Organization of plasma membrane functional rafts upon T cell activation.

Raft microdomains have been shown to play a key role in T cell activation. We found that in human T lymphocytes the formation of functional rafts at the plasma membrane was induced by T cell priming. In resting T cells from peripheral blood Lck and the raft glycosphingolipid GM1 resided in intracellular membranes. T cell activation induced synthesis of GM1 and effector cells showed very high levels of this lipid, which became predominantly plasma membrane associated. TCR triggering also induced targeting of the cytosolic Lck to the plasma membrane. Thus, effector cells acquire an improved signaling machinery by increasing the amount of rafts at the plasma membrane. The fact that, when compared with naive T cells, memory T cells showed higher GM1 levels suggests that raft lipid synthesis may be developmentally regulated and tune T cell responsiveness.

[Bacterial perihepatitis]. / Périhépatites bactériennes.

The Fitz-Hugh-Curtis syndrome is a peri-hepatitis following a genital infection. It usually occurs in young women. Chlamydia trachomatis is the most frequent causal agent. Clinical signs include acute or recurrent pain in the right hypochondria. Liver tests are not modified and the sonographic examination is normal. Diagnosis can be suspected on the basis of serology, and formally established by laparoscopy showing violin string-like adhesions. Prolonged antibiotic treatment is effective.

[Collagen gastroenterocolitis]. / Une gastroentérocolite collagène.

INTRODUCTION: Collagenous gastroenterocolitis is a recently known rare cause of chronic diarrhoea, that raises numerous nosological and diagnostic problems. OBSERVATION: A 41 year-old woman was hospitalised for severe diarrhoea, diagnosed as collagenous gastroenterocolitis. Gastroscopy and ileocolonoscopy were macroscopically normal, but a 20 to 40 microns thick sub-epithelial collagenous band was revealed in the gastric, duodenal and colic biopsies. Parenteral nutrition and treatment with salazopyrine and prednisolone progressively normalised the transit. Three months later, only a 30 microns colic mucosa collagenous band persisted. All the biopsies taken during control gastro-colonoscopy 2 years later were histologically normal. After 5 years follow-up and absence of treatment, the patient no longer presented diarrhoea or biological abnormality. COMMENTS: This exceptional observation is a reminder that sub-epithelial collagen deposits are not always limited to the colon and therefore justify, in patients with collagenous colitis, systematic gastro-duodenal and ileum biopsies.

Expression of interleukin 3 and granulocyte-macrophage colony-stimulating factor receptor common chain betac, betaIT in normal haematopoiesis: lineage specificity and proliferation-independent induction.

Interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 5 (IL-5) exert their biological activities through interaction with cell-surface receptors that consist of two subunits, a specific alpha subunit and a common beta transducing subunit (betac). We have evaluated the expression of betac on purified haematopoietic progenitor cells (HPCs) induced to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk) or monocytic (Mo) lineage. HPCs displayed low betac expression, which increased during the initial stages of HPC differentiation along the E, G, Mo or Mk lineages. At later stages of differentiation, betac chain expression increased in both G and Mo lineages, was expressed at low levels in the Mk lineage and declined to undetectable levels in the E lineage. Analysis of the full-length betac and intracytoplasmically truncated betac (betaIT) mRNAs showed that the former was predominant in the G and Mo lineages, whereas the latter was prevalent in the E and Mk lineages. The betac induction takes place even in the absence of cell cycling. Thus, incubation of HPCs with graded amounts of IL-3 showed that the initial induction of betac expression is unrelated to cell proliferation. Furthermore, circulating monocytes and granulocytes exhibit a low level of betac expression that is greatly stimulated following incubation with either IL-3 or GM-CSF.

Extramitochondrial porin: facts and hypotheses.

Mitochondrial porin, or VDAC, is a pore-forming protein abundant in the outer mitochondrial membrane. Several publications have reported extramitochondrial localizations as well, but the evidence was considered insufficient by many, and the presence of porin in nonmitochondrial cellular compartments has remained in doubt for a long time. We have now obtained new data indicating that the plasma membrane of hematopoietic cells contains porin, probably located mostly in caveolae or caveolae-like domains. Porin was purified from the plasma membrane of intact cells by a procedure utilizing the membrane-impermeable labeling reagent NH-SS-biotin and streptavidin affinity chromatography, and shown to have the same properties as mitochondrial porin. A channel with properties similar to that of isolated VDAC was observed by patch-clamping intact cells. This review discusses the evidence supporting extramitochondrial localization, the putative identification of the plasma membrane porin with the "maxi" chloride channel, the hypothetical mechanisms of sorting porin to various cellular membrane structures, and its possible functions.

Glycosphingolipid domains on cell plasma membrane.

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains.

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.

Expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane. Retention of caveolin-2 at the level of the golgi complex.

Caveolins-1 and -2 are normally co-expressed, and they form a hetero-oligomeric complex in many cell types. These caveolin hetero-oligomers are thought to represent the assembly units that drive caveolae formation in vivo. However, the functional significance of the interaction between caveolins-1 and -2 remains unknown. Here, we show that caveolin-1 co-expression is required for the transport of caveolin-2 from the Golgi complex to the plasma membrane. We identified a human erythroleukemic cell line, K562, that expresses caveolin-2 but fails to express detectable levels of caveolin-1. This allowed us to stringently assess the effects of recombinant caveolin-1 expression on the behavior of endogenous caveolin-2. We show that expression of caveolin-1 in K562 cells is sufficient to reconstitute the de novo formation of caveolae in these cells. In addition, recombinant expression of caveolin-1 allows caveolin-2 to form high molecular mass oligomers that are targeted to caveolae-enriched membrane fractions. In striking contrast, in the absence of caveolin-1 expression, caveolin-2 forms low molecular mass oligomers that are retained at the level of the Golgi complex. Interestingly, we also show that expression of caveolin-1 in K562 cells dramatically up-regulates the expression of endogenous caveolin-2. Northern blot analysis reveals that caveolin-2 mRNA levels remain constant under these conditions, suggesting that the expression of caveolin-1 stabilizes the caveolin-2 protein. Conversely, transient expression of caveolin-2 in CHO cells is sufficient to up-regulate endogenous caveolin-1 expression. Thus, the formation of a hetero-oligomeric complex between caveolins-1 and -2 stabilizes the caveolin-2 protein product and allows caveolin-2 to be transported from the Golgi complex to the plasma membrane.

Phorbol ester-induced disruption of the CD4-Lck complex occurs within a detergent-resistant microdomain of the plasma membrane. Involvement of the translocation of activated protein kinase C isoforms.

Recent studies have highlighted the existence of discrete microdomains at the cell surface that are distinct from caveolae. The function of these microdomains remains unknown. However, recent evidence suggests that they may participate in a subset of transmembrane signaling events. In hematopoietic cells, these low density Triton-insoluble (LDTI) microdomains (also called caveolae-related domains) are dramatically enriched in signaling molecules, such as cell surface receptors (CD4 and CD55), Src family tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins, and gangliosides (GM1 and GM3). Human T lymphocytes have become a well established model system for studying the process of phorbol ester-induced down-regulation of CD4. Here, we present evidence that phorbol 12-myristate 13-acetate (PMA)-induced down-regulation of the cell surface pool of CD4 occurs within the LDTI microdomains of T cells. Localization of CD4 in LDTI microdomains was confirmed by immunoelectron microscopy. PMA-induced disruption of the CD4-Lck complex was rapid (within 5 min), and this disruption occurred within LDTI microdomains. Because PMA is an activator of protein kinase C (PKC), we next evaluated the possible roles of different PKC isoforms in this process. Our results indicate that PMA induced the rapid translocation of cytosolic PKCs to LDTI microdomains. We identified PKCalpha as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells.

Mild proteolysis induces a ready-to-fuse state on Sendai virus envelope.

The Sendai virus fuses with host cell membranes in a pH-independent manner through an unknown mechanism. Here we report that mild trypsin pre-treatments of Sendai virions, for example 15 min at 4 degrees C, give Sendai virions the ability to fuse at a rate up to 10-fold higher than control. By using human erythrocytes as host cell membranes, viral fusion was assessed by hemolysis as well as fluorescence dequenching of octadecyl rhodamine B chloride. The mild protease treatment strikingly shortens the lag time taken by the virus to start the fusion process. Similar data were obtained on reconstituted Sendai virus envelope. Among proteases, tested as fusion enhancer, trypsin is more effective than either endoproteinase Lys-C, chymotrypsin, or endoproteinase Arg-C. After removal of trypsin from treated virions the fusion rate enhancement remains for hours at room temperature. The lack of protease specificity, together with the impossibility to detect any new N-terminal products, suggests that only a small percentage of viral envelope components are cleaved, still a large enough number to set the envelope in a ready-to-fuse state.

Evidence for the existence of ganglioside-enriched plasma membrane domains in human peripheral lymphocytes.

In human peripheral blood lymphocytes (PBL) monosialoganglioside GM3 appears to be the major ganglioside on the cell plasma membrane. We have analyzed the expression and distribution pattern of GM3 molecules on the lymphocyte plasma membrane by flow cytometry, immunofluorescence, and immunoelectron microscopy, using an anti-GM3 monoclonal antibody. Both CD4+ and CD8+ T lymphocyte subpopulations showed substantial GM3 expression, as determined by thin-layer chromatography and flow cytometric analysis. A clustered distribution of GM3 molecules on the cell surface, revealed by immunofluorescence and immunogold electron microscopy, clearly indicated the presence of GM3 molecule-enriched plasma membrane domains. To better define these domains, we analyzed the ganglioside and protein composition of buoyant low-density Triton-insoluble (LDTI) lymphocyte fractions. The results show that GM3 is enriched approximately 20-fold in LDTI fraction, as compared with total cell lysates. In addition, CD4 and lck molecules are selectively recovered in the same LDTI fraction isolated from human PBL. These findings, together with the observation that anti-CD4 co-immunoprecipitated GM3, support the hypothesis of a possible GM3-CD4 interaction and suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in T-cell activation, antigen recognition, and other dynamic lymphocytic plasma membrane functions.

Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program.

In preliminary studies, we have analyzed the hematopoietic growth factor (HGF) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of erythropoietin (Epo) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous HGFs, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative ATRA receptor (RAR) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the RAR/RXR heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.

Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes.

Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin ("caveolae"). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low-density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes. We provide a detailed analysis of the two most highly expressed NRPTK, p53/p56lyn and p56lck, which are involved in the transduction of signals for proliferation and differentiation of monocytes/B lymphocytes and T lymphocytes, respectively. We show that lyn is selectively recovered in LDTI complexes isolated from human leukemic cell lines (promyelocytic [HL-60], erythroid [K562] and B-lymphoid [697]) and from normal human granulocytes, and that lck is recovered from LDTI fractions of leukemic T- and B-lymphoid cell lines (CEM, 697). In LDTI fractions of leukemic cells, lck and lyn are enriched 100-fold as compared with the total cell lysates. Analysis of these fractions by electron microscopy shows the presence of 70- to 200-nm vesicles: lyn and lck are homogenously distributed in the vesicles, as revealed by an immunogold labeling procedure. These novel results propose a role for these vesicles in signal transduction mechanisms of normal and neoplastic hematopoietic cells. In support of this hypothesis, we further observed that molecules participating in B- and T-cell receptor activation cofractionate in the LDTI fractions, CD45/lyn (B cells) and CD45/lck/CD4 (T cells).

c-fes expression in ontogenetic development and hematopoietic differentiation.

The c-fes protein (NCP92) is a tyrosine-specific protein kinase, capable of both autophosphorylation and phosphorylation of other substrates. We have analysed c-fes RNA expression in human/murine ontogenetic development and in homogeneous populations of embryonic and adult human hematopoietic cells. c-fes expression has been observed in rapidly proliferating embryonic-fetal tissues originating from different germinal layers, but not in adult non-hematopoietic tissues. In particular, a spatially and temporally regulated transcription was observed in the central nervous system and in developing cartilage. Expression in hematopoietic cells was evaluated in progenitors purified from embryonic-fetal liver and adult peripheral blood differentiating gradually and specifically along the erythroid or granulomonocytic lineage. In both embryonic and adult hematopoietic cells c-fes was abundantly expressed in undifferentiated progenitors of both lineages, as well as in differentiated granulomonocytic precursors, but not in erythroblasts. This expression pattern correlates with that of GM-CSF and in part IL-3 receptors (Testa et al., 1993 and our unpublished results). Altogether, these results suggest a possible role for c-fes in signal transduction, in both embryonic non-hematopoietic tissues and embryonic/adult hematopoietic cells, following interaction of growth factors with their tyrosine-kinase negative receptors (i.e., GM-CSF and IL-3 receptors in adult hematopoietic cells and other hypothetical growth factor(s) receptors during embryonic development.

Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture.

All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.

Replication and multiplication of hepatitis C virus genome in human foetal liver cells.

The ability to obtain primary long-term cultures of human foetal hepatocytes maintaining liver differentiation characteristics in serum-free medium prompted us to test their susceptibility to hepatitis C virus infection. Using PCR, we detected the presence of the HCV RNA-positive strand in the supernatants and in the cells of the virus-infected hepatocyte cultures, at various times post-infection. Evidence of effective virus genome replication and multiplication was also based on the time-dependent appearance of the putative HCV RNA-negative strand, the detection of virus replicative intermediates and an increase in HCV genomic templates in the HCV-infected cells.