular matrix-protein, tenascin-R (Restrictin, J1-160-180, Janusin), associates preferentially with the parvalbumin-positive subpopulation of interneurones within the cerebral cortex. In situ-hybridization indicated that tenascin-R-mRNA was expressed in a subpopulation of nerve cells distinct from that containing parvalbumin, suggesting that this protein's association with the latter is receptor mediated. These nerve cells thus modulate at a distance the composition of the extracellular matrix around parvalbuminneurons.

Immunocytological localization of the HNK-1 carbohydrate in murine cerebellum, hippocampus and spinal cord using monoclonal antibodies with different epitope specificities.

The HNK-1 carbohydrate, an unusual 3'-sulfated glucuronic acid epitope characteristic of many neural recognition molecules, serves as a ligand in neural cell interactions and is differentially expressed in the quadriceps and saphenous branches of the femoral nerve in the PNS of adult mice. Based on these observations, we investigated the possibility that the HNK-1 carbohydrate may be differentially distributed in neurons and fiber tracts also in the CNS thereby contributing to different targeting and guidance mechanisms. We have used antibodies with different HNK-1 epitope specificities to probe for subtle differences in expression patterns. In the adult mouse cerebellum the HNK-1 carbohydrate is detectable in stripe-like compartments in the molecular and Purkinje cell layers, whereas N-CAM and its associated alpha2,8 polysialic acid does not show this compartmentation. In the adult hippocampus, the HNK-1 carbohydrate localizes to perineuronal nets of inhibitory interneurons and marks the inner third of the molecular layer of the dentate gyrus. In the adult spinal cord, HNK-1 labeling is most pronounced in gray matter areas. White matter enriched regions show differential labeling with regard to fiber tracts and antibody specificity. Whereas the different antibodies do not show differences in staining in the cerebellum and the hippocampus, they show differences in staining pattern of fiber tracts and motoneurons in the spinal cord. The HNK-1 expression pattern also differed in the adult spinal cord from that observed at embryonic day 14 and postnatal day 14. Our observations suggest a functional role in the specification of functionally discrete compartments in different areas of the CNS and during development.

Apoptosis induced by oxidized low density lipoprotein in human monocyte-derived macrophages involves CD36 and activation of caspase-3.

Macrophage death may play a crucial role in the progression of atherosclerotic lesions. Here we present evidence that CD36 is involved in oxidized LDL (OxLDL)-induced apoptosis in human monocyte-derived macrophages. Anti-CD36 mAb SMO and OKM-5 reduced the number of apoptotic cells in OxLDL-treated macrophages by more than 94%, but they did not block ceramide-triggered apoptosis. Thrombospondin inhibited the induction of apoptosis by OxLDL in a dose-dependent manner with an IC50 of 10-30 microM. OxLDL did not induce apoptosis in CD36-negative macrophages, demonstrating the essential role of this scavenger receptor in OxLDL-triggered programmed cell death. Neither anti-CD36 Ig nor thrombospondin triggered programmed cell death suggesting that binding to CD36 alone is not sufficient to initiate apoptosis. However, inhibitors of OxLDL-induced apoptosis did not block the uptake of 3H-labeled OxLDL. In contrast, acetylated LDL and polyinosinic acid, ligands of scavenger receptor A (SRA), inhibited uptake of 3H-labeled OxLDL by 65 and 49%, respectively, but did not block OxLDL-induced apoptosis, indicating that SRA is not involved in this process. OxLDL also stimulated caspase-3 activity in human macrophages. Activation of caspase-3 was blocked by anti-CD36 Ig and the caspase-3 inhibitor Z-DEVD-FMK. These results suggest that binding of OxLDL to CD36 initiates a yet unknown OxLDL-specific signaling event, which leads to the rapid activation of caspase-3 resulting in apoptosis of human macrophages. Our data demonstrate a novel role for CD36 in macrophage biology with likely consequences for the development of atherosclerotic lesions.

Selective distribution of calretinin in adenocarcinomas of the human colon and adjacent tissues.

The expression of calretinin, a calcium-binding protein, has been studied in a series of 82 human colorectal adenocarcinomas. In 22.5% of the cases, part of the tumor cells were calretinin-positive, whereas the cells of the normal and paratumoral mucosa were always negative. Two types of cells from the tumoral mass reacted positively and selectively with calretinin-antisera: the tumor cells and giant fibroblasts. The neurons of enteric ganglia and reactive mesothelial cells also reacted positively to the same antibody. The results obtained by immunochemistry have been confirmed by Western blot analysis and in situ hybridization for calretinin mRNA. There is a correlation between the expression of calretinin and the degree of differentiation of the tumor. Well-differentiated tumors express calretinin in only 5% of the cases, whereas this percentage is 20% for moderately differentiated tumors and 66.6% for poorly differentiated or undifferentiated tumors. We conclude that calretinin is expressed by most undifferentiated colorectal adenocarcinomas, but only by a limited number of cells in well-differentiated tumors. The degree of its expression coincides also with additional signs of malignancy, such as an increase in the number of metastases in the regional lymph nodes and in other organs.

Comparative studies of calretinin expression by WiDr cell line in vivo in xenografts in nude mice and in vitro.

WiDr cells from a human colon adenocarcinoma cultivated in vitro express the calcium binding protein calretinin. The immunoreactivity is present in some interphasic cells and decreases after seven days in culture together with the augmentation of the cell number. Calretinin expression is maintained in the undifferentiated cells of the tumoral mass developed in nude mice and in recultivated isolated tumour cells from the xenograft. From the experiments here described, the protein expression is quantitatively influenced in vitro by the addition of drugs, such as colchicine and taxol, which intervene in cytoskeleton organisation. The percentage of the calretinin immunoreactive cells increases after the addition of colchicine to the medium while the immunoblot analysis shows a higher calretinin content in the cells treated with taxol.

Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity.

Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36+CD14-LDL-R-(58+/-12%), CD36+CD14+LDL-R+(18+/-5%), the remaining cells being CD36-CD14- LDL-R-. The first two subsets decreased in size during further differentiation (51+/-12% and 8+/-3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis.

Dehydroascorbic acid prevents apoptosis induced by oxidized low-density lipoprotein in human monocyte-derived macrophages.

Human low-density lipoprotein (LDL) oxidized with Cu2+ or the radical generator 2,2'-azobis(2-methyl-propionamidine) hydrochloride (AAPH) induces apoptosis in mature human monocyte-derived macrophages as assessed by staining with fluorescein-isothiocyanate-labeled annexin V, by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and by staining of the 7A6 mitochondrial antigen. Oxidized LDL-induced apoptosis was dose and time dependent and clearly distinct from apoptosis induced by serum deprivation. Human autologous serum and lipoprotein-deficient human serum prevented apoptosis induced by oxidized LDL. Supplementation of serum-free culture medium with 25 microM ascorbic or isoascorbic acid only partially protected macrophages from apoptosis, whereas dehydroascorbic acid (DHAA) completely inhibited apoptosis induced by either Cu2+- or AAPH-oxidized LDL. Apoptosis was also inhibited by the structural analogue alloxan. Both cyclic multiketones dose-dependently inhibited oxidized LDL-induced apoptosis with IC50 in the submicromolar range. Prior loading of macrophages with ascorbic acid did not prevent the induction of apoptosis. Apoptosis was reduced by more than 90% after treatment of oxidized LDL with DHAA, whereas after incubation with either ascorbic or isoascorbic acid there was no such reduction. Removal of free DHAA by gel filtration did not reverse the inactivation. Parameters of LDL oxidation such as electrophoretic mobility, alpha-tocopherol content, thiobarbituric-acid-reactive subtances and lipid peroxide levels did not correlate to apoptotic activity. Also, binding and uptake of Texas-red-labeled oxidized LDL was not prevented by DHAA. Dithiothreitol-treatment of oxidized LDL, however, reduced the apoptotic activity by 76%. Our results suggest that oxidized thiols on apoB may be essential for the induction of apoptosis by oxidized LDL in human macrophages.

Changes in the expression of the extracellular matrix molecules tenascin-C and tenascin-R after 3-acetylpyridine-induced lesion of the olivocerebellar system of the adult rat.

