Rabbit M cells and dome enterocytes are distinct cell lineages.

We have studied the M cell origin and differentiation pathway in rabbit gut-associated lymphoid tissues. Micro-dissected domes and epithelium isolated by ethylene diamine tetra acetic acid detachment allowed us to view the whole epithelial surface from the bottom of crypts to the top of domes. We used monoclonal antibodies specific to the apex of either M cells or dome enterocytes, lectins, and antibodies to vimentin in appendix, distal Peyer's patches and caecal patches. The earliest vimentin-labeled M cells were observed in the BrdU-positive proliferative zone of dome-associated crypts. Gradual differentiation of the M cell vimentin cytoskeleton started at this site to progressively give rise to the first pocket-forming M cells in the upper dome. Therefore, these mitotic cells of the crypts appear as the direct precursors of M cells. In addition to an early appearance of M cell markers, a regular mosaic-like relative distribution of M cells and dome enterocytes was already detected in the vicinity of crypts, similar to that observed on the lateral surface of domes where functional M cells lie. This constant distribution implies that there is no trans-differentiation of enterocytes to M cells along the crypt-dome axis. Together, these observations provide very strong evidence in favor of an early commitment in crypts of M cell and enterocyte distinct lineages.

A neuronal isoform of nitric oxide synthase expressed in pancreatic beta-cells controls insulin secretion.

Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Double-immunofluorescence studies demonstrated the presence of nNOS in insulin-secreting beta-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the beta-cell response to arginine and glucose after nNOS blockade with N(G)-nitro-L-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzyme-substrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that beta-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal beta-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in beta-cell dysregulation involved in certain pathological hyperinsulinic states.

Glycocalyx on rabbit intestinal M cells displays carbohydrate epitopes from Muc2.

It is essential to investigate the apical surface properties of both M cells and dome enterocytes to understand the mechanisms involved in the binding of pathogens to M cells. In rabbit appendix tissue, monoclonal antibodies (MAbs) highlight differences between M cells (MAb 58) and dome enterocytes (MAb 214). Such antibodies ultimately recognized intestinal mucin-related epitopes. To further characterize these differences, the labeling patterns obtained with these MAbs were compared to those obtained with other antibodies to intestinal mucins on dissected domes from all gut-associated lymphoid tissues. A glycoprotein recognized by MAb 58 was purified on a CsCl isopycnic density gradient and microsequenced, and its mRNA expression was localized by in situ hybridization. It was identified as the rabbit homologue of human Muc2, i.e., the major mucin secreted in intestine tissue. Two other Muc2 carbohydrate epitopes were also expressed on M cells, although Muc2 mRNA was not detected. All results indicated that M cells express, on their apical membrane, glycoconjugates bearing at least three glycosidic epitopes from Muc2. MAb 214 and MAb 6G2, which recognized a partially characterized mucin expressed on dome enterocytes, were negative markers for M cells in rabbit gut-associated lymphoid tissues. We propose that the presence, on the surface of M cells, of carbohydrates also expressed on Muc2, together with the absence of an enterocyte-associated mucin, could favor pathogen attachment and accessibility to the M-cell luminal membrane.

Mucin-related epitopes distinguish M cells and enterocytes in rabbit appendix and Peyer's patches.

The biochemical composition of the apical membranes of epithelial M cells overlying the gut-associated lymphoid tissues (GALT) is still largely unknown. We have prepared monoclonal antibodies (MAbs) directed against carbonate-washed plasma membranes from epithelial cells detached with EDTA from rabbit appendix, a tissue particularly rich in GALT. As determined by immunofluorescence microscopy, several MAbs specifically recognized either M cells or enterocyte-like cells of the domes from rabbit appendix, sacculus rotundus, and Peyer's patches. M cells were identified by their large ventral pocket containing lymphoid cells and by specific labeling with antivimentin. Among various characterized MAbs, MAb 104 recognized rabbit immunoglobulins and was used as an apical marker for M cells in the rabbit appendix, MAb 58 selectively stained an integral membrane glycoprotein of greater than 205 kDa located at the apex of M cells, and MAb 214 stained a smaller soluble glycoprotein associated with the apical surfaces from neighboring enterocytes. In addition, both MAbs 58 and 214 also labeled luminal mucus and secretory granules in goblet cells. The selective association of mucin-related molecules at the surfaces of either M cells or enterocyte-like cells of the follicle-associated epithelium suggests that specific carbohydrate antigens are differentially expressed by epithelial cells and could account for the differential binding properties of pathogens.

Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during In vitro maturation of reticulocytes.

Exosomes are membrane vesicles released by reticulocytes during their maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, eg, acetylcholinesterase (AChE) and transferrin receptor (TfR), respectively. To better understand the molecular events leading to protein sorting in exosomes, we analyzed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins on the exosome surface through a technique involving bead coupling and flow cytometry immunodetection. The presence of AChE, decay-accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and lymphocyte function-associated antigen 3 (LFA-3) on the surface of exosomes obtained from normal and paroxysmal nocturnal hemoglobinuria (PNH) reticulocytes, suggests that (1) the GPI anchor is efficiently sorted during exosome formation, (2) exosome release could account for the observed discrepancy in GPI-protein expression between reticulocytes and erythrocytes from PNH patients, and (3) exosomes could have another physiologic function related to controlling membrane attack complex formation.

Involvement of ATP-dependent Pseudomonas exotoxin translocation from a late recycling compartment in lymphocyte intoxication procedure.

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125I-labeled PE was transported after 2 h at 37 degrees C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20 degrees C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired 125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane.

Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N-terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.

Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein.

It is thought that residents of the Golgi stack are localized by a retention mechanism that prevents their forward progress. Nevertheless, some early Golgi proteins acquire late Golgi modifications. Herein, we describe GPP130 (Golgi phosphoprotein of 130 kDa), a 130-kDa phosphorylated and glycosylated integral membrane protein localized to the cis/medial Golgi. GPP130 appears to be the human counterpart of rat Golgi integral membrane protein, cis (GIMPc), a previously identified early Golgi antigen that acquires late Golgi carbohydrate modifications. The sequence of cDNAs encoding GPP130 indicate that it is a type II membrane protein with a predicted molecular weight of 81,880 and an unusually acidic lumenal domain. On the basis of the alignment with several rod-shaped proteins and the presence of multiple predicted coiled-coil regions, GPP130 may form a flexible rod in the Golgi lumen. In contrast to the behavior of previously studied type II Golgi proteins, overexpression of GPP130 led to a pronounced accumulation in endocytotic vesicles, and endogenous GPP130 reversibly redistributed to endocytotic vesicles after chloroquine treatment. Thus, localization of GPP130 to the early Golgi involves steps that are saturable and sensitive to lumenal pH, and GPP130 contains targeting information that specifies its return to the Golgi after chloroquine washout. Given that GIMPc acquires late Golgi modifications in untreated cells, it seems likely that GPP130/GIMPc continuously cycles between the early Golgi and distal compartments and that an unidentified retrieval mechanism is important for its targeting.

Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.

Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.

[Clinical importance of the determination of the hydrophobic forms of serum carcinoembryonic antigen (CEA) in metastatic cancer of the colon]. / Intérêt clinique de la détermination des formes hydrophobes de l'antigène carcinoembryonnaire (ACE) sérique dans les cancers métastatiques du côlon.

Thirty-four patients with metastatic colon cancer were treated with 5 fluorouracil and folinic acid. The follow-up of disease was evaluated by tomodensitometric CT-scan analysis and by serum CEA determination. In addition, a study of the different CEA molecular forms separated by Triton X114 partitioning, immunoprecipitation and immunoblotting was completed. Concerning the aqueous phase, no relationship appeared between the pattern of CEA species and the outcome of chemotherapy. Opposingly, the analysis of the hydrophobic phase gave results closely correlated to chemotherapeutic response. In 19/34 patients, the hydrophobic CEA forms were absent or weakly expressed; out of these patients, 16/19 underwent a successful response to chemotherapy regimen. Opposingly, all of the remaining 15 patients expressing high levels of hydrophobic CEA species were non-responders. The present study thus gives new means for predicting the outcome of 5 fluorouracil-folinic acid chemotherapy by screening the molecular CEA forms expressed in the serum of patients with metastatic colon cancer.

GPI membrane anchor is determinant in intracellular accumulation of apical plasma membrane proteins in the non-polarized human colon cancer cell line HT-29 18.

We have compared the intracellular localization of plasma membrane proteins anchored either with a transmembrane segment or with a glycosylphosphatidylinositol moiety to estimate the effects of membrane anchor on protein segregation in the non-polarized form of the human colon cancer cell line HT-29 18. We have monitored two endogenous proteins: the carcinoembryonic antigen, a glycosylphosphatidylinositol protein and the transmembrane protein dipeptidyl peptidase IV, and two transfected proteins: the glycosylphosphatidylinositol protein Thy-1 and an engineered transmembrane form of Thy-1. Using immunocytochemistry on ultra-thin cryosections and confocal microscopy, we detected a carcinoembryonic antigen-rich vesicular compartment, excluding classical pre-lysosomal and lysosomal markers such as mannose 6-phosphate receptor, lamp-1 and cathepsin D. This compartment, where carcinoembryonic antigen accumulated, excluded the transmembrane protein dipeptidyl peptidase IV and was reduced during the polarization of the cells. Moreover, the glycosylphosphatidylinositol form of Thy-1 also accumulated in the carcinoembryonic antigen-rich compartment whereas the transmembrane form of Thy-1 was excluded. We proposed that, in the non-polarized HT-29 18 cells, accumulation of glycosylphosphatidylinositol proteins independently of transmembrane proteins reveals different intracellular fates for proteins according to their anchor in the plasma membrane.

Ezrin has properties to self-associate at the plasma membrane.

Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J. G. and Mangeat, P. (1991). EMBO J. 10, 2363-2373). Human ezrin was expressed in and purified from Escherichia coli. It possesses all the major biophysical, immunological and physiological properties of natural ezrin. Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized. By coimmunoprecipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins. In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay. The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin. In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane. Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family. In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). J. Cell Biol. 120, 129-139), both domains interacted synergistically in a salt-dependent manner to trigger self-association of ezrin. Ezrin's self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites.

The invariant chain induces compact forms of class II molecules localized in late endosomal compartments.

The invariant chain (Ii) binds to newly synthesized major histocompatibility complex (MHC) class II molecules and is targeted to an acidic compartment where it is degraded. To evaluate its role on the conformation and the subcellular distribution of murine MHC class II molecules we have established stable L cell transfectants expressing class II IAk heterodimers alone or in conjunction with p31 and p41 Ii chains. In these cells, class II molecules were present under three forms: alpha beta heterodimers bearing high mannose carbohydrate moieties, and fully glycosylated alpha beta heterodimers that are sensitive or resistant to sodium dodecyl sulfate dissociation at 20 degrees C. The latter class II molecules called compact heterodimers, were here highly induced in Ii-positive cells. Using in situ iodination of endosomal compartments, class II heterodimers were detected in late endosomal compartments essentially as compact forms in Ii-positive cells, and as non-compact forms in Ii-negative cells. Using confocal microscopy, IAk molecules were located in compartments distinct from early endosomes labeled with transferrin, but partially coincident with vesicles containing fluid-phase markers, and highly coincident with compartments containing large amounts of cathepsins B, D, H, and L in Ii-positive and Ii-negative cells. At the ultrastructural level, class II molecules were mostly present in multivesicular bodies, even without Ii expression. But Ii chains were needed to induce an efficient presentation of the hen egg lysozyme antigen and were sufficient to promote a major conformational change of the late endosomal, and/or lysosomal resident, class II molecules. Ii molecules are presumably playing a chaperoning function favoring the association of peptides with class II molecules in endosomal compartments.

GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells.

In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or basolateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical transmembrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.

Carcinoembryonic antigen has a different molecular weight in normal colon and in cancer cells due to N-glycosylation differences.

Carcinoembryonic antigen, an apical membrane glycoprotein expressed in normal human colonic epithelial cells, colonic polyps, tumor, and tissue culture cell lines originating from colonic adenocarcinomas, is generally considered to have a molecular weight of 180,000. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis associated with immunoprecipitation or immunoblotting with both monoclonal (Mab 517 and Mab 601) and polyclonal antibodies, we observed that carcinoembryonic antigen was actually expressed as two discrete apparent molecular weight forms in normal tissues: a broad band averaging at Mr 200,000 and a sharp band at Mr 130,000. This constituted the phenotype of the normal colon. In cancer cells we detected a single band at Mr 170,000 or lower. This variation was mainly the consequence of a modification of the glycosylation pattern of the molecule since deglycosylation by N-glycanase or biosynthesis in the presence of tunicamycin always produced a single molecular weight form, whether or not the source of tissue was normal or cancerous. By close inspection of benign, moderately transformed, and carcinomatous human colonic polyps we noticed that this shift in the molecular weight of carcinoembryonic antigen preceded the detection of other cancer markers such as nonspecific cross-reacting antigen at Mr 95,000 or the histological modifications leading to malignant diagnosis. Carcinoembryonic antigen constitutes, therefore, an important model with which to study the modifications of the glycosylation pattern induced during cancer biogenesis.

The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.

Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K-ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F-actin-containing, microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP-mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921-930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin-containing cell surface structures.

T cell receptor/CD3 complex internalization following activation of a cytolytic T cell clone: evidence for a protein kinase C-independent staurosporine-sensitive step.

The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti-TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA). After binding of the anti-TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti-TcR mAb. PMA-induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC-depleted cells or in the presence of the PKC inhibitor staurosporine. Anti-TcR mAb-induced internalization was maintained in PKC-depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti-TcR mAb Fab fragment is non-stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC-depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA-induced phosphorylation of gamma and epsilon subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross-linking of the TcR via plastic-coated anti-TcR mAb led to phosphorylation of CD3 gamma and epsilon and also of zeta, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti-TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.

Endocytosis and recycling of MHC-encoded class II molecules by mouse B lymphocytes.

The movements of mouse MHC-encoded class II (H-2E) and class I (H-2K), transferrin receptor and surface Ig molecules of B lymphocytes were studied using radiolabeled mAb and electron microscopy. A total of 10 to 20% of antibodies specific for H-2E molecules were gradually internalized with a t 1/2 of 15 min, reaching a plateau after 30 min at 37 degrees C. Equivalent results were obtained either with the whole antibody or Fab' fragments, suggesting that the internalization of class II molecules was spontaneous. Similar results were obtained with antibodies specific for the transferrin receptor, of which 50% were internalized with t 1/2 of 5 min, reaching a plateau after 30 min. In contrast to antibodies specific for H-2E molecules and the transferrin receptor, antibodies specific for H-2K were not internalized. Reappearance of internalized H-2E-specific antibodies at the cell surface was observed at 37 degrees C. When compared to antibodies specific for surface Ig, degradation of antibodies specific for H-2E molecules was limited even after 5 h incubation. Neither ammonium chloride nor cycloheximide inhibited internalization and recycling. Electron microscopy showed that internalization of H-2E molecules occurred via coated pits/coated vesicles. These results indicate that class II molecules are spontaneously internalized and recycled by B lymphocytes.

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein, in mammal heart and brain.

According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314-322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrahin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions. The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43. Proteins of Mr 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.

Mouse thymic epithelial cell lines interact with and select a CD3lowCD4+CD8+ thymocyte subset through an LFA-1-dependent adhesion--de-adhesion mechanism.

Differentiation of bone-marrow-derived precursor cells into mature mouse T lymphocytes occurs in the thymus and involves sequential interactions with MHC-positive hemopoietic and epithelial stromal cells. To study the in vitro molecular mechanisms at play during the lympho-epithelial cell adhesion, we derived thymic stromal cell lines which were shown to possess cytokeratin filaments and tight junctions. These mouse thymic epithelial (MTE) cell lines did not express the classical hemopoietic stromal cell surface markers (i.e. LFA-1, Mac-1, and CD45) but expressed ICAM-1, NCAM, J11d, CD44, and MHC molecules. A quantitative cell adhesion assay was used to evaluate the interaction of various lymphoid cell subsets with MTE cells. Two cell interaction patterns could be defined: first, a rapid adhesion of a fraction of CD4+CD8+ and of a few CD4-CD8- immature thymocytes to MTE cells was observed at 4 degrees C. The CD8 molecule was shown to be partially involved in this initial contact. The strength of adhesion between MTE cells and distinct thymocyte subsets was evaluated and found to be maximal with neonatal thymocytes. Second, a temperature-dependent adhesion step characterized by a rapid and active stabilization of the interaction of MTE cells with 20% of CD4+CD8+CD3low thymocytes was seen, followed by a more progressive de-adhesion step. This active process of engagement was highly LFA-1-dependent, involved the CD4 and CD8 molecules, and required protein kinase C activation and cytoskeletal integrity. The results are consistent with the involvement of LFA-1 in a transient and regulated cell adhesion under the control of the TCR-CD3 complex that progressively appears on maturing cells. This phenomenon might contribute to the selection of a subset of immature thymocytes by epithelial cells occurring during the process of maturation of these cells.