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The activation, expansion, and survival of regulatory T cells (Tregs) as well as the expression of their suppressive capacities result from distinct signaling pathways involving various membrane receptors and cytokines. Multiple studies have shown that thymus-derived naturally occurring Tregs constitutively express the forkhead/winged helix transcription factor FoxP3 in addition to high levels of CD25, the negative co-stimulatory molecule CTLA-4, and the glucocorticoid-induced TNF receptor-related protein GITR. At variance, adaptive or induced Tregs acquire these phenotypic markers as they differentiate in the periphery, following adequate stimulation in the appropriate environment, together with their capacity to produce immunomodulatory cytokines (mainly, IL-4, IL-10 and TGF-beta) and to display regulatory capacities. However, none of these molecules but FoxP3 are restricted to Tregs since they may also be expressed and upregulated on activated effector T cells. This explains why different hypotheses were proposed to interpret interesting reports showing that in vivo abrogation of CTLA-4 signaling using neutralizing CTLA-4 antibodies triggers different autoimmune or immune-mediated manifestations. Thus, an effect on pathogenic T cell effectors and/or Tregs has been proposed. Here we present and discuss recent results we obtained in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes, arguing for a key role of CTLA-4 in the functional activity of Tregs. Moreover, data are presented that simultaneous blockade of CTLA4 and TGF-beta further impairs immunoregulatory circuits that control disease progression.

Growth Hormone (GH), Insulin-like Growth Factors (IGFs) and IGF-Binding Proteins which modulate the IGFs' bioavailability (e.g. IGFBP-3, -4, -5), are essential regulators of bone remodeling. In this study, MG-63 human osteosarcoma cells were used as a model system to investigate the mechanism(s) whereby IGF-I and GH control IGFBP-3 gene expression. Physiological concentrations of IGF-I (1-20 nM) induced a dose-dependent increase in the steady-state amount of IGFBP-3 mRNA (maximal stimulation: approximately 9-10-fold). This increase was detectable 3 h after the onset of IGF-I treatment, was enhanced over a 24 h period, then plateaued until at least 30 h. Consistently, a dose-dependent increase in IGFBP-3 secretion ( approximately 40-50-fold at IGF-I concentrations/=16 nM) was observed by western ligand- and immuno-blot analysis of MG-63 cells conditioned medium, and its time course was similar to that observed for IGFBP-3 transcripts. IGFBP-3 mRNA stability (t(1/2) approximately 20 h) was identical in the presence or absence of IGF-I treatment. By contrast, human (h) GH treatment (24-72 h) of MG-63 cells did not increase IGFBP-3 secretion in the conditioned medium. Ectopic expression of recombinant rat GH-R resulted in hGH-enhanced expression of GH-responsive reporter gene constructs, but did not increase endogenous IGFBP-3 gene expression, suggesting that the GH unresponsiveness was not only due to the very low level of GH binding sites at the plasma membrane level. Altogether, these results support the conclusions that in MG-63 cells (i) transcriptional rather post-transcriptional mechanisms are involved in the IGF-I-induced increase of IGFBP-3; (ii) the abundance of GH-R is very low at the plasma membrane level; (iii) the dowstream GH-signaling cascade is fully functional in this human osteosarcoma cell line; and (iv) the endogenous IGFBP-3 gene is not responsive to hGH in human MG-63 osteosarcoma cells.

