Metabotropic P2 receptor activation regulates oligodendrocyte progenitor migration and development.

To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in cultured rat oligodendrocyte progenitors and investigated the effects of ATP and its breakdown products on the migration and proliferation of this immature glial cell population. Using Western blot analysis, we show that oligodendrocyte progenitors express several P2X (P2X(1,2,3,4,7)) and P2Y (P2Y(1,2,4)) receptors. Intracellular Ca(2+) recording by Fura-2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADPbetaS = ADP = Benzoyl ATP > ATP > ATPgammaS > UTP, alpha,beta-meATP ineffective. Based on the above findings, on pharmacological inhibition by the antagonists oxATP and MRS2179, and on the absence of alpha,betameATP-induced inward current in whole-cell recording, P2X(7) and P2Y(1) were identified as the main ionotropic and metabotropic P2 receptors active in OPs. As a functional correlate of these findings, we show that ATP and, among metabotropic agonists, ADP and the P2Y(1)-specific agonist ADPbetaS, but not UTP, induce oligodendrocyte progenitor migration. Moreover, ATP and ADP inhibited the proliferation of oligodendrocyte progenitors induced by platelet-derived growth factor, both in purified cultures and in cerebellar tissue slices. The effects of ATP and ADP on cell migration and proliferation were prevented by the P2Y(1) antagonist MRS2179. By confocal laser scanning microscopy, P2Y(1) receptors were localized in NG2-labeled oligodendrocyte progenitors in the developing rat brain. These data indicate that ATP and ADP may regulate oligodendrocyte progenitor functions by a mechanism that involves mainly activation of P2Y(1) receptors.

Intracerebral regulation of immune responses.

Major progress has been made over the last years in our understanding of the mechanisms underlying immune privilege and immune surveillance of the central nervous system (CNS). Once considered a passive process relying only on physical barriers, immune privilege is now viewed as a more complex phenomenon, which involves active regulation of immune reactivity by the CNS microenvironment. Evidence has also emerged that the immune system continuously and effectively patrols the CNS and that dysregulated immune responses against CNS-associated (exogenous or self) antigens are involved in the pathogenesis of various neurological diseases. In this article we shall briefly review current knowledge of how the immune response is regulated locally in the CNS and which cell types and molecular mechanisms are involved in shaping intracerebral immune responses.

Intracerebral recruitment and maturation of dendritic cells in the onset and progression of experimental autoimmune encephalomyelitis.

Dendritic cells (DCs) are thought to be key elements in the initiation and maintenance of autoimmune diseases. In this study, we sought evidence that DCs recruited to the central nervous system (CNS), a site that is primarily devoid of resident DCs, play a role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE). After immunization of SJL mice with proteolipid protein 139-151 peptide, process-bearing cells expressing the DC markers DEC-205 and CD11c appeared early in the spinal cord. During acute, chronic, and relapsing EAE, DEC-205(+) DCs expressing a lymphostimulatory phenotype (including the mature DC marker MIDC-8, major histocompatibility complex class II, CD40, and CD86 molecules) accumulated within the CNS inflammatory cell infiltrates. More prominent infiltration of the spinal cord parenchyma by mature DCs was observed in mice with relapsing disease. Macrophage inflammatory protein 3alpha, a chemokine active on DCs and lymphocytes, and its receptor CCR6 were up-regulated in the CNS during EAE. These findings suggest that intracerebral recruitment and maturation of DCs may be crucial in the local stimulation and maintenance of autoreactive immune responses, and that therapeutic strategies aimed at manipulating DC migration could be useful in the treatment of CNS autoimmune disorders.

Glia-T cell dialogue.

Interactions of CD4(+) T helper (Th) cells with microglia and astrocytes are likely to play an important role in regulating immune responses as well as tissue damage and repair during infectious and autoimmune central nervous system (CNS) diseases. T cells secreting Th1-type cytokines provide inducing signals for microglia to mature into functional antigen presenting cells (APC). The ability of microglia to act as efficient APC for the restimulation of Th1 cells suggests a role for these cells in the local amplification of pro-inflammatory immune responses. Conversely, the Th2-inducing capacity of microglia and astrocytes together with their ability to produce anti-inflammatory mediators could play a role in providing counter-regulatory signals limiting CNS inflammation. In this article, we review recent studies addressing the functional significance of T cell-CNS glia interactions and present new data on the expression of cyclooxygenase-2, the inducible enzyme involved in prostanoid biosynthesis, in microglia and astrocytes during the course of experimental allergic encephalomyelitis.

Short-lived immunization site inflammation in self-limited active experimental allergic encephalomyelitis.

