Release and intercellular transfer of cell surface CD81 via microparticles

The human tetraspan molecule CD81 is a coreceptor in B and T cell activation and a candidate receptor for hepatitis C virus infection. We examined the surface expression of CD81 on B and T lymphocytes by quantitative flow cytometry. Upon cellular activation, CD81 surface levels were rapidly reduced. This reduction occurred as early as 1 h after activation and was linked to the release of CD81-positive microparticles into the cell culture medium. CD81 mRNA levels were not affected early after activation, but the release of CD81-positive microparticles was rapidly enhanced. In addition, intercellular transfer of CD81 was observed upon coculture of CD81-positive donor cells (Jurkat T cell line) with CD81-negative acceptor cells (U937 promonocytic cell line). This transfer was rapidly increased upon T cell activation, coinciding with enhanced CD81 release from activated Jurkat cells. We propose that the release and intercellular trafficking of CD81-positive microparticles regulate the expression of CD81 surface receptors in lymphocytes and play a role in the immune response during infections.     

Effects of aldehydes on CD36 expression

INTRODUCTION: During the oil frying process lipid peroxidation compounds are formed. These products can modulate gene expression and alter cellular behaviour. The cellular uptake of oxidized LDL, a key step in the development of atherosclerosis, is mediated by the CD36 scavenger receptor, whose expression is down-regulated by alpha-tocopherol.OBJECTIVE: To determine the effects of water-soluble aldehydes, obtained from thermally oxidized sunflower oil on the expression of CD36 scavenger receptor in human monocytes (THP-1 cells). We also wanted to study the effects of alpha-tocopherol on CD36 expression in the presence of water-soluble aldehydes.MATERIALS AND METHODS: Sunflower oil was heated in a frying pan, at 180--200 degrees C for 40 min, water-soluble aldehydes were isolated, and the content of thiobarbituric acid reacting substances (TBARS) was determined. THP-1 monocytes were cultured in RPMI medium during 24 h and incubated with increasing concentrations of the water-soluble aldehydes (ranging from 0.05 to 1 microM) and with or without 50 microM of alpha-tocopherol. In parallel, THP-1 cells were cultured with the same volume of an extract obtained from non-oxidized oil or distilled water. The CD36 expression at the cell surface was studied with fluorescence-activated cell sorting (FACS).RESULTS: Monocytes incubated in a medium containing water-soluble aldehydes, showed a dose dependent increase in the expression of the CD36 protein on the cell surface, compared to with the control groups. When the cells were treated simultaneously with 50 microM of alpha-tocopherol a significant reduction in the expression of the CD36 protein was observed.CONCLUSION: Water-soluble aldehydes, extracted from thermally oxidized culinary oil, increase the expression of CD36. This effect is partially decreased by the presence of alpha-tocopherol.     

Heparan sulfate modification of the transmembrane receptor CD47 is necessary for inhibition of T cell receptor signaling by thrombospondin-1

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent M(r) > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent M(r) 230,000) and CD47 (apparent M(r) > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser(64) and Ser(79). Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser(64).     

Serum increases the CD13 receptor expression, reduces the transduction of fluid-mechanical forces, and alters the metabolism of HL60 cells cultured in agitated bioreactors

The effects of serum medium concentration on the CD13 receptor surface content and mRNA levels of HL60 (human promyelocytic leukemia) cells were examined using flow cytometry and Northern blotting. Increasing the serum concentration from 2.5% to 10% and from 5% to 10% increased the CD13 receptor surface content of HL60 cells by 100% and 25%, respectively, in spinner flasks agitated at 60 rpm. In bioreactors at 80 rpm, increasing the serum concentration from 2.5% to 10% and from 5% to 10% increased the CD13 receptor surface content by 60% and 35%, respectively. This increase in CD13 receptor surface content was correlated with a 30% and a 20% increase in CD13 mRNA levels. Increasing serum concentrations also increased the average HL60 cell size under non-damaging conditions (60 rpm in spinner flasks, 80 rpm in bioreactors). Under conditions of agitation at 300 rpm in 2 L bioreactors, increasing serum concentrations (2.5% vs. 10%, 5% vs. 10%) allowed for higher HL60 apparent growth rates, but decreased the CD13 receptor surface content and mRNA levels. In view of our earlier findings on the effects of agitation on the CD13 antigen, these data suggest that serum reduces the transduction of mechanical forces that affect CD13 expression. At 300 rpm, HL60 cells cultured in 10% serum exhibited glucose consumption and lactate production rates that were approximately 50% and 60% lower than the values of cells cultured in 5% and 2.5% serum, respectively.     

NK and LAK activities from human marrow progenitors. I. The effects of interleukin-2 and interleukin-1.

We have investigated the role of interleukin-2 (IL2) as a differentiation factor for human marrow-derived NK cell progenitors and have assessed the effects of interleukin-1 (IL1) on this activity. The effects of these cytokines on early NK cell precursors was determined by testing marrow which had been depleted of mature cells and of CD2+ cells by treatment with soybean agglutinin and sheep erythrocytes (SBA-E-BM). The cytolytic activities of the SBA-E-BM were tested in 51Cr release assays following 7-8 days of liquid culture. K562 targets were used to assess NK activity and NK-resistant Daudi targets were used to measure lymphokine-activated killer (LAK) cell activity. Neither NK nor LAK activity were measurable in marrow incubated in medium without cytokines, or in medium containing IL1 alone. In contrast, culture in medium containing IL2 resulted in a dose-dependent development of lytic activity. NK and LAK activities could be differentiated by the percentage of cultures in which the activity developed, the dose of IL2 required, the time kinetics of induction, and the effect of depletion of residual cells with NK phenotype prior to culture. The most lytically active effectors of both activities, however, were CD56+. Immunofluorescence analyses before and after culture with IL2 revealed that Leu19+ (CD56) cells increased from less than 2% to as much as 17% of the total marrow cells and showed the appearance of a population of CD56+CD16- cells. The addition of IL1 to the marrow cultures increased NK activity when suboptimal amounts of IL2 were used (less than or equal to 100 U/ml), but did not increase LAK activity at any concentration of IL2. A higher number of NK cells, as well as MY7+(CD13+) myeloid cells were recovered from cultures containing IL1 plus IL2, indicating that NK cells as well as myeloid cells had a growth advantage in the presence of IL1. IL2 receptor (CD25) expression was low in all cultures but was consistently higher in cultures containing IL1 and IL2, however, CD25 was not coexpressed on NK cells. These studies indicate that early NK cell precursors can grow and differentiate in response to IL2 and that NK and LAK lytic activities may be acquired at different developmental stages. IL1 may serve to promote the responsiveness of NK cell progenitors to low concentration of IL2 by a mechanism which may not require expression of CD25.     

AP2 adaptor complex-dependent internalization of CD5: differential regulation in T and B cells

CD5 is a key regulator of Ag receptor-mediated activation, selection, and differentiation in both T and B cells. Accumulating evidence indicates that lymphocyte activation and selection are sensitive to variations in levels of CD5 on the cell surface. We now show that CD5 expression on the surface of B and T cells is regulated posttranslationally by direct interaction with the mu(2) subunit of the AP2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD5 is rapidly internalized from the cell surface in lymphoid cell lines, mature splenic T and B cells, and peritoneal CD5(+) B cells following monovalent or bivalent ligation of the receptor. We mapped the mu(2) subunit binding site on CD5 to Y(429) and determined that the integrity of this site was necessary for CD5 internalization. Cross-linking of the Ag receptor with intact Abs inhibited CD5 internalization in B cells, but had the opposite effect in T cells. However, if F(ab')(2) Abs were used to stimulate the Ag receptor in B cells, the effect on CD5 internalization was now similar to that observed in T cells, indicating that signals through the Ag receptor and FcR regulate CD5 endocytosis in B cells. This was confirmed using an FcgammaRIIB1-deficient B cell line. The ability to differentially alter posttranslational CD5 expression in T and B cells is likely to be key in regulation of Ag receptor signaling and generation of tolerance in T and B lymphocytes.     

A Molecular Model of CD86 and Analysis of Mutations which Disrupt Receptor Binding

CD86 and its homologue CD80 are type I transmembrane proteins expressed on antigen presenting cells. CD80 and CD86 specifically interact with CD28 and CD152 on T cells. This interaction results in T cell costimulation and complements T cell receptor signaling. The extracellular regions of CD80 and CD86 contain two immunoglobulin-like domains. In the presence of low sequence similarity to proteins with known three-dimensional structure, a molecular model of the N-terminal receptor-binding domain of human CD86 was built based on consensus residue analysis and structure-oriented sequence comparison. The model was assessed by energy profile analysis and regions of high, medium, and low prediction confidence were identified. Several CD86 point mutations which abolish receptor binding map to high confidence regions of the model. This has made it possible to rationalize their effects on binding or structure.     

Biochemical and morphological differentiation of the human colonic epithelial cell line SW620 in the presence of dimethylsulfoxide.

In vitro models of intestinal cell differentiation provide an important adjunct for studying normal and abnormal intestinal epithelial cell differentiation. The studies reported herein describe morphologic and biochemical changes in the colonic epithelial cell line SW620 following dimethylsulfoxide (DMSO) incubation. Cells cultured in the presence of DMSO showed striking changes in morphology characterized by enlargement, elongation, and formation of process-like structures by light microscopy and a propensity to form microvillus-like structures by electron microscopy. These changes were accompanied by significant differences in the expression of the cell surface markers CD4 (HIV gp120 receptor), CD44 (hyaluronate receptor), and KS1 (adenocarcinoma/epithelial specific antigen). There was a marked decrease in CD4 expression (38% to 2%), an increase in CD44 expression (4% to 50%) and a decrease in KS1 expression (98% to 66%) as detected by flow cytometry following incubation of SW620 cells in DMSO. Parallel changes in the expression of these markers were seen by metabolic and surface labeling studies. Although SW620 cells were infected by HIV-1, DMSO-treated SW620 cells could not be infected. DMSO-induced changes in surface expression of CD4, CD44, and KS-1 were reversible over time upon removal of DMSO from the culture medium. Secretory component, sucrase, neuron-specific enolase, chromogranin-A, and mucin were not detectable in SW620 cells with or without DMSO treatment. SW620 cells provide a useful model for studying specific biochemical and molecular events involved in intestinal epithelial cell differentiation and function.     

Regulation of choline deficiency apoptosis by epidermal growth factor in CWSV-1 rat hepatocytes.

Previous studies show that acute choline deficiency (CD) triggers apoptosis in cultured rat hepatocytes (CWSV-1 cells). We demonstrate that prolonged EGF stimulation (10 ng/mL x 48 hrs) restores cell proliferation, as assessed by BrdU labeling, and protects cells from CD-induced apoptosis, as assessed by TUNEL labeling and cleavage of poly(ADP-ribose) polymerase. However, EGF rescue was not accompanied by restoration of depleted intracellular concentrations of choline, glycerphosphocholine, phosphocholine, or phosphatidylcholine. In contrast, we show that EGF stimulation blocks apoptosis by restoring mitochondrial membrane potential (Delta Psi(m)), as determined using the potential-sensitive dye chloromethyl-X-rosamine, and by preventing the release and nuclear localization of cytochrome c. We investigated whether EGF rescue involves EGF receptor phosphorylation and activation of the down-stream cell survival factor Akt. Compared to cells in control medium (CT, 70 micromol choline x 48 hrs), cells in CD medium (5 micromol choline) were less sensitive to EGF-induced (0-300 ng/mL x 5 min) receptor tyrosine phosphorylation. Compared to cells in CT medium, cells in CD medium treated with EGF (10 ng/mL x 5 min) exhibited higher levels of phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of AktSer473. Inactivation of PI3K was sufficient to block EGF-stimulated activation of Akt, restoration of mitochondrial Delta Psi(m), and prevention of cytochrome c release. These studies indicate that stimulation with EGF activates a cell survival response against CD-apoptosis by restoring mitochondrial membrane potential and preventing cytochrome c release and nuclear translocation which are mediated by activation of Akt in hepatocytes.     

Effects of aldehydes on CD36 expression

Introduction: During the oil frying process lipid peroxidation compounds are formed. These products can modulate gene expression and alter cellular behaviour. The cellular uptake of oxidized LDL, a key step in the development of atherosclerosis, is mediated by the CD36 scavenger receptor, whose expression is down-regulated by α-tocopherol.

Objective: To determine the effects of water-soluble aldehydes, obtained from thermally oxidized sunflower oil on the expression of CD36 scavenger receptor in human monocytes (THP-1 cells). We also wanted to study the effects of α-tocopherol on CD36 expression in the presence of water-soluble aldehydes.

