Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.
Chlamydia species are the causative agents of trachoma, various forms of pneumonia, and the most common sexually transmitted diseases. Although the infection cycle has been extensively characterized in epithelial cells, where the Chlamydia entry-vacuoles avoid fusion with host-cell lysosomes, the cellular immune response has received less attention. Moreover, despite the abundant presence of dendritic cells (DC) in the sites of infection, the interaction between Chlamydia and DC has never been studied. We observe that DC kill Chlamydia trachomatis and Chlamydia psittaci. The chlamydiae are internalized by the DC in a nonspecific manner through macropinocytosis, and the macropinosomes fuse subsequently with DC lysosomes expressing MHC class II molecules. The interaction induces maturation of the DC, since presentation of an exogenous Ag is severely inhibited after a 1-day incubation, although chlamydial Ags are still presented and recognized by Chlamydia-specific CD4+ T cells. Thus, DC most likely play a role in initiating the T cell response in vivo and could potentially be used in adoptive transfer therapies to vaccinate against Chlamydia.
Plasma membrane receptors are retrieved continually from the cell surface by endocytosis and transported to intracellular organelles. They are internalized at various rates depending on their ability to interact with endocytic structures of the plasma membrane. The interleukin-2-receptor beta chain is endocytosed constitutively and efficiently. Here we show that different motifs in its cytosolic tail promote entry in an additive way, each of them acting as a weak internalization signal. The transmembrane domain of beta also participates in endocytosis. In conclusion, several weak endocytic determinants can be responsible for the rapid internalization of a membrane protein.
ABSTRACT Chlamydiae are bacterial pathogens which develop strictly inside the epithelial cells of their hosts. The mechanism used by chlamydiae to enter cells is not well characterized; however, it is thought to consist of a receptor-mediated process. In addition, the formation of clathrin-coated pits appears to be dispensable for chlamydiae to be internalized by host cells. Clathrin-independent endocytosis has recently been shown to occur through cholesterol-rich lipid microdomains, which are characterized by detergent insolubility. In the present study, we investigated whether these lipid domains play a role in Chlamydia trachomatis serovar L2 internalization by host cells. Our results show that after binding to HeLa cells, chlamydiae are associated with detergent-resistant lipid microdomains (DRMs), which can be isolated by fractionation of infected HeLa cells and flotation on a sucrose gradient. After internalization by HeLa cells, chlamydiae were still found in DRMs. In addition, extraction of plasma membrane cholesterol inhibited infection of HeLa cells by C. trachomatis . Many of the proteins associated with DRMs are glycosylphosphatidylinositol (GPI)-anchored proteins; however, our results could not identify a role for GPI-anchored proteins in the entry process. The same results were obtained for Chlamydia psittaci strain GPIC. We propose that cholesterol-rich domains participate in the entry of chlamydiae into host cells. Chlamydia binding to cholesterol-rich domains may lead to coalescence of the bacterial cells, which could trigger internalization by host cells.
Abstract The effect of the immunosuppressor cyclosporin A (CsA) on the expression of interleukin (IL) 2 receptors was investigated in a human T cell line IARC301 which constitutively expresses such receptors. This cell line also spontaneously secretes IL 2 which supports its autocrine growth. We have previously shown that CsA prevents the constitutive transcription of the IL 2 gene in these cells. Here we show that as soon as 4 h after CsA addition, the transcription of the gene encoding the α chain (p55) of IL 2R was inhibited. IL 2 can transiently increase the expression of this gene. CsA did not prevent this transient IL 2‐dependent induction of IL 2Rα, but could still partially inhibit it. Once IL 2 induction was over, CsA exerted its full inhibition. Thus, CsA does not seem to inhibit IL 2Rα gene transcription simply by inhibition of IL 2 synthesis. However, no modification of IL 2Rα expression on the cell surface could be detected after 48 h in the presence of CsA. This discrepancy between the effect of CsA on IL 2Rα expression as probed at the mRNA or the protein level can be accounted for by the stability of the IL 2Rα protein after synthesis. Indeed, the half‐life of IL 2Rα chain is longer than 40 h. This suggests that the α chain, after it is endocytosed together with the β chain as a component of high‐affinity IL 2R, might recycle back to the cell surface.
