Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites

Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membrane anchorage is required. Soluble betaglycan can bind TGF-beta but does not enhance binding to membrane receptors. In fact, recombinant soluble betaglycan acts as potent inhibitor of TGF-beta binding to membrane receptors and blocks TGF-beta action, this effect being particularly pronounced with the TGF-beta 2 isoform. The results suggest that release of betaglycan into the medium converts this enhancer of TGF-beta action into a TGF-beta antagonist.     

Neutrophil Fc gamma and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fc gamma receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fc gamma and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1 receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1 receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+ influx/mobilization demonstrated that the platelet P2X1 receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.     

Associations between TGF-beta1 receptors in human bone marrow stromal cells

Bone marrow stromal cells are required for sustained haemopoiesis. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine present in the bone marrow microenvironment which regulates the expression of several cytokines, cytokine receptors and cell adhesion elements. The TGF-beta receptors type I and II, and endoglin, mediate TGF-beta1 binding to the membrane of human bone marrow stromal cells. [125I]TGF-beta1-affinity labelling experiments showed that three different anti-endoglin monoclonal antibodies co-immunoprecipitated a 68 kD TGF-beta1-labelled polypeptide together with TGF-beta1/endoglin complexes. Here, we have shown that the 68 kD receptor corresponds to the type I receptor, indicating that endoglin and the type I receptor associate on the membrane of these cells upon ligand binding. The expression of endoglin by stromal cells was found to be up-regulated by TGF-beta1, but not by IL-1beta. The association of endoglin with signalling components of the TGF-beta receptor system on the membrane of bone marrow stromal cells might modulate TGF-beta1 access to the signalling pathways, and therefore it could regulate TGF-beta1-mediated stromal cellular responses.     

TGF-beta receptor expression on human keratinocytes: a 150 kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with type I and type II receptors

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-beta has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-beta receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-beta receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-beta1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-beta isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-beta receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-beta1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-beta receptors on keratinocytes, supporting the heterotetrameric model of TGF-beta signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-beta by markedly downregulating all four TGF-beta binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with the TGF-beta signalling receptors suggests that this GPI-anchored protein may modify TGF-beta signalling in human keratinocytes.     

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis"

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

TNF-alpha, unlike other pro- and anti-inflammatory cytokines, induces rapid release of the IL-1 type II decoy receptor in human myelomonocytic cells

The present study was designed to investigate the effect of a series of cytokines on the release of the type II IL-1 decoy receptor, which represents a unique pathway of negative regulation of the IL-1 system. After 20 min, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-gamma, granulocyte-CSF, macrophage-CSF, and TGF-beta had little or no effect on IL-1 binding by human polymorphonuclear cells. In contrast granulocyte-macrophage-CSF and, to a greater extent, TNF markedly reduced IL-1 binding. The action of TNF was rapid, reaching 50% of its maximum (80%) at 2 min, and plateauing at 20 min with decrease in receptor number and no significant change in affinity. Loss of surface receptor was associated to rapid release of a 45-kDa IL-1-binding molecule identified as the decoy RII. TNF-induced release of the decoy RII was independent of protein synthesis and reactive oxygen intermediates. Monocytes showed a similar response to TNF, except for the size of the released molecule (approximately 60 kDa). TNF induced rapid release of its own receptors. In contrast IL-1beta affected neither its own receptors nor the TNF-R. TNF and, more efficiently, PMA caused release of the decoy RII in fibroblasts transfected with the full-length decoy RII or with a cytoplasmatic deletion mutant. TNF-induced decoy RII release represents an unidirectional pathway of communication in the interplay between the IL-1 and TNF system.     

Human platelets display high-affinity receptors for thrombopoietin

Thrombopoietin (Tpo) is a major regulator of megakaryopoiesis both in vivo and in vitro. Tpo initiates its biologic effects by binding to the Mpl receptor, which is a member of the hematopoietin receptor family. To define the Tpo binding characteristics of the Mpl receptor, we iodinated purified 70-kD recombinant human Tpo using the Bolton-Hunter reagent. Autoradiographic analysis of (125)I-Tpo binding to normal human marrow mononuclear cells showed many grains specifically associated with megakaryocytes; there were no grains specifically associated with myeloblasts or erythroblasts. Equilibrium binding experiments with (125)I-Tpo and normal human platelets showed a single class of high-affinity receptors (kd, 190 pmol/L) with approximately 30 Mpl receptors per platelet. Affinity cross-linking with (125)I-Tpo showed that the Mpl receptor on platelets is of molecular weight approximately 98 kD. Despite their sequence similarity, erythropoietin and Tpo did not cross-compete for binding to BaF3 cells engineered to coexpress Mpl receptor and erythropoietin receptor. Progeny of normal human burst-forming units-erythroid (BFU-E) contained Mpl receptor mRNA, and flow cytometric analysis showed the presence of Mpl receptor protein on the surface of these cells. These data indicate that display of the Mpl receptor is not limited to the megakaryocytic lineage, but also includes progeny of BFU-E. Like receptors for other hematopoietic cytokines, the binding affinity of the Mpl receptor for Tpo is high, with relatively few receptors displayed per cell. These results suggest that the effects of Tpo to speed red blood cell recovery after myelosuppressive therapy in vivo and to enhance colony-forming unit-erythroid generation in vitro may be mediated by direct interaction of Tpo and erythroid progenitor cells.     

Insulin-induced expression of prostacyclin receptors on platelets is mediated through ADP-ribosylation of Gi alpha protein

The binding of insulin in physiological amounts to human blood platelets, which increases adenylate cyclase-linked prostacyclin receptor numbers on the cell surface, was found to be directly related to the ADP-ribosylation of the Gi alpha. Conversely, resuspension of the insulin-treated platelets in the hormone-free medium decreased both the prostaglandin receptor numbers and ADP-ribosylation of Gi alpha. Furthermore, incubation of platelets with pertussis toxin or its A-protomer, which ADP-ribosylates Gi alpha, also stimulated the binding of the prostanoid. These results suggest that the increase of prostacyclin receptor numbers in platelets is mediated through the ADP-ribosylation of Gi alpha.     

Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas

Transforming growth factor beta (TGF-beta) is an extracellular ligand that binds to a heterodimeric receptor, initiating signals that regulate growth, differentiation, and apoptosis. Many cancers, including pancreatic cancer, harbor defects in TGF-beta signaling and are resistant to TGF-beta-mediated growth suppression. Genetic alterations of DPC4, which encodes a DNA binding protein that is a downstream component of the pathway, most frequently occur in pancreatic and biliary carcinomas. We searched for other targets of mutation of the TGF-beta pathway in these cancers. We report somatic alterations of the TGF-beta type I receptor gene ALK-5. Homozygous deletions of ALK-5 were identified in 1 of 97 pancreatic and 1 of 12 biliary adenocarcinomas. A germ-line variant of ALK-5, presumably a polymorphism, was identified, but no somatic intragenic mutations were identified upon sequencing of all coding regions of ALK-5. Somatic alterations of the TGF-beta type II receptor gene (TGFBR2) were identified in 4 of 97 (4.1%) pancreas cancers, including a homozygous deletion in a replication error-negative cancer and three homozygous frameshift mutations of the poly(A) tract of the TGF-beta type II receptor in replication error-positive cancers. We also studied other related type I receptors of the TGF-beta superfamily. In a panel of pancreas cancers preselected for loss of heterozygosity at the ALK-1 locus, sequencing of all coding exons of the ALK-1 gene revealed no alterations. No homozygous deletions were detected in the ALK-1, ALK-2, ALK-3, or ALK-6 genes in a panel of 86 pancreatic cancer xenografts and 11 pancreatic cancer and 22 breast cancer cell lines. The rate of genetic inactivation of TGF-beta pathway members was determined in 45 pancreatic cancers. Eighty-two % of these pancreatic cancers had genetic inactivation of the DPC4, p15, ALK-5, or TGFBR2 genes. Our results indicate that the TGF-beta type I and type II receptor genes are selective targets of genetic inactivation in pancreatic and biliary cancers.     

Trabecular cells express receptors that bind TGF-beta 1 and TGF-beta 2: a qualitative and quantitative characterization

PURPOSE: To quantitate the receptors for transforming growth factor (TGF)-beta 1 on trabecular cells in culture and to determine the relative affinities of TGF-beta 1 and TGF-beta 2 for these receptors. METHODS: We quantitated the receptors for TGF-beta 1 by Scatchard analysis of radioligand binding of 125I-TGF-beta 1 to cultured porcine trabecular cells. We established the relative affinities of TGF-beta 1 and TGF-beta 2 for the receptors by competitive binding of 125I-TGF-beta 1 with increasing concentrations of the unlabeled TGF-beta 1 or TGF-beta 2. We also investigated the binding of 125I-TGF-beta 1 after pre-treatment of trabecular cells with heparinase. RESULTS: Trabecular cells expressed approximately 4,000 high-affinity receptors per cell for TGF-beta 1, with a dissociation constant (Kd) of 15.8 +/- 7.6 pmol/l. By varying the concentrations of the unlabeled growth factors, we determined that the relative affinities of TGF-beta 1 and TGF-beta 2 for the receptors were 16 pmol/l and 50 pmol/l, respectively. Heparinase treatment of the trabecular cells did not change the binding affinity of the receptor for 125I-TGF-beta 1. CONCLUSIONS: Our findings show that trabecular cells express heparinase-insensitive TGF-beta receptors that have an approximately threefold greater affinity for TGF-beta 1 than for TGF-beta 2. Based on the present investigation, together with our previous data on the molecular weights of the binding sites, we conclude that trabecular cells do possess types II and III receptors but not type I receptors.     