Materials and Methods: Sunflower oil was heated in a frying pan, at 180–200°C for 40?min, water-soluble aldehydes were isolated, and the content of thiobarbituric acid reacting substances (TBARS) was determined. THP-1 monocytes were cultured in RPMI medium during 24?h and incubated with increasing concentrations of the water-soluble aldehydes (ranging from 0.05 to 1?μM) and with or without 50?μM of α-tocopherol. In parallel, THP-1 cells were cultured with the same volume of an extract obtained from non-oxidized oil or distilled water. The CD36 expression at the cell surface was studied with fluorescence-activated cell sorting (FACS).

Results: Monocytes incubated in a medium containing water-soluble aldehydes, showed a dose dependent increase in the expression of the CD36 protein on the cell surface, compared to with the control groups. When the cells were treated simultaneously with 50?μM of α-tocopherol a significant reduction in the expression of the CD36 protein was observed.

Conclusion: Water-soluble aldehydes, extracted from thermally oxidized culinary oil, increase the expression of CD36. This effect is partially decreased by the presence of α-tocopherol.     

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.     

Negative Regulation of Toll-like Receptor-4 Signaling through the Binding of Glycosylphosphatidylinositol-anchored Glycoprotein,CD14, with the Sialic Acid-binding Lectin,CD33

When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

A murine thymic stromal cell line which may support the differentiation of CD4-8- thymocytes into CD4+8- alpha beta T cell receptor positive T cells.

A fibroblastoid cell line TSt-4 was established from fetal thymus tissue of C57BL/6 mice. When fetal thymus (FT) cells or CD4-8- (DN) cells of adult thymuses were cultured on the monolayer of TSt-4, a considerable proportion of lymphocytes expressed CD4 or both CD4 and CD8 within 1 day, and the CD4+CD8- cells were maintained further while the CD4+8+ cells disappeared by Day 5. A large proportion of cells generated from DN cells but not FT cells was shown to express CD3 and T cell receptor alpha beta. Addition of recombinant interleukin (IL)-7 into the cultures resulted in a marked increase of cell recovery without virtual change in differentiation process of alpha beta lineage. The present work strongly suggests that thymic fibroblasts play an important role in T cell differentiation and IL-7 contributes to supporting proliferation of differentiated cells.     

Hyaluronan uptake by adult human skin fibroblasts in vitro

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.     

IL-3 promotes basophilic differentiation of KU812 cells through high affinity binding sites

The myeloid precursor cell line KU812 exhibits a constitutive potential to differentiate into basophilic cells. In the present study, the influence of recombinant human (rh)IL-2, rhIL-3, and recombinant human granulocyte-macrophage-CSF on basophilic differentiation of KU812-F cells was studied. Of all cytokines tested, rhIL-3 induced a significant increase in formation of metachromatically granulated cells (from 10% in control cultures up to 30% in cultures supplemented with 100 U/ml of rhIL-3) as well as dose-dependent (1.5- to 3 fold) increase in cellular histamine in KU812-F cell cultures. In addition, KU812-F cells exposed to rhIL-3 bound more IgE antibody than cells cultured in control medium with up to 3.3-fold increases in the mean fluorescence intensity on days 2 and/or 5 compared with control (p less than 0.001). RhIL-3 failed to induce significant changes in expression of the Tac-reactive subunit of the IL-2R (CD25), surface aminopeptidase N (CD13), ICAM-1 Ag (CD54), or CD40 Ag on KU812-F cells. To investigate the mechanism of IL-3 action on KU812-F cells, receptor analyses were performed by using 125I-radiolabeled rhIL-3. Quantitative binding studies and Scatchard plot analyses revealed the presence of a single class of 1910 to 2460 high affinity IL-3-binding sites per KU812-F cell with an apparent dissociation constant of 1.22 to 2.35 x 10(-9) M. Together, these results show that rhIL-3 promotes basophilic differentiation of KU812-F cells through a specific receptor.     

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3 gamma subunit of the TCR. To investigate the role of CD3 gamma phosphorylation in PKC-mediated TCR down-regulation, point mutated CD3 gamma cDNA was transfected into the CD3 gamma-negative T cell line JGN and CD3 gamma transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3 gamma was required for PKC-mediated down-regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131 and L132) in the cytoplasmic tail of CD3 gamma was required for PKC-mediated TCR down-regulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKC-mediated TCR down-regulation in non-mutated T cells, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucine- or tyrosine-based motifs is proposed.     

Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.