395 The common cytokine receptor γc, shared by interleukin receptors 2, 4, 7, 9 and 15, plays a major role in lymphocyte proliferation and differentiation. Endocytosis and degradation of these cytokine receptors reduce the number of receptors on the cell surface, and together with biosynthesis, control cell responsiveness. The role of γc in the endocytosis of these receptors and their ligands is not known. The purpose of this study was to analyze the endocytic behavior of the γc chain and to look for cytosolic sequences involved in intracellular trafficking. Using confocal microscopy and indirect immunofluorescence, we first showed thatγc is internalized in the human lymphoid cell line YT12881. Cell surface iodination of these cells and immunoprecipitation of γc after cell incubation for different times at 37°C showed that γc is degraded after its internalization and that its half-life on the cell surface is short (120 minutes). These results show that γc is internalized and degraded in lymphoid cells without any ligand. To identify the γc regions involved in sorting along the endocytic pathway, we constructed a chimeric protein, αγγwt, composed of the extracellular part of the interleukin 2-receptor α chain and transmembrane and intracellular domains of γc. This protein was stably transfected in the human T cell line Jurkat. Internalization studies were performed by indirect immunofluorescence and flow cytometry. To calculate the half-life of cell surface proteins, cells were incubated at 37°C with the protein synthesis inhibitor cycloheximide, and the amount of proteins on the cell surface was determinated by indirect immunofluorescence and flow cytometry. Whenαγγwt was transfected in Jurkat T cells, it was internalized and degraded as efficiently as γc, thus demonstrating that the transmembrane region and the cytosolic tail of γc carry sequences involved in these processes. To identify these sequences, we investigated the trafficking of chimeric proteins with serial truncations in their cytosolic tail. Internalization studies showed that the cytosolic tail of γc contains three regions which are involved in γc endocytosis and are located between cytosolic amino acids 1-35, 35-40, and 40-65. Successive deletions of these sequences reduce endocytosis. In contrast, only one region containing the 5 cytosolic amino-acids 36-40 seems to direct γc to the degradation pathway. In conclusion, γc is internalized and degraded in lymphoid cells. Its cytosolic tail contains three sorting sequences involved in endocytosis and one sequence involved in degradation. By participating in the fine tuning of cell surface γc expression, these sequences might regulate cell responsiveness to interleukins whose receptors share the γc chain.
Staphylococcus enterotoxin superantigens are potent T cell activators. To gain new insights into the mechanism of T cell activation induced by these superantigens, we investigated the recruitment of signaling molecules in this process. Here, we show that enterotoxin superantigen activation can be transmitted to TCR-CD3 complexes that did not interact with their ligand. Indeed, by studying cells expressing two distinct TCRs, we found that enterotoxin superantigens induced tyrosine phosphorylation of TCRzeta subunits, the recruitment and tyrosine phosphorylation of the protein tyrosine kinase ZAP-70, and an increase in protein tyrosine kinase activity of both directly stimulated and unstimulated TCR-CD3 complexes. As the involvement of unstimulated TCR-CD3 complexes in signal transduction would increase the number of signaling molecules and, therefore, the efficiency of T cell activation, these data provide a novel explanation for the ability of enterotoxin superantigens to potently activate T lymphocytes.
We previously demonstrated that long term treatment of the Ag-specific CD4+ T cell clone P28D with soluble HIV envelope glycoprotein gp120 results in a marked impairment of CD3/TCR-mediated responses. In this report, to further understand these inhibitory effects, the binding properties and internalization of gp120 have been investigated, in parallel with functional studies, in long term incubations of P28D cells with gp120. Immunofluorescence studies show that surface-bound gp120 level is maximal within 1 h of incubation at 37 degrees C and then gradually decreases. This decrease is accompanied by a progressive down-modulation of membrane CD4 (30-35% loss over a 18-h incubation period) without concomitant alteration of the CD4 mRNA steady-state level. Similar experiments performed with 125I-labeled gp120 demonstrate that the glycoprotein is progressively internalized (up to 35% internalized material after 18 h) and that it accumulates inside the cells. Confocal microscopy studies show that internalized gp120 is concentrated in localized intracellular compartments. CD4 also accumulates in compartments with a similar localization and is stained with mAb OKT4 but not with mAb OKT4a. Concomitantly to internalization of gp120 and disappearance of membrane CD4, a correlated loss of the CD4-associated tyrosine kinase p56lck is evidenced. Interestingly, a progressive impairment of the P28D responses to specific Ag or to anti-CD3 mAb is also observed. Inhibitions of T cell proliferation increase with the degree of both CD4 and p56lck down-modulation. Removal of exogenous gp120 results in a rapid and spontaneous release of internalized gp120 into a degraded form. A progressive restoration of CD4 and p56lck levels is also noticed. In parallel, CD3/TCR-mediated responses of clone P28D are fully recovered. Altogether, our results suggest that HIV-1 glycoprotein gp120 is able to down-modulate membrane CD4 presumably by a cointernalization process and to further down-modulate the associated p56lck. This dual phenomenon is presumably involved in the direct immunosuppressive effect of gp120 on the CD3/TCR-mediated activation pathway.