Insulin-like growth factor binding proteins (IGFBPs) are essential mediators of the bioavailability and biological effects of the IGFs. Liver expression of the rat (r) IGFBP-1 and rIGFBP-2 genes has been characterized between day 16 in utero (16 diu) and 16 days postnatally (+16 dpn). Run-on experiments showed transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth (B) to be 25 and 5 times that at 16 diu, respectively. After B, transcriptional activity of the rIGFBP-1 gene remained high (140% B at +6 dpn), but that of the rIGFBP-2 gene dropped to 70% B by +6 dpn. Northern blot analysis done simultaneously showed rIGFBP-1 messenger RNA (mRNA) levels to increase approximately 50-fold between 16 diu and B, whereas rIGFBP-2 mRNA increased only 5- to 10-fold. rIGFBP-1 mRNA levels decreased after birth, reaching about 20% B by +6 dpn; rIGFBP-2 mRNA, however, remained stable (about 80% B) at least up to +6 dpn. Parallel Western ligand blot and immunoblot analyses of serum rIGFBPs revealed rIGFBP-1 and rIGFBP-2 concentrations to be increased 3- and 2-fold, respectively between 20 diu and B. Maximal expression of rIGFBP-1 was at +1 dpn (220% B), and of rIGFBP-2, at B. Both rIGFBPs then decreased, reaching about 5% B at adulthood. All these data indicate that increased transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth would determine the increased synthesis in the liver and circulating levels of these proteins. In addition, it would seem that post-transcriptional events (reduced half-life of the rIGFBP-1 messenger after birth, translation efficiency of the rIGFBP-2 messenger) modulate transcriptional regulation.

BACKGROUND: The cross-talk between pathogenic T lymphocytes and regulatory T cells (Tregs) plays a major role in the progression of autoimmune diseases. Our objective is to identify molecules and/or pathways involved in this interaction and representing potential targets for innovative therapies. Glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand are key players in the T effector/Treg interaction. GITR is expressed at low levels on resting T cells and is significantly up-regulated upon activation. Constitutive high expression of GITR is detected only on Tregs. GITR interacts with its ligand mainly expressed on antigen presenting cells and endothelial cells. It has been suggested that GITR triggering activates effector T lymphocytes while inhibiting Tregs thus contributing to the amplification of immune responses. In this study, we examined the role of GITR/GITRLigand interaction in the progression of autoimmune diabetes. METHODS AND FINDINGS: Treatment of 10-day-old non-obese diabetic (NOD) mice, which spontaneously develop diabetes, with an agonistic GITR-specific antibody induced a significant acceleration of disease onset (80% at 12 weeks of age). This activity was not due to a decline in the numbers or functional capacity of CD4(+)CD25(+)Foxp3(+) Tregs but rather to a major activation of 'diabetogenic' T cells. This conclusion was supported by results showing that anti-GITR antibody exacerbates diabetes also in CD28(-/-) NOD mice, which lack Tregs. In addition, treatment of NOD mice, infused with the diabetogenic CD4(+)BDC2.5 T cell clone, with GITR-specific antibody substantially increased their migration, proliferation and activation within the pancreatic islets and draining lymph nodes. As a mirror image, blockade of the GITR/GITRLigand pathway using a neutralizing GITRLigand-specific antibody significantly protected from diabetes even at late stages of disease progression. Experiments using the BDC2.5 T cell transfer model suggested that the GITRLigand antibody acted by limiting the homing and proliferation of pathogenic T cells in pancreatic lymph nodes. CONCLUSION: GITR triggering plays an important costimulatory role on diabetogenic T cells contributing to the development of autoimmune responses. Therefore, blockade of the GITR/GITRLigand pathway appears as a novel promising clinically oriented strategy as GITRLigand-specific antibody applied at an advanced stage of disease progression can prevent overt diabetes.

Two unusual but similar cases of very high serum iron, extremely high transferrin concentrations (5.4 to 6.5 g/L), and increased transferrin saturation are reported. No anemia or microcytosis was observed. The ferritin concentration remained within the normal range, and no iron overload was observed. In one case, the in vivo half-life of 59Fe-labeled transferrin was shown to be prolonged (206 minutes versus 75 minutes for controls). In both patients, a monoclonal IgG was observed in the serum. The association between serum transferrin and monoclonal IgG was demonstrated by Western blot analysis and affinity chromatography. We suggest that the transferrin bound to the monoclonal IgG molecule has a prolonged half-life in the circulation, leading to high transferrin concentrations, and that the increased serum iron values prevent the onset of anemia. The antitransferrin activity of monoclonal antibody should be added to the list of situations accounting for elevated serum iron with elevated transferrin saturation.