Direct or C5a-induced activation of heterotrimeric Gi2 proteins in human neutrophils is associated with interaction between formyl peptide receptors and the cytoskeleton

The binding of ligands to N-formyl peptide chemoattractant receptors in human neutrophils results in a rapid association of these receptors with a cytoskeletal fraction and a specific activation and release of Gi2 alpha-subunits from this fraction. In the present study we could show that pretreating neutrophils with GDPbetaS prevented the fMet-Leu-Phe-induced association of its receptor with a cytoskeletal fraction and also blocked the release of Gi2 alpha-subunits from the same cytoskeletal fraction. In contrast, direct activation of Gi2 proteins by addition of GTPgammaS or AlF4- not only caused a release of Gi2 alpha-subunits from the cytoskeleton but also an association of formyl peptide receptors with the cytoskeleton. The receptor for complement fragment 5a, which transduces its signaling through the same Gi2 protein, triggers both a release of Gi2 alpha-subunits from the cytoskeleton fraction and, of even greater interest, an association between formyl peptide receptors and the cytoskeleton. The close relationship between the activation and release of Gi2 alpha-subunits from the cytoskeleton and the association of formyl peptide receptors with the cytoskeleton might, however, not be a matter of protein-protein exchange, since the increased binding of formyl peptide receptors to the cytoskeleton occurs more rapidly than the release of Gi2 alpha-subunits from the cytoskeleton. The present findings suggest a possible mechanism for the initiation of formyl peptide receptor desensitization during neutrophil locomotion.     

Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-beta superfamily

Endoglin (CD105) is a transmembrane glycoprotein that binds transforming growth factor (TGF)-beta1 and -beta3, and coprecipitates with the Ser/Thr kinase signaling receptor complex by affinity labeling of endothelial and leukemic cells. The present study shows that in addition to TGF-beta1 and -beta3, endoglin interacts with activin-A, bone morphogenetic protein (BMP)-7, and BMP-2 but requires coexpression of the respective ligand binding kinase receptor for this association. Endoglin cannot bind ligands on its own and does not alter binding to the kinase receptors. It binds TGF-beta1 and -beta3 by associating with the TGF-beta type II receptor and interacts with activin-A and BMP-7 via activin type II receptors, ActRII and ActRIIB, regardless of which type I receptor partner is coexpressed. However, endoglin binds BMP-2 by interacting with the ligand binding type I receptors, ALK3 and ALK6. The formation of heteromeric signaling complexes was not altered by the presence of endoglin, although it was coprecipitated with these complexes. Endoglin did not interact with BMP-7 through complexes containing the BMP type II receptor, demonstrating specificity of its action. Our data suggest that endoglin is an accessory protein of multiple kinase receptor complexes of the TGF-beta superfamily.     

Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2

Most evidence points toward there being a shared binding site in complement receptor type 2 (CR2, CD21) for the complement ligand C3dg and the EBV surface envelope glycoprotein gp350/220. Indeed, synthetic peptide studies have suggested that the CR2-binding sites in human C3dg and EBV gp350/220 share a similar sequence motif. The proposed CR2-binding sequence in C3dg is EDPGKQLYNVEA (residues 1199-1210 of mature C3), whereas that in EBV gp350/220 is EDPGFFNVEI (residues identical to C3dg are underlined). To further examine the role of amino acids 1199-1210 in the binding of the C3 fragments iC3b and C3dg to CR2, the following alanine-substitution variants of human C3 were tested in two independent CR2-binding assays: ED1199,1200AA; KQ1203,1204AA; L1205A; Y1206A; NV1207,1208AA; E1209A; and ED-KQ-NV1199,1200-1203,1204-1207,1208AA-AA-AA. Also engineered and tested was a chimeric C3 molecule in which the 1199-1210 sequence (PVPGGYQLTLEA) from the non-CR2-binding trout C3 molecule was grafted onto a human C3 background. Recombinant C3 proteins were expressed transiently in COS-1 cells, deposited as C3b on C3 convertase-bearing sheep erythrocytes and finally converted to cell-bound iC3b or C3dg using factors H and I. Binding of EAC423bi and EAC423dg to CR2 on Raji cells or EAC423dg to soluble CR2 was assessed. In most cases, the substitutions had little effect on CR2-binding activity and even in the case of the most highly substituted variants, the decrease in CR2-binding activity was less than twofold. Thus, contrary to the results anticipated from synthetic peptide studies, the single and multiple substitutions to the C3 sequence tested failed to corroborate a role for the 1199-1210 sequence in the C3dg-CR2 interaction.     