To understand the mechanisms underlying spontaneous remission of proteolipid protein (PLP) 139-151 peptide-induced experimental allergic encephalomyelitis (EAE), an acute autoimmune disease of SJL mice resembling human multiple sclerosis, we examined both the effector response site in the central nervous system (CNS) and the immunization site at different phases of the disease. In the CNS, the frequency of PLP 139-151 peptide-specific IFN-gamma-producing T cells as well as the amount of infiltrating CD4(+) and CD11b(+) cells decreased with recovery. However, IL-4-producing cells were always rare and cyclooxygenase-2(+) cells were numerous only at disease peak in the CNS, suggesting that T(h)2 cytokines and prostaglandins did not determine remission of EAE. By looking at the s.c. site of PLP 139-151 peptide plus adjuvant injection, we found that, although the inflammatory infiltrate was abundant, CD11b(+) cells started to decrease already during disease acute phase and DEC-205(+) cells were numerous only at early time points. We propose that immunization site inflammation is short-lived in PLP 139-151 peptide-induced EAE, and this leads to a temporary autoreactive T cell stimulation and to a self-limited disease.

[Computerized histological analysis in chronic viral hepatitis]. / Analisi isotologica computerizzata di epatiti croniche virali.

Chronic liver diseases evolve towards cirrhosis by means of the progressive substitution of hepatic tissue with fibrotic areas. In this work we measured the percentage of fibrotic areas with respect to the total area of the bioptic specimens, utilizing a histomorphometric computerized evaluation. The specimens were obtained by 39 patients affected to chronic viral liver diseases, before and after a therapy with alpha interferon. The aim of this study is to determine the architectural sprain degree at the different stages of chronic liver disease, and to verify if the response to the therapy with interferon is associated with a reduction of the fibrotic areas within the hepatic tissue. The comparison between our computerized index and the Knodell index indicates that the first can be considered an useful system for the evaluation of hepatic biopsies from patients affected by chronic viral hepatitis.

'Tissue' transglutaminase release from apoptotic cells into extracellular matrix during human liver fibrogenesis.

Enhanced apoptosis characterizes several pathologies affecting human liver. This study sought to determine whether apoptosis is involved in the formation of fibrotic lesions occurring in hepatic disease. The expression of Bcl-2 was analysed, and of 'tissue' transglutaminase (tTG), a cross-linking enzyme which recent evidence suggests plays a role in the formation of fibrotic lesions in experimental settings. Regardless of the degree of liver injury, tTG abnormally accumulated in the liver cells adjacent to fibrotic tissue. Many cells showing DNA fragmentation and morphological features of apoptosis were also observed near fibrotic lesions. Bcl-2 was detected predominantly in infiltrating lymphocytes within the liver tissue. Marked staining for both tTG protein and chromatin was also observed in the acellular fibrotic tissue, which suggested an active release of intracellular macromolecules from the dying cells into the extracellular matrix. This study indicates that fibrogenesis in the liver is associated with the release of tTG from dying cells. By cross-linking extracellular matrix proteins, this enzyme might play a role in the formation of fibrotic lesions.

Lysosomal involvement in the removal of clofibrate-induced rat liver peroxisomes. A biochemical and morphological analysis.

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate-treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug-induced peroxisomes was investigated under physiological conditions, i.e. in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal beta-oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.

Morphometric analysis of liver and kidney peroxisomes in lactating rats and their pups after treatment with the peroxisomal proliferator di-(2-ethylhexyl)phthalate.

Di-(2-ethylexyl)phthalate (DEHP) administered to adult lactating rats from delivery to weaning induces age- and organ-specific modifications of the peroxisomal morphometric parameters (VV, NA and D) in the liver and kidney of both rats and their pups. In both tissues, peroxisomal relative volume and catalase biochemical activity show a similar pattern during the development, as well as under DEHP treatment. Morphometric results suggest that two modalities of peroxisomal proliferation exist, involving: a) increases in both number and mean diameter of the organelles; b) a purely numerical increase of the organelles, accompanied by a remarkable decrement in their mean diameter. A peroxisomal population proliferated through the latter model appears unable to return to normal conditions, following treatment withdrawal. These two proliferation systems, the first implying a swelling and the latter a fragmentation of pre-existing peroxisomal profiles, are supposed to be tissue-specific in the adult animal. In particular, in the liver the 'swelling' model appears more suitable to explain peroxisome proliferation, while the kidney this process would follow the 'fragmentation' model. Immature animals might instead show in both organs intermediate features of peroxisomal proliferation modalities.

Effects of Di-(2-ethylhexyl)phthalate on peroxisomes of liver, kidney and brain of lactating rats and their pups.

Di-(2-ethylhexyl)phthalate administered to adult lactating rats, from delivery to weaning, induces modifications of the peroxisomal enzymatic pattern in the liver, kidney and brain of both dams and pups. These modifications are age- and organ-dependent. Biochemical analysis shows that: 1) catalase specific activity is two-fold increased in the liver of both adult and newborn animals, in the kidney of newborns and in the brain of adults. 2)D-amino acid oxidase doubles in all newborn organs and in adult brain; it increases, although to a lesser extent, also in adult kidney, while it is half-reduced in adult liver. 3) Dihydroxyacetone phosphate acyl transferase only doubles in newborn liver, remaining fairly unchanged in all the other tested tissues. 4) Palmitoyl-CoA oxidase is greatly induced in the liver of both dams and litters, doubled in the kidneys and slightly increased or not at all in the brain of pups and mothers, respectively. The effect of the drug on enzyme activities is reversible, with different time courses depending on the considered enzyme and organ. Western blottings confirm the biochemical data. Electron microscopy shows proliferated peroxisomes in the liver and kidney of treated animals but not in the brain, where high catalase-like immunoreactivity is observed in the cytosol of neurons. Taken together, our data demonstrate that the response of peroxisomal enzymes to DEHP treatment is age- as well as tissue-dependent and specific for each enzyme studied.

