GARP: a key receptor controlling FOXP3 in human regulatory T cells.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

Modulation of NCAM expression by transforming growth factor-beta, serum, and autocrine factors.

The expression of NCAM (neural cell adhesion molecule) is precisely regulated in terms of cell type specificity and developmental control. We searched for extracellular factors that may be involved in this regulation using N2A neuroblastoma and NIH 3T3 fibroblastic cells. Factors contained in FBS promoted a two- to threefold increase in NCAM protein and mRNA abundance in both cell lines. This increase in NCAM expression in high serum could be entirely attributed to enhanced levels of the NCAM-140 message. Modulation of NCAM synthesis via an autocrine mechanism is suggested by the observation that medium conditioned by N2A cells stimulated NCAM mRNA expression by 3T3 and N2A cells. Among the pure factors tested, transforming growth factor-beta (TGF beta) was found to act as an inducer of NCAM expression in 3T3 but not in N2A cells. 3T3 cells responded to exposure to TGF beta with a two- to threefold increase in NCAM protein and mRNA. Exposure of early-passage embryonic cells to TGF beta resulted in four- and twofold increases in NCAM protein and mRNA abundance, respectively, suggesting a role for TGF beta in modulating NCAM expression in the embryo. TGF beta seems to act by stimulating the transcriptional activity of the NCAM gene because it did not affect transcript stability and stimulated transcription from a proximal promoter element of the NCAM gene.

Characterization and purification of glycosaminoglycans from crude biological samples.

Chondroitin sulfate (CS) is a glycosaminoglycan derived from cartilage and commonly used to treat osteoarthritis, psoriasis, and other conditions. The dimethylmethylene blue (DMMB) assay has been used often to measure glycosaminoglycan levels in relatively pure samples. In this study, we verified the accuracy of the DMMB assay in measuring CS levels in unpurified extract from bovine trachea and shark cartilage, despite potential interference from salts, proteins, and DNA. We found that the glycosaminoglycan signal obtained was due to CS and not to other glycosaminoglycan species. This was confirmed using fluorophore-assisted carbohydrate electrophoresis, which also revealed that the majority of the CS was monosulfated at the C4 or C6 position. Finally, we used anion-exchange chromatography to purify the bovine extract and obtained complete recovery of the glycosaminoglycans, with no contaminating protein. The results of this study should be very useful for future purification and analysis of this common supplement.

Evaluation of the effect of suramin on neural cell growth and N-CAM expression.

Suramin, a polysulfonated naphthylurea, is currently under investigation for treatment of advanced malignancy and has been shown to exhibit antiproliferative effects on some cells. We investigated its action on two cell lines of neural origin, one with neuronal (N2A) and the other with glial (C6) phenotype, as well as on brain primary cultures. We showed that suramin completely inhibited astrocytoma proliferation for an optimal dose of 1000 micrograms/ml but had the opposite effect on neuroblastoma cells. For these cells, doses as low as 12.5 micrograms/ml first increased cell proliferation and then led to massive cell death. This cytotoxic effect, which could be compatible with an internalization of the drug by the cells, was also observed for postmitotic neurons in brain primary cultures. In both cell lines, suramin was responsible for an accumulation of the neural cell adhesion molecule at the cell surface. One of the causes was the inhibition by suramin on the liberation processes of the phosphatidylinositol anchored Mr 120,000 isoform. At the mRNA level, suramin (12.5 to 50 micrograms/ml) induced an increase of all neural cell adhesion molecule transcripts in N2A but not in C6 cells. Suramin did not have an overall effect on transcription rates or RNA stability as the levels of transcripts coding for PrPc, another cell surface molecule, and actin were not affected. Our data demonstrated pleiotropic action of suramin. The neurotoxic effect exerted on neurons needs to be considered as possible outcomes for the use of suramin in humans.

let-756, a C. elegans fgf essential for worm development.