In the central nervous system of rodents, the extracellular matrix glycoproteins tenascin-C and tenascin-R are expressed predominantly by astrocytes and oligodendrocytes respectively. Both molecules support neurite outgrowth from several neuronal cell types when presented as uniform substrates. When offered as a sharp boundary with a permissive substrate, however, both molecules prevent neurite elongation. On the basis of these observations it has been suggested that tenascin-C and tenascin-R may be relevant in determining the cellular response after injury in the adult rodent central nervous system. To investigate whether tenascin-C and tenascin-R may play important functional roles in the lesioned central nervous system, we have analysed their expression in the olivocerebellar system of the adult rat after 3-acetylpyridine-induced degeneration of nerve cells in the inferior olivary nucleus. Tenascin-C mRNA was not detectable at any time in the unlesioned or lesioned inferior olivary nucleus by in situ hybridization. In the cerebellar cortex, tenascin-C mRNA in Golgi epithelial cells was down-regulated 3 days after the lesion and returned to control values 80 days after the lesion. Tenascin-R mRNA was expressed by distinct neural cell types in the unlesioned olivocerebellar system. After a lesion, the density of cells containing tenascin-R transcripts increased significantly in the inferior olivary nucleus and in the white matter of the cerebellar cortex. Immunohistochemical and immunochemical investigations confirmed these observations at the protein level. Our data thus suggest differential functions of tenascin-C and tenascin-R in the injured central nervous system.

The proteoglycan DSD-1-PG occurs in perineuronal nets around parvalbumin-immunoreactive interneurons of the rat cerebral cortex.

Proteoglycans involved in the shaping of the developing brain are often preserved in the adult brain in more restricted locations. We have studied the fate of DSD-1-PG, a chondroitin sulfate proteoglycan containing the hybrid epitope DSD-1. DSD-1-PG exerts neurite outgrowth promoting activity and has been shown to occur in the developing brain during late brain development and into adulthood. In the adult rat brain, monoclonal and polyclonal antibodies against DSD-1-PG labelled only the circumference of a selected subpopulation of neurons. These nerve cells invariably expressed the calcium-binding protein parvalbumin. The label occupied the extracellular space in close vicinity to the cell body, surrounding axon terminals and glial end feet, but was absent from synaptic clefts. DSD-1-PG is thus shown to be an additional representative of the growing list of substances found in perineuronal locations in the adult mammalian brain.

Temporal and spatial appearance of the membrane cytoskeleton and perineuronal nets in the rat neocortex.

Parvalbumin-immunoreactive interneurons are surrounded by perineuronal nets, containing molecules of the extracellular matrix (e.g. tenascin-R). Furthermore, they seem to have a special cytoskeleton composed of, among others, ankyrinR and beta Rspectrin. In the present developmental study we showed that the intracellular markers parvalbumin, ankyrinR and beta Rspectrin as well as Vicia Villosa agglutinin, an extracellular marker for perineuronal nets, appeared in the second postnatal week. In the third postnatal week, ankyrinR and beta R spectrin were present in the parvalbumin-positive interneurons. Tenascin-R appeared in a similar topographic distribution as the intracellular markers. The adult pattern was established upon the end of the fourth postnatal week. Our results indicate that cytoskeletal maturity maybe a prerequisite for the organization of perineuronal nets of extracellular matrix.

CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the alpha 6/beta 1 integrin and the neural adhesion molecule L1.

We describe here a novel monoclonal antibody (mab H6) which recognizes CD9, an integral cell surface constituent previously described in cells of the hematopoietic lineage and involved in the aggregation of platelets. Mab H6 was raised against membranes of immature mouse astrocytes and reacted with a protein of 25-27 kD in detergent extracts of adult mouse brain membranes. Sequence analysis of the N-terminal amino acids revealed an identity of 96% with CD9 from mouse kidney. CD9 was localized in the central and peripheral mouse nervous systems: in the spinal cord of 11-day-old mouse embryos, CD9 was strongly expressed in the floor and roof plates. In the adult mouse sciatic nerve, myelin sheaths were highly CD9-immunoreactive. Mab H6 reacted with the cell surfaces of both glial cells and neurons in culture and inhibited migration of neuronal cell bodies, neurite fasciculation and outgrowth of astrocytic processes from cerebellar microexplants. Neurite outgrowth from isolated small cerebellar neurons was increased in the presence of mab H6 on substrate-coated laminin, but not on substrate-coated poly-L-lysine. Addition of mab H6 elicited an increase in intracellular Ca2+ concentration in these cells on substrate-coated laminin. Immunoprecipitates of CD9 from cultured mouse neuroblastoma N2A cells contained the alpha 6/beta 1 integrin. Moreover, preparations of CD9 immunoaffinity-purified from adult mouse brain using a mab H6 column contained the neural adhesion molecule L1, but not other neural adhesion molecules. CD9 bound to L1, but not to NCAM or MAG. Both the alpha 6/beta 1 integrin and L1 could be induced to coredistribute with CD9 on the surface of cultured neuroblastoma N2A cells. The combined observations suggest that CD9 can associate with L1 and alpha 6/beta 1 integrin to influence neural cell interactions in vitro.