A preparative method for isolation of peroxisomes from rat kidney.

A new method for the isolation of peroxisomes from rat kidney cortex is described. The L fraction obtained according to Wattiaux-De Coninck et al. (1965) was layered on a discontinuous Nycodenz gradient (density = 1.15-1.21 g/ml) and then centrifuged in a fixed angle rotor for 45 min. at 136,000 g. On the basis of the morphological and biochemical analysis, the fraction recovered at the bottom of the tube was composed mainly by peroxisomes enriched in fatty acyl beta-oxidation system, whereas lower enrichment was found for other peroxisomal marker enzymes. Negligible contamination by mitochondria (marker enzyme cytochrome oxidase), lysosomes (marker enzyme acid phosphatase) and microsomes (marker enzyme NADPH cytochrome c reductase) was found.

Differentiation of kidney cortex peroxisomes in fetal and newborn rats.

Peroxisomal enzyme assays as well as cytochemical detection of catalase were carried out on fetal and newborn rat kidney cortex throughout the last 3 days of prenatal life and the first month of postnatal development. Concerning the patterns of peroxisomal enzymes, catalase activity, hardly detectable in the fetus, shows the strongest increment after the second week of postnatal life; beta-oxidation system and D-amino acid oxidase increase soon after birth; urate oxidase activity, detected in fetal life, rapidly decreases after birth; dihydroxyacetone phosphate-acyltransferase activity doubles at birth, remaining constant thereafter. Since by cytochemistry no catalase particles were detected in fetal kidneys, morphometric parameters were studied only postnatally. The numerical density shows only minor variations, mainly at day 3; the mean diameter remains practically unchanged between birth and day 14 but strongly increases later. The volume density pattern correlates in the early phase with the numerical density and later with the profile mean diameter. The results suggest that enzymes are asynchronously incorporated into pre-existing peroxisomes; that this import is faster in smaller organelles than in the larger, adult ones; that catalase increases after the H2O2-producing oxidases; and that the abrupt rise of beta-oxidation capacity and DH-APAT is related to the increased renal work immediately after birth.

Purification of peroxisomal fraction from rat brain.

A purification procedure to obtain peroxisomes (microperoxisomes) from the brain of suckling rats is reported. A P2 fraction, (crude light mitochondria) frozen and thawed seven times, was subfractionated yielding a P4 fraction, 4-fold enriched in catalase activity with respect to the cytoplasmic extract S1. The P4 fraction was used for further purification of peroxisomes by isopicnic centrifugation on Nycodenz gradient (1.10-1.20 g/ml). When the cerebellum was not included in the starting material, the equilibrium density of peroxisomes was 1.152-1.162 g/ml. In this case the overall yield of catalase in the most enriched fraction was 7% and its relative specific activity more than 50. When the cerebellum was included in the total homogenate, the equilibrium density shifted towards higher values (1.177 g/ml) and in this case the catalase relative specific activity in the peroxisomal enriched fraction was extremely high (> 100). The biochemical results, together with the electron microscope examination of the purified fractions, demonstrate that our procedure allows the best purification of brain peroxisomes so far obtained. The different equilibrium densities of peroxisomes observed in the two sets of experiments are interpreted in terms of size heterogeneity of these organelles in different brain portions and cell types.

31P-NMR of liver peroxisome membranes from normal and clofibrate-treated rats.

Normal and clofibrate-induced rat liver peroxisomes were studied in order to correlate the drug-elicited modifications of membrane permeability and fluidity with changes of the phospholipid component. The phospholipid composition and membrane fluidity of purified peroxisomal fractions from control and test animals were evaluated by 31P-NMR and fluorescence polarization spectroscopic techniques. The ultrastructural, enzymatic and electrophoretic analyses confirmed that the drug was able to induce: i) peroxisome proliferation; ii) increase of the catalase and fatty acyl CoA beta-oxidation system (FAOS) activities, and iii) neosynthesis of the 69 kDa peroxisomal membrane protein (PMP). The distribution pattern of the peroxisomal enzymes and the fluorescence polarization values of anilinonaphthalene-8-sulfonate (ANS)-labelled peroxisomes, suggests that the membrane permeability and fluidity are altered, while 31P-NMR spectroscopy seems to indicate that the phospholipid composition and the phospholipid/lysophospholipid ratio do not vary between test and control samples.