Integrin alpha IIb beta 3 mediates binding of the Lyme disease agent Borrelia burgdorferi to human platelets

Lyme disease is a chronic, multisystemic infection caused by the tick-borne spirochete Borrelia burgdorferi. Attachment of the spirochete to host cells via specific receptors is likely to be important in the establishment of infection. B. burgdorferi have previously been shown to bind to a variety of mammalian cells in vitro. Here we demonstrate that binding of B. burgdorferi to human platelets is mediated by the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa), a critical receptor in thrombosis and hemostasis. Functional expression of this receptor requires platelet activation, and binding of the spirochete was observed only to activated platelets. Binding was inhibited by a synthetic Arg-Gly-Asp peptide that blocks ligand interaction with many integrins and by a synthetic peptide based on the gamma chain of fibrinogen that blocks binding to alpha IIb beta 3. In addition, attachment of the spirochete to platelets was inhibited by monoclonal antibodies directed against alpha IIb beta 3 that are known to block ligand-receptor interaction. No inhibition was seen with control peptides or with antibodies directed against other platelet receptors. B. burgdorferi bound efficiently to purified alpha IIb beta 3 but did not bind to platelets deficient in this integrin. Efficient platelet binding was displayed by a cloned, infectious B. burgdorferi strain, whereas a cloned noninfectious strain did not bind to platelets. Binding to integrins may be important for the ability of B. burgdorferi to establish infection in the diverse tissues affected by Lyme disease.     

Cloning of a type I TGF-beta receptor and its effect on TGF-beta binding to the type II receptor

Transforming growth factor-beta (TGF-beta) affects cellular proliferation, differentiation, and interaction with the extracellular matrix primarily through interaction with the type I and type II TGF-beta receptors. The type II receptors for TGF-beta and activin contain putative serine-threonine kinase domains. A murine serine-threonine kinase receptor, Tsk 7L, was cloned that shared a conserved extracellular domain with the type II TGF-beta receptor. Overexpression of Tsk 7L alone did not increase cell surface binding of TGF-beta, but coexpression with the type II TGF-beta receptor caused TGF-beta to bind to Tsk 7L, which had the size of the type I TGF-beta receptor. Overexpression of Tsk 7L inhibited binding of TGF-beta to the type II receptor in a dominant negative fashion. Combinatorial interactions and stoichiometric ratios between the type I and II receptors may therefore determine the extent of TGF-beta binding and the resulting biological activities.     

Studies on the biological effects of ozone: 6. Production of transforming growth factor 1 by human blood after ozone treatment

After exposing human whole blood from normal volunteers to ozone concentrations ranging from 22 to 156 micrograms/ml, we have shown that, upon incubation of up to 8 hours, there is a significant release of transforming growth factor beta (TGF-beta 1). In comparison to TGF-beta 1, TGF-beta 2 production is not influenced by ozone concentrations. In line with our previous findings it appears that blood, in the presence of heparin and 5mM Ca,2+ allows a consistent production of tumor necrosis factor a (TNF alpha) and the release of low and non-hazardous levels of free hemoglobin. These data support the contention that autohemotherapy performed after treating blood with ozone followed by reinfusion into the donor, may represent a valuable therapeutic approach for achieving immunoregulatory effects.     

Pep34, a synthetic peptide whose sequence corresponds to the intracytoplasmic domain of the Epstein-Barr virus receptor (CR2, CD21), regulates human B lymphocyte proliferation triggered through CR2

CR2 is involved in regulation of human B lymphocyte proliferation by interacting, through distinct domains, with extracellular, cell surface or intracellular components. Contribution of CR2 intracytoplasmic domain in CR2 regulatory functions remains unclear. Thus, we used pep34, a 34 amino acid synthetic peptide whose sequence corresponds to CR2 intracytoplasmic domain. Pep34 was incorporated into B lymphocytes which were then activated by EBV or C3d through CR2. Our data demonstrate that pep34 inhibits 100% B lymphocyte proliferation triggered by EBV or C3d. Irrelevant peptide had no effect. When B lymphocyte proliferation was triggered by a multipotent B cell activator as SAC, pep34 did not exert any inhibitory effect. Our data demonstrate that pep34 inhibits B lymphocyte proliferation only when lymphocytes are triggered through CR2. Thus, this strongly supports that despite its short length. CR2 intracytoplasmic domain participates to regulatory functions of this receptor.