Mice deficient for the myelin-associated glycoprotein show subtle abnormalities in myelin.

Using homologous recombination in embryonic stem cells, we have generated mice with a null mutation in the gene encoding the myelin-associated glycoprotein (MAG), a recognition molecule implicated in myelin formation. MAG-deficient mice appeared normal in motor coordination and spatial learning tasks. Normal myelin structure and nerve conduction in the PNS, with N-CAM overexpression at sites normally expressing MAG, suggested compensatory mechanisms. In the CNS, the onset of myelination was delayed, and subtle morphological abnormalities were detected in that the content of oligodendrocyte cytoplasm at the inner aspect of most myelin sheaths was reduced and that some axons were surrounded by two or more myelin sheaths. These observations suggest that MAG participates in the formation of the periaxonal cytoplasmic collar of oligodendrocytes and in the recognition between oligodendrocyte processes and axons.

Localization of janusin mRNA in the central nervous system of the developing and adult mouse.

Janusin (formerly termed J1-160/180) is an oligodendrocyte-derived extracellular matrix molecule which is restricted to the central nervous system and which is expressed late during development (Pesheva et al., J. Cell Biol., 1765-1778, 1989). To gain insights into the molecule's morphogenetic functions and to identify its cellular source in vivo, we have studied the localization of janusin messenger RNA in the optic nerve, retina and spinal cord and the expression of janusin protein in the spinal cord of developing and adult mice. Moreover, we have analysed optic nerve cell cultures and retinal cell suspensions in double-labelling experiments using a janusin-specific anti-sense complementary RNA probe and cell type-specific antibodies to identify the cell types containing janusin transcripts. In developing animals, oligodendrocytes were strongly labelled with the janusin anti-sense cRNA probe during the period of myelination. The number of labelled cells and intensity of the hybridization signal decreased significantly with increasing age. Interestingly, expression of janusin was not confined to oligodendrocytes. Some neuronal cell types and type-2 astrocytes present in optic nerve cell cultures also contained janusin transcripts. In contrast to oligodendrocytes, the number and labelling intensity of neurons containing janusin transcripts remained constant during postnatal development and into adulthood. Expression of janusin protein in the spinal cord was developmentally regulated, with a peak of expression in 2- or 3-week-old animals. The molecule was visible in the white and grey matter. In myelinated regions, it was associated with myelinated fibres and accumulated at nodes of Ranvier. These observations suggest that janusin may be of functional relevance for myelination.

Molecular characterization and in situ mRNA localization of the neural recognition molecule J1-160/180: a modular structure similar to tenascin.

The oligodendrocyte-derived extracellular matrix glycoprotein J1-160/180 is a recognition molecule expressed exclusively in the central nervous system. J1-160/180 has been shown to be adhesive for astrocytes and repellent towards neurons and growth cones. We report here the complete nucleotide sequence of J1-160/180 in the rat. The predicted amino acid sequence showed a structural architecture very similar to tenascin: a cysteine-rich amino terminal region is followed by 4.5 epidermal growth factor-like repeats, 9 fibronectin type III homologous repeats and a domain homologous to fibrinogen. Sequence comparison analysis revealed highest homology of rat J1-160/180 to mouse tenascin and chicken restrictin with a similarity of 66% and 85%, respectively. The J1-160/180-coding mRNA is derived from a single copy gene. Using the polymerase chain reaction we could show that two J1-160/180 isoforms are generated by alternative splicing of the sixth fibronectin type III homologous repeat. Localization of J1-160/180 mRNA by in situ hybridization in the cerebellum, hippocampus and olfactory bulb confirmed the expression of J1-160/180 by oligodendrocytes with a peak of transcription at 7-14 d after birth, indicating a functional role during myelination. In addition, J1-160/180-specific RNA was found in a small subset of neurons in all three structures of the CNS analyzed. These neurons continue to express J1-160/180 in the adult.