In vertebrates, Fibroblast Growth Factors (FGFs) and their receptors are involved in various developmental and pathological processes, including neoplasia. The number of FGFs and their large range of activities have made the understanding of their precise functions difficult. Investigating their biology in other species might be enlightening. A sequence encoding a putative protein presenting 30-40% identity with the conserved core of vertebrate FGFs has been identified by the C. elegans sequencing consortium. We show here that this gene is transcribed and encodes a putative protein of 425 amino acids (aa). The gene is expressed at all stages of development beyond late embryogenesis, peaking at the larval stages. Loss-of-function mutants of the let-756 gene are rescued by the wild type fgf gene in germline transformation experiments. Two partial loss-of-function alleles, s2613 and s2809, have a mutation that replaces aa 317 by a stop. The truncated protein retains the FGF core but lacks a C-termins portion. These worms are small and develop slowly into clear and scrawny, yet viable and fertile adults. A third allele, s2887, is inactivated by an inversion that disrupts the first exon. It causes a developmental arrest early in the larval stages. Thus, in contrast to the other nematode fgf gene egl-17, let-756/fgf is essential for worm development.

Lipid-mediator synthesis in peritoneal macrophages from mice injected with immunostimulants.

The metabolism of bioreactive lipid mediators was studied in two types of activated macrophages (M phi). We compared the capacity of resident and activated M phi to release, upon a zymosan challenge, cyclooxygenase and lipoxygenase products as well as PAF-acether (platelet-activating factor) and its 2-lyso precursor. Activated M phi were obtained from mice injected intraperitoneally either with nonviable C74 streptococci (St-M phi) or with trehalose dimycolate, a defined immunostimulant isolated from Mycobacterium tuberculosis (TDM-M phi). Both activated populations exhibited common features: conversion of endogenous [14C]arachidonic acid into prostaglandin E2 and thromboxane A2 rather than into prostaglandin I2 and low biosynthesis of PAF-acether, probably due to an impairment of the acetylation step. However, contrary to St-M phi, TDM-M phi did not display a marked overall reduction of arachidonate metabolism. In addition, as compared to resident M phi, TDM-M phi presented a ratio of thromboxane B2/6-ketoprostaglandin F1 alpha 30-fold higher, a better conversion of leukotriene C to leukotriene D and a higher capacity to release the PAF-acether they synthesize. These macrophages thus seem to be valuable tools for studying the formation of mediators and for determining specific markers of an activated state.

Structure and developmental expression of mouse Garp, a gene encoding a new leucine-rich repeat-containing protein.

Proteins with leucine-rich repeats (LRR) constitute a large family of molecules playing a role in protein-protein interactions and signal transduction. They are involved in various cellular processes in different species. We characterized the organization and pattern of expression of the mouse Garp gene. It is composed of two coding exons, expressed as a major 4.3 kb mRNA, and encodes a putative LRR transmembrane protein with an extracellular region almost entirely made of 20 repeats, and a short intracytoplasmic region. The mouse GARP deduced amino-acid sequence is highly similar to that of the human protein. The Garp gene is expressed in various areas in the mid-gestation developing embryo, including skin, lens fibre cells, nasal cavity, smooth and skeletal muscles, lung, and megakaryocytes of the fetal liver. In the adult it is expressed in the megakaryocytes of the spleen and in endothelial cells of the placenta. The data suggests that GARP might be involved in platelet-endothelium interactions.

Lymphoid cells in lymph nodes and peripheral blood of patients with squamous cell carcinoma of the head and neck.

Fifty-five patients with squamous cell carcinoma of the head and neck were evaluated immunologically by measuring the level of T cells (E-RFC) and high affinity subset T cells (E-29) in the peripheral blood and peritumorous lymph nodes. A significant decrease (p less than 0.05) in mean percentage of E-29 was observed in cancer patient peripheral blood. In peritumorous lymph nodes, there was no difference in terms of total T cells or of high affinity subset T cells, as compared to non-malignant lymph nodes, or between tumor-free and metastatic lymph nodes. Macrophage content was much higher in metastatic than in tumor-free lymph nodes (p less than 0.05) and these macrophages frequently appeared to be more active when tested in phagocytosis of sheep red blood cells sensitized with IgG or IgM + C.

Formation of prostaglandins, leukotrienes and paf-acether by macrophages.

Prostaglandins (PG) and leukotrienes (LT)--arachidonic acid-dependent metabolites--and paf-acether (platelet-activating factor)--an ether phospholipid--are potent mediators of allergic and inflammatory reactions. Their structures, chemical synthesis and biosynthetic pathways have been recently described. These mediators are produced by various cells with proinflammatory activities including the macrophages upon interaction with a specific secretagogue stimulus (phagocytosis of zymosan particles, immune-complexes); in IgE-dependent hypersensitivity reactions; upon interaction with one of these mediators. Formation of these mediators by macrophages depends upon their local environment. Qualitative and/or quantitative variations in their synthesis are observed depending on the tissue they are derived from (alveole or peritoneum) and on the type of inflammation (immunologic specific or not). Their potent biological activities (increase of vascular permeability, smooth muscle contraction, cardiac and vascular effects and/or chemotactism) suggest a role for these mediators in various pathologies.

The role of immunomodulators in the production of lipid mediators by macrophages (M phi).

There is evidence indicating that bioreactive lipid mediators, PAF-acether (platelet-activating factor: 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and arachidonic acid (AA) metabolites, are formed upon deacylation of ether lipids. M phi obtained from mice treated with the sterile irritant thioglycolate exhibited an impaired formation of PAF-acether, leucotrienes (LT) C4 and prostaglandins (PG). In order to assess whether the impaired formation in lipid mediators is a general feature of M phi found at inflammatory sites, we have compared the capacity of resident (R-M phi) and immunostimulant-activated M phi to release, upon a zymosan challenge, PAF-acether and cyclooxygenase and lipoxygenase products. Activated M phi was obtained from mice injected intraperitoneally with non-viable C74 streptococci (St-M phi), bacilli Calmette-Guerin (BCG-M phi) or trehalose dimycolate (TDM-M phi), a defined immunostimulant isolated from Mycobacterium tuberculosis. All populations were capable of releasing PAF-acether. However, the amount of cell-associated PAF-acether was reduced by 75-90% in activated M phi populations as compared to R-M phi. Although the acetyltransferase level was comparable, acetyl-CoA supplementation restored the formation of PAF-acether by activated M phi to control (R-M phi) level. The amount of 14C-AA metabolites released by St-M phi was much lower compared with TDM-M phi or R-M phi, as was the amount of LTC4 detected as SRS contractile activity after HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional activation of the NCAM gene during myogenic differentiation.

Earlier work has shown that in vitro myogenesis of muscle cells is accompanied by important changes in the expression of NCAM mRNAs and proteins, consisting mainly in an upregulation of the smaller transcripts and the 120- to 125-kD NCAM isoforms they encode. By measuring run-on transcription rates in nuclei isolated from C2 myoblasts and myotubes, we show in this report that transcriptional control can account for the increase in NCAM mRNA levels during myogenesis. Hence, the quantitative changes in overall abundance of NCAM transcripts and the concomitant qualitative changes in the mRNA pattern are regulated by independent mechanisms. Moreover, as transcriptional activity was stimulated to similar extents in the 5' and 3' region of the gene, we can exclude premature transcription termination as a mechanism involved in the preferential synthesis of shorter transcripts. Further support for transcriptional regulation of the NCAM gene is provided by experiments showing increased NCAM promoter activity in differentiating cells. As this increase was observed with 4.5 kb but not with 426 bp of upstream sequence, it seems to involve one or more upstream regulatory elements.

Release of leukotrienes C (LTC) and D (LTD) from inflammatory macrophages during phagocytosis of zymosan and bacteria.

Release of slow-reacting substance (SRS) was obtained from resident mouse peritoneal macrophages (R-M phi) upon stimulation with phagocytic stimuli (zymosan, bacteria). The release of SRS from thioglycollate elicited M phi was impaired, whereas that from BCG-elicited M phi was quantitatively unaffected. However, using a high pressure liquid chromatography separation procedure, qualitative variations between SRS released from R-M phi and BCG-M phi were observed. In both cases, LTC was the major component released from M phi, but greater amounts of LTD were released from BCG-M phi than from R-M phi. These data indicate that local environment alters leukotriene generation by M phi.

Paf-acether generates chemiluminescence in human neutrophils in the absence of cytochalasin B.

In the absence of cytochalasin B, synthetic Paf-acether (0.1-10 microM) induced oxygen radical production in polymorphonuclear neutrophils as measured by the luminol-dependent chemiluminescence ( LDCL ) test. This effect was observed after a lag period of 10 s and was maximal between 5 and 15 min. In the presence of cytochalasin B, the kinetics were shortened, but the lag period was not modified and the same concentrations of the agonist had to be used to induce LDCL . None of the structural analogs tested (2-lyso Paf-acether, Paf-acether enantiomer, 1 ester analog of Paf-acether, lyso-phosphatidylcholine) were active, irrespective of the presence of cytochalasin B. Paf-acether (10 microM) shortened the kinetics of opsonized zymosan (10 micrograms/ml)-induced LDCL and enhanced it by 550% and 250% at 5 min and 10 min respectively, without affecting the peak value. Similar results were obtained using non-opsonized zymosan (100 micrograms/ml). Lower concentrations of Paf-acether (0.1 microM) were also able to increase oxygen radical production induced by low doses of zymosan and opsonized zymosan. The triggering and enhancing effects of Paf-acether on oxygen radical production by resting and stimulated polymorphonuclear neutrophils support the role of Paf-acether in inflammation.

Markers of macrophage heterogeneity. I. Studies of macrophages from various organs of normal mice.

A unique subpopulation of macrophages (M phi) was identified among the spleen and bone marrow M phi of normal mice. After 24 h of culture, approximately 2.5% of the adherent cells cluster into "foci" of 10-30 cells. On the basis of their phagocytic and morphologic characteristics, these focus-forming M phi (FF-M phi) appeared to be highly activated. Uncoated sheep erythrocytes (E) were ingested by FF-m phi indicating that opsonization was not a prerequisite for phagocytosis. However, IgM-coated E (EIgM) were more readily phagocytosed by FF-M phi than were E suggesting that IgM is recognized as an effective opsonin by these cells. EIgM and E coated with IgM and complement (C) (EIgMC) were ingested by approximately the same percentage of FF-M phi; thus, if these cells possess complement receptors in addition to structures which bind EIgM, the C receptors do not enhance the ability of FF-M phi to ingest opsonized particles. The non-focus-forming M phi, e.g. individual M phi (I-M phi), in the spleen and bone marrow can, themselves, be divided into various subpopulations distinguished by their ability to bind and ingest E, EIgM ana EIgMC. These may represent various subpopulations of M phi or M phi at various stages of activation or differentiation. While spleen and bone marrow M phi contained FF-M phi and I-M phi which vary in their ability to ingest E, EIgM and EIgMC, theM phi of the peritoneum and blood of normal mice were far more homogeneous. Peritoneal and blood M phi did not form foci, ad did not ingest E or EIgM in significant amounts although a small percentage were able to ingest EIgMC. These data suggest that the population of M phi in the spleen and bone marrow are far more heterogenous than those found in the peritoneum or blood and that binding and phagocytosis of various coated and uncoated erythrocytes can be studied to elucidate this heterogeneity.

Release of platelet-activating factor (PAF-acether) and leukotrienes C and D from inflammatory macrophages.

Macrophages (M phi) isolated from the peritoneal cavity of C57BL/6 mice were either untreated or treated with various eliciting agents (thioglycollate, sodium caseinate) or with an activating agent bacillus Calmette Guérin (BCG). The various populations were assessed for their ability to release platelet-activating factor (PAF-acether), an ether phospholipid mediator, and slow-reacting substance (SRS), a lipoxygenase arachidonic acid derivative. PAF-acether was recovered in higher amounts form BCG M phi than from resident M phi, whereas elicited M phi exhibited a marked decreased ability to release this mediator. Such variations were only quantitative as evidenced by the similar enzyme sensitivity and high pressure liquid chromatography (HPLC) retention times of the various PAF-acether-containing supernatants. Resident, BCG- and sodium caseinate-induced M phi released similar amounts of SRS, whereas thioglycollate M phi exhibited once again a marked decreased ability to release this mediator. Comparing retention times on HPLC of resident and BCG M phi SRS with those of synthetic leukotrienes C and D, molecular variations were noted. Even though both M phi populations released higher amounts of leukotrienes C than D, the D/C ratio was higher in BCG M phi than in resident M phi. These results show that different environmental factors can influence the release of PAF-acether and leukotrienes from M phi.

Characterization of the mononuclear cell infiltrate in human malignant melanoma.

In the skin infiltrate of superficial spreading melanoma, non phagocytosing mononuclear cells (NPMC) represent a major cell component. The number of NPMCs decreases as a function of tumour progression. In addition, when an NPMC is in contact with a malignant melanocyte, the latter cell exhibits ultrastructural degenerative changes. In the blood of healthy donors and of melanoma patients, atypical mononuclear cells (AMC) can be identified. AMCs have been shown to participate in antibody-dependent cellular cytotoxicity (ADCC) reactions against melanoma cells in vitro. In this paper, it is reported that NPMCs and AMCs have in common their size, and some ultrastructural features such as indented nuclei, dispersed organelles, rough endoplasmic reticulum profiles and surface microvilli. The two cell types are negative for non-specific esterase. They also fail to react for peroxidase at either the light or the electron microscopic level. They do not adhere to glass. AMCs do not form spontaneous E rosettes, they have no surface IgG and they have no receptors for complement. However, they do form rosettes with EAIgG. On frozen sections firm binding of EAIgG has been seen on the skin infiltrate in three cases out of 10. It is concluded that NPMCs might react with tumour cells in vivo, in the same manner as do AMCs in vitro.

Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families.

FGFs (fibroblast growth factors) play major roles in a number of developmental processes. Recent studies of several human disorders, and concurrent analysis of gene knock-out and properties of the corresponding recombinant proteins have shown that FGFs and their receptors are prominently involved in the development of the skeletal system in mammals. We have compared the sequences of the nine known mammalian FGFs, FGFs from other vertebrates, and three additional sequences that we extracted from existing databases: two human FGF sequences that we tentatively designated FGF10 and FGF11, and an FGF sequence from Caenorhabditis elegans. Similarly, we have compared the sequences of the four FGF receptor paralogs found in chordates with four non-chordate FGF receptors, including one recently identified in C. elegans. The comparison of FGF and FGF receptor sequences in vertebrates and nonvertebrates shows that the FGF and FGF receptor families have evolved through phases of gene duplications, one of which may have coincided with the emergence of vertebrates, in relation with their new system of body scaffold.

Human peripheral Nullymphocytes: ultrastructural aspects of the "K" cell effect against a melanoma target cell.

A null cell fraction was isolated from human peripheral blood by Ficoll density gradient centrifugation followed by removal of mononuclear phagocytes, passage through Ig-antiIg columns and sedimentation of E-rosette forming T cells. In contrast to the T cell fraction in the Null cell population exhibited cytotoxic activity against antibody-sensitized human melanoma target cells (ADCC reaction). This activity could be attributed to a low affinity Fc-receptor bearing cells present in the Null cell preparation. At the ultrastructural level in 2 h ADCC reaction most tumor cells were surrounded by up to 10 lymphoid cells which showed narrow junctions with parallel plasma membranes and local evaginations of lymphoid cells into recessus of the target melanoma cells. The mononuclear cell type found in close contact with the antibody-sensitized target cells had the following morphological criteria: 8 to 10 mu diameter, irregular nucleus, discrete rough endoplasmic reticulum, isolated ribosomes, cytoplasmic organelles concentrated under a ruffling membrane in the contact area with the tumor cell.

Biosynthesis of paf-acether. Paf-acether but not leukotriene C4 production is impaired in cultured macrophages.

After adherence for 24 or 48 h mouse peritoneal macrophages, upon a zymosan challenge, synthesized 114 +/- 55 and 82 +/- 31 pmol of paf-acether (paf)/mg of protein respectively, as compared with 513 +/- 195 pmol of paf/mg of protein in 2 h-adherent macrophages (means +/- S.D., n = 10). By contrast, 24 h- and 48 h-adherent macrophages exposed to zymosan produced more leukotriene C4 (2.7 +/- 1.1 and 1.4 +/- 0.2 nmol/mg of protein respectively, n = 5) than did 2 h-adherent macrophages (0.5 +/- 0.2 nmol/mg of protein, n = 5). Paf production was not altered when 2 h- and 24 h-adherent cells were cultured and/or stimulated in the presence of 5 microM-indomethacin, 10 microM-nordihydroguaiaretic acid or 100 microM-BW755C as compared with untreated cells. These results indirectly exclude the regulation of paf production by arachidonic acid metabolites. We investigated the efficiency of the enzymic steps which govern paf synthesis. We showed that the anabolic process was not impaired since (1) the amounts of alkylacylglycerophosphocholine and lyso-paf were similar in 2 h-, 24 h- and 48 h-adherent macrophages; (2) adding synthetic lyso-paf or acetyl-CoA to intact cells did not increase paf production in zymosan-stimulated 24 h- and 48 h-adherent macrophages; (3) the basal level of acetyltransferase was comparable in 2 h-, 24 h- and 48 h-adherent macrophages and in all cases was increased by 2-3 times upon zymosan challenge. We also showed that impaired paf production in 24 h- and 48 h-cultured macrophages was not due to the nature of the stimulus used to induce its synthesis.

Is platelet-activating factor (PAF-acether) synthesis by murine peritoneal cells (PC) a two-step process?

PAF-acether (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from several cell sources simultaneously with an inactive non-acylated compound (1-O-alkyl-glyceryl-3-phosphorylcholine) (lyso-PAF-acether). Formation of the latter probably results from the activation of a phospholipase A2 (PLA2). Indeed, the PLA2 inhibitors, bromophenacyl bromide (BPB), mepacrine, 874CB (100 microM), and EDTA (5 mM), blocked the zymosan-induced release of PAF-acether from PC. EDTA and BPB also markedly reduced the release of lyso-PAF-acether. To verify this hypothesis, acetyl coenzyme A (acetyl-CoA) was added to stimulated PC. This enhanced the release of PAF-acether in a dose-dependent fashion from 1 microM acetyl-CoA to reach a maximal increase - 200% - at 100 microM. Furthermore, using 3H acetyl-CoA, incorporation of labelled acetate into PAF-acether was suggested by (1) identical chromatographic patterns of biological activity and radioactivity; (2) disappearance of these activities after treatment with PLA2, but not after exposure to lipase from Rhizopus arrhizus. PAF-acether was also obtained when both acetyl-CoA (100 microM) and synthetic lyso-PAF-acether (0.2 microM) were added to unstimulated PC previously treated with BPB (100 microM for 10 min). These results suggest that the release of PAF-acether is the consequence of at least two different steps: (1) hydrolysis of 1-O-alkyl-2-acyl-glyceryl phosphorylcholine by PLA2; (2) enzymatic acetylation of the hydrolysis product.