Resume Les IGF-I et -II presents dans les milieux biologiques sont lies a des proteines vectrices de haute affinite (IGFBP-1 a -6) qui modulent leurs effets cellulaires. Parmi les mecanismes regulant la biodisponibilite des IGF, la proteolyse limitee de certaines IGFBP joue un role majeur. L'IGFBP-3 est proteolysee in vivo et dans diverses lignees cellulaires par des proteases a serine, dont la plasmine, dans des conditions que nous avons reproduites in vitro avec de l'IGFBP-3 recombinante humaine non glycosylee (rh IGFBP-3). Deux principaux fragments ont ete isoles : le plus abondant, de 22–25 kDa garde une faible affinite pour IGF-I et n'inhibe que faiblement, compare a FIGFBP-3 intacte, ses effets mitogenes. L'autre de 16 kDa, bien que sans affinite pour IGF, se comporte neanmoins comme un inhibiteur de croissance. Dans le present travail, les fragments proteolytiques ont ete isoles par HPLC et leur sequence en acides amines N-terminale a ete determinee. Les deux principaux fragments appartiennent a la region N-terminale de l'IGFBP-3, celui de 22–25 kDa comprend la sequence 1–160, et celui de 16 kDa la sequence 1–95. Des experiences sur la lignee de cellules osteoblastiques MG-63 qui produisent l'IGFBP-3 et de Purokinase induisant sa proteolyse en presence de plasminogene, montrent que l'apparition du peptide 1–160 precede celle du peptide 1–95 et que ce dernier resulte probablement d'une proteolyse secondaire du plus gros fragment. L'ensemble des donnees suggerent que la proteolyse de l'IGFBP-3 est intimement associee a ses fonctions. Nous proposons un modele rendant compte de son mode d'action cellulaire.

Limited proteolysis in vivo of insulin-like growth factor-binding protein-3 (IGFBP-3) by as yet unidentified serine proteases plays a key role in controlling the bioavailability of IGFBP-3-associated insulin-like growth factors (IGFs). Both the IGF system and the system of plasminogen activators (PAs) and their inhibitors (PAIs) are involved in bone remodeling, and plasmin has been shown to provoke dissociation of IGFBP-IGF complexes in cultured MG-63 human osteoblasts. The aim of this work was to investigate interactions between IGF-I and the PA/PAI system and their influence on IGFBP-3 production and proteolysis in this cell model. At confluency, MG-63 cells maintained for 3 days in serum-free medium constitutively secreted IGFBP-2 and small amounts of IGFBP-3 and IGFBP-4. As shown by Western ligand and immunoblot analyses of the culture medium and Northern blot analysis of IGFBP-3 messenger RNA, production of these IGFBPs, and of IGFBP-3 in particular, was dose dependently stimulated by the addition of 12.5-100 ng/ml recombinant human (rh) IGF-I. Increasing concentrations of plasminogen (0.05-3.5 micrograms/ml) added during the final 12 h of culture reduced the amounts of IGFBP detectable by Western ligand blotting, especially IGFBP-3. This reduction reflected proteolysis, as shown by immunoblotting, which revealed 30-, 20-, and 16-kilodalton fragments of IGFBP-3. In the presence of 25 ng/ml IGF-I, which stimulated IGFBP-3 production, proteolysis was reduced by approximately half. Incubation of glycosylated [125I]rh-IGFBP-3 as substrate in cell-free conditioned medium gave the same results. Addition of 50 ng/ml rhIGF-I to conditioned medium (to promote IGFBP-3-rhIGF-I complex formation) failed to diminish plasmin-induced proteolysis of IGFBP-3. Urokinase PA activity in the conditioned medium decreased significantly when the cells were cultured with rhIGF-I, indicating a direct action of IGF-I on urokinase PA and/or PAI production. Our results support the notion of a regulation loop whereby IGF-I controls its own bioavailability via its action on both IGFBP-3 production and the PA/PAI system, which regulates IGFBP-3 proteolysis. The proteolytic cleavages of IGFBP-3 caused by plasmin were the same as those caused in vivo by serine protease acting on this IGFBP.

Western ligand blot analysis of the different molecular forms of insulin-like growth factor-binding protein IGF-BP) in serum and plasma samples from 89 pregnant women has revealed a marked decrease, after the second month of pregnancy, in the 41.5 and 38.5K species (which are the binding units of the 150K complex) as well as in the 24K form. There was also a slight decrease in the 34K form, the 30K form was unaffected, and additional 21.5 and 20K bands appeared. Cross-linking experiments demonstrated the disapperance of a 49K band which is characteristic of the 150K complex. The alterations of the electrophoretic profile of the BPs were accompanied by a decrease in binding activity of up to 90%. Gel filtration at pH 7.4 confirmed that the decrease was essentially attributable to changes in the 150K complex BPs: 1) material eluting in the 150K zone contained only one third of the binding activity, as opposed to three quarters in reference material; 2) radiocompetition experiments illustrated the loss of affinity for IGF-I and IGF-II of the BPs extracted from the 150K complex; 3) ligand blot analysis revealed, in contrast with the virtual disappearance of the 41.5 and 38.5K forms, the appearance of a broad indistinct band at 30K and additional bands at 21.5 and 20K. With immunoblotting, the anti-IGF-BP-3 antibody, which specifically recognizes the 41.5 and 38.5K species, cross-reacted with this 30K material. The alterations of the BPs appeared to be enzymatic. When pregnancy serum was mixed with reference serum, the 41.5, 38.5, and 24K forms contributed by the reference serum were markedly reduced after 30 min of incubation at 37 C. However, these alterations could be prevented by incubation at either 0 or at 37 C in the presence of EDTA or aprotinin and could be curbed in the presence of high concentrations of phenylmethylsulfonylfluoride. Unmixed reference serum incubated at 37 C yielded an unchanged BP profile. Incubation of pregnancy serum with hypopituitary serum, which has elevated levels of the 34 and 30K BPs, resulted in a marked decrease in the 41.5 and 38.5K forms, a slight alteration of the 34K form, and no change in the 30K form. These findings suggest that during pregnancy, enzymatic (probably protease) activity either appears or is significantly increased in the circulation, which specifically degrades some of the IGF-BPs.(ABSTRACT TRUNCATED AT 400 WORDS)

Circulating insulin-like growth factors (IGFs) are bound to specific, high-affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150000 (‘large’ complex) and 40000 (‘small’ complex). The large complex appears to be under growth-hormone control and its proportions vary with those of the IGFs. Molecular heterogeneity among the binding proteins was revealed by polydcrylamide gel electrophoresis (SDS-PAGE) of serum in which they were cross-linked to 125I-labelled IGF I or II. Out of the six specific bands observed, of 150000, 120000, 49000, 46000, 40000 and 37000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49000–37000-Mr species may constitute the subunits of 150000-Mr and/or 120000-Mr IGF-BP complexes. With electrophoresis followed by transfer onto nitrocellulose and incubation with either 125I-labelled IGF I or II (western blot), the different binding proteins were identified per se. There were five molecular forms with Mr of 41 500, 38 500, 34000, 30000 and 24000. In normal serum the 41 500 and 38 500-Mr forms were the major binding proteins. They appeared in both complexes, but were predominant in the large complex where they constitute the elementary binding units. These two proteins therefore bind to IGFs to form both ‘monomeric’ IGF-BP and ‘oligomeric’ (IGF-BP)n complexes. The 34000, 30000 and 24000-Mr forms, by contrast, were visible only in the small complex. Different mechanisms appear to regulate the different binding proteins: in acromegalic serum the 41 500 and 38 500-Mr forms were augmented and the 34000-Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30000-Mr form was augmented. With chromatofocusing, the 34000, 30000 and 24000-Mr forms eluted in three peaks between pH 6.0 and 4.0, whereas the 41 500 and 38 500-Mr forms eluted throughout the gradient, principally at pH 7.5 and 7.0. Competitive binding studies, done on binding proteins separated either by chromatofocusing or by SDS-PAGE and transfer onto nitrocellulose, revealed different affinities for the IGFs among the different molecular forms. The 41 500 and 24 000-Mr binding proteins preferentially bound IGF I and the 38 500, 34 000 and 30 000-Mr proteins preferentially bound IGF II. Our findings demonstrate the molecular heterogeneity of the binding proteins and the existence of a relationship between their structure and their affinities for the IGFs. They also suggest that, apart from their function as IGF carriers, the binding proteins also play a modulating role in the interaction between IGFs and their target cells.

A nitrocellulose gel transfer technique has been adapted to study the insulin-like growth factor (IGF) binding proteins of human serum. Normal and hypopituitary sera were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose or nylon membrane. Nonidet-P40 (3%) and Tween 20 (0.1%) were required for quenching and to allow detection of the IGF binding proteins by autoradiography after overlay with either 125I-labeled IGF I or IGF II. Several forms of IGF binding protein have been identified with molecular weights of 41,500, 38,500, 34,000, 30,000, and 24,000. Titration and competitive binding studies with IGF were performed on the transferred IGF binding proteins, indicating that binding proteins isolated by this technique can be characterized.

Human cerebrospinal fluid (CSF) has been found to contain several different molecular forms of IGF-specific binding proteins (BPs). Qualitatively, they are similar to those present in serum, although their relative proportions are very different, as well as to those present in the culture media of brain tissue from which these BPs presumably arise. One particular form of BP is predominant in CSF. It has an M r of 34 000, as estimated by SDS-polyacrylamide gel electrophoresis followed by transfer onto nitrocellulose, and an isoelectric point around 5.0 based on chromatofocusing. It has a selective affinity for IGF-II (~4 × 10 10 M −1 ) as shown by competitive binding experiments in which biosynthetic IGF-I was about 40-times less potent than native IGF-II in displacing 125 I-labelled IGF-II. These findings are in agreement with the preponderance of IGF-II in nervous tissue and in CSF and suggest that this BP plays an important role in the interaction of IGF-II with its target cells.

Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins. In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes. In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40,000, which is similar to that of the 'small' complex of serum. Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S. Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF I revealed molecular heterogeneity. Three specific bands corresponding to Mr 46,000, 40,000 and 37,000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum. SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000, identical to those seen in serum. The relative concentrations of the 41,500 and 38,500-Mr forms varied from one medium to another, but the 34,000 and 30,000-Mr forms were regularly more abundant in the liver culture media than in normal serum. The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources). The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41,500 and 38,500-Mr forms, which are the only binding units of the serum 'large' complex (150,000 Mr), indicates that these two binding proteins are capable of binding IGFs to form 'monomeric' IGF-BP complexes. Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43,000 and 39,000 and three between 32,000 and 24,000. Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41,500 and 38,500-Mr forms were more abundant in the adult and the 34,000 and 30,000-Mr forms more abundant in the foetus.(ABSTRACT TRUNCATED AT 400 WORDS)

IGFs are synthesized especially by the liver, and circulate in the blood bound to specific, high-affinity binding proteins. After SDS-PAGE of serum followed by transfer onto nitrocellulose, incubation with labelled IGF and autoradiography, five molecular forms of BP with MWs of 42, 39, 34, 30 and 24K have been identified. In normal serum the 42 and 39K BPs predominate and constitute the binding units of the “large complex”. In acromegalic serum, the 42 and 39K BPs strongly enhance and the 24K BP less so, whereas the 34K BP is barely detectable. With treatment, clinical remission is accompanied by a return to normal of IGF levels and the electrophoretic profile of the BPs. In hypopituitary serum, the 42 and 39K BPs significantly diminish and the 34K BP strongly enhances. With GH therapy, the normal doses restoring growth induce an increase in the levels of the IGFs and 42 and 39K BPs and a reduction of the 34K BP, although normalization is not complete. In the foetus, whose growth is not under GH control, the BP profile between 19 - 37 weeks of gestation is stable, with particularly depressed levels of the 42 and 39K BPs and a predominant 34K BP. The 34K BP is also the major in cerebrospinal fluid. From our results we infer that 1) the 42 and 39K BPs are regulated by GH, their abundance being related to IGF levels 2) the 34K BP may be negatively controlled by GH 3) western blot analysis of the BPs may constitute a complementary index in the surveillance of acromegaly and hypopituitarism.