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1.
J Mater Sci Mater Med ; 18(2): 255-63, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17323156

ABSTRACT

Bone morphogenetic proteins (BMPs) and 45S5 Bioglass microspheres (bioactive GM) can increase the differentiation of osteoblasts. Recombinant human BMP-2 (rhBMP-2) is presently the BMP most frequently used in delivery systems and it has already been used in clinical bone healing studies. We have developed a delivery system that combines a collagen Type I gel, BMP and bioactive GM. Since BMP-9 seems to be more osteogenic than BMP-2, we compared the differentiation of MC3T3-E1 preosteoblasts induced by our delivery system containing either a peptide derived from BMP-9 (pBMP-9), or rhBMP-2, both at 100 ng/mL. After 5 days, alkaline phosphatase staining showed that pBMP-9 induced more differentiation than rhBMP-2 in all experimental conditions. Also, bioactive GM increased this BMP effect. Since preosteoblasts secreted matrix metalloproteinases (MMPs) that can degrade collagen, we then studied the influence of the delivery system on MMPs production. We observed that MMP-2 was the major MMP involved in all experimental conditions. In addition, pBMP-9 with bioactive GM generated less MMP-2 than did rhBMP-2 on days 3 and 5. Thus, a delivery system using collagen Type I gel with pBMP-9 and bioactive GM seems to be a promising system for bone regeneration.


Subject(s)
Bone Morphogenetic Proteins/administration & dosage , Drug Delivery Systems/methods , Glass/chemistry , Osteoblasts/cytology , Osteoblasts/physiology , Osteogenesis/physiology , Transforming Growth Factor beta/administration & dosage , 3T3 Cells , Animals , Bone Morphogenetic Protein 2 , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Ceramics , Growth Differentiation Factor 2 , Growth Differentiation Factors , Humans , Materials Testing , Mice , Microspheres , Osteoblasts/drug effects , Osteogenesis/drug effects , Particle Size , Surface Properties , Tissue Engineering/methods
2.
Blood ; 95(8): 2610-6, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10753841

ABSTRACT

In this report we present a transgenic mouse model in which we targeted gene expression specifically to B-lymphocytes. Using the human CD19 promoter, we expressed major histocompatibility complex class II I-E molecules specifically on B cells of all tissues, but not on other cell types. If only B cells expressed I-E in a class II-deficient background, positive selection of CD4(+) T cells could not be observed. A comparison of the frequencies of I-E reactive Vbeta5(+) and Vbeta11(+) T cells shows that I-E expression on thymic B cells is sufficient to negatively select I-E reactive CD4(+) T cells partially, but not CD8(+) T cells. Thus partial negative but no positive selection events can be induced by B-lymphocytes in vivo. (Blood. 2000;95:2610-2616)


Subject(s)
B-Lymphocytes/immunology , Dendritic Cells/immunology , Histocompatibility Antigens Class II/genetics , Thymus Gland/immunology , Animals , Gene Expression Regulation/immunology , Gene Transfer Techniques , Histocompatibility Antigens Class II/immunology , Humans , Mice , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/immunology
3.
Eur J Immunol ; 29(5): 1729-39, 1999 05.
Article in English | MEDLINE | ID: mdl-10359128

ABSTRACT

The effects of whole genome duplications that characterize the evolution of vertebrates have been studied on the gene of the Xenopus thymocyte molecule CTX and its mammalian relatives. CTX, with an extracellular part consisting of one V and one C2 external domain, defines a new subset of the immunoglobulin superfamily and is conserved from amphibians to mammals. The number of CTX loci, their polymorphism, and their genetic linkages have been studied in several Xenopus species and in humans. In the genetically simplest species, X. tropicalis (2n = 20), the unique CTX locus is linked to the MHC. In the polyploid species, all CTX genes, unlike many other immune system genes, have remained in the genome; i.e. there are two CTX loci in the tetraploid species X. laevis (2n = 6) and six CTX loci in the dodecaploid species X. ruwenzoriensis (2n = 108). In X. laevis, one CTX gene is linked to the MHC and the other not, presumably because one set of MHC class I and II has been deleted from the corresponding linkage group. The various mammalian homologues are less related to each other than are the Xenopus CTX genes among each other, and they do not cross-hybridize with each other because they stem from the ancient polyploidization. Some human CTX homologies are on chromosomes 11 and 21, but others are on chromosomes 1, 6 and 19, which contain MHC paralogous regions; this suggests that a very ancient linkage group has been preserved.


Subject(s)
Genetic Linkage , Major Histocompatibility Complex , Membrane Proteins/genetics , Xenopus Proteins , Amino Acid Sequence , Animals , Antigens, Differentiation, T-Lymphocyte , Base Sequence , Chromosome Mapping , Gene Duplication , Humans , Mammals , Molecular Sequence Data , Polymorphism, Genetic , Sequence Homology, Amino Acid , Xenopus laevis/genetics
4.
Eur J Immunol ; 28(12): 4094-104, 1998 12.
Article in English | MEDLINE | ID: mdl-9862345

ABSTRACT

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) member comprising one variable and one constant C2-type Igsf domain, a transmembrane segment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, the variable domain is not encoded by somatic rearrangement of the gene but by splicing of two half-domain exons. The C2 domain, also encoded by two exons, has an extra pair of cysteines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70 amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chicken homologue of CTX, has the same structural and genetic features, and both molecules are expressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNA and genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similarity with recently described human cell surface molecules, A33 antigen and CAR (coxsackie and adenovirus 5 receptor), and a number of expressed sequence tags leads us to propose that CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extending beyond the immune system. Strong homologies within vertebrate sequences suggest that the V and C2 CTX domains are scions of a very ancient lineage.


Subject(s)
Membrane Proteins/genetics , T-Lymphocytes/immunology , Xenopus Proteins , Amino Acid Sequence , Animals , Antigens, Differentiation, T-Lymphocyte , Antigens, Surface/genetics , Antigens, Surface/immunology , Conserved Sequence , Humans , Immunoglobulins/genetics , Immunoglobulins/immunology , Membrane Proteins/immunology , Mice , Molecular Sequence Data , Sequence Alignment , Sequence Analysis , Xenopus
5.
Eur J Immunol ; 27(3): 763-71, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9079820

ABSTRACT

cDNA of the T cell receptor beta (TCRB) have been isolated from the anuran amphibian Xenopus and they show strong structural homology to TCRB sequences of other vertebrates. Ten BV families, two D segments, ten J segments, and a single C region have been defined so far. Each V family consists of one to two members per haploid genome. A unique feature of the Xenopus TCRB constant region is the lack of N-linked carbohydrate glycosylation sites. The recombination signal sequences suggest that the mechanism of rearrangements are identical to those of mammals. The locus is inherited in a diploid manner despite the pseudotetraploidy of the Xenopus laevis and X. gilli used in this study.


Subject(s)
Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Receptors, Antigen, T-Cell, alpha-beta/genetics , Xenopus laevis/genetics , Amino Acid Sequence , Animals , Blotting, Southern , Cloning, Molecular , Cysteine/chemistry , Gene Expression , Genes , Glycosylation , Molecular Sequence Data , Multigene Family , Phylogeny , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid , Sequence Alignment , Sequence Homology, Amino Acid
6.
Mol Immunol ; 34(2): 133-43, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9188846

ABSTRACT

CTX, a new Xenopus Ig superfamily molecule present on some cortical thymocytes and lymphoid tumor cells, is expressed at the cell surface under six differently glycosylated isoforms as shown by two-dimensional gel analysis and by endo F glycosidase treatment. Following chemical cross-linking before immunoprecipitation, a large fraction of surface CTX forms non-covalently linked dimers at the cell surface. This finding, which is consistent with the presence of a J segment with diglycine beta bulge in the V region of the molecule, suggests that this dimer has the same conformation as a T-cell receptor (TCR) or an Ig molecule. The V8 digest patterns of the monomers and dimers are identical. While this suggests that the dimer is a homodimer of two CTX chains, it does not distinguish whether each CTX chain is encoded by the same or different gene loci. When tumor cells were added to culture wells that had been coated with the anti-CTX monoclonal antibody X71, 30-50% underwent rapid (within 30 min) morphological changes followed by growth inhibition as determined by a decrease in thymidine incorporation and by direct cell counting. No apoptosis, calcium flux or external calcium requirement was noted after cross-linking of CTX. These results suggest that CTX can function as a receptor, and that its interaction with a ligand influences the control of cell proliferation through a signalling pathway that is distinct from the TCR machinery.


Subject(s)
Antigens, Surface/metabolism , Antineoplastic Agents/metabolism , Membrane Proteins/metabolism , Xenopus Proteins , Animals , Antibodies, Monoclonal/immunology , Antigens, Differentiation, T-Lymphocyte , Antigens, Surface/immunology , Antineoplastic Agents/immunology , Apoptosis , Calcium/metabolism , Cell Differentiation , Cell Division , Cell Survival , Cross-Linking Reagents/pharmacology , DNA Replication , Dimerization , Electrophoresis, Gel, Two-Dimensional , Glycosylation , Membrane Proteins/immunology , Surface Properties , Tumor Cells, Cultured , Xenopus laevis
9.
Eur J Immunol ; 26(4): 780-91, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8625968

ABSTRACT

CTX, a novel developmentally regulated type-I transmembrane protein is expressed specifically by a large fraction of cortical thymocytes in the amphibian Xenopus. This apparently monomeric 55-kDa glycoprotein is composed of two immunoglobulin domains, one variable (V) and one constant (C2 type), followed by a transmembrane and a 64-amino acid cytoplasmic domain. The first immunoglobulin domain is a V-J segment that is generated without gene rearrangement. In the genome, the V and C2 domains are both encoded by two half-domain exons. Two CTX loci are found per haploid genome, and they exhibit sequence differences with a high replacing/silent ratio in the CDR1-like region of the V domain, suggesting that these differences were selected. The cytoplasmic domain contains a motif that is highly conserved evolutionarily in several types of proteins, including adenylyl cyclases. Based on its unique tissue distribution, the variability of its V region and the motif of its cytoplasmic domain, CTX is a candidate for a new type of specific molecule involved in thymocyte selection.


Subject(s)
Antigens, Surface/biosynthesis , Membrane Proteins/isolation & purification , T-Lymphocytes/metabolism , Thymus Gland/metabolism , Xenopus Proteins , Xenopus laevis/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigens, Differentiation, T-Lymphocyte , Base Sequence , Biomarkers , Clonal Deletion , Consensus Sequence , DNA, Complementary/genetics , Exons/genetics , Gene Rearrangement, T-Lymphocyte , Genes , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Polymorphism, Genetic , Protein Conformation , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction
10.
J Immunol ; 154(11): 5620-9, 1995 Jun 01.
Article in English | MEDLINE | ID: mdl-7751615

ABSTRACT

The MHC class II-associated invariant chain (Ii) is involved in Ag processing and presentation. Physical association of MHC class II molecules with Ii and an effect of Ii on peptide loading to class II have been demonstrated, but to date these functions have not been related to a particular region of Ii. We investigated luminal deletion mutants of Ii and their role in Ag processing and presentation. IAk-expressing L cells were transfected with deletion mutants of the Ii gene and assayed for their ability to present hen egg lysozyme to three different T cell hybridomas. It is shown that the sequence aa 131-191 of Ii is important for the presentation of native hen egg lysozyme. In addition, this C terminal region is shown to be responsible for Ii oligomer formation. It is therefore conceivable that oligomer formation of Ii is a prerequisite for class II-restricted Ag processing and presentation.


Subject(s)
Antigen Presentation/immunology , Antigens, Differentiation, B-Lymphocyte , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Mutation/genetics , Animals , Base Sequence , Biopolymers/metabolism , DNA/biosynthesis , Egg Proteins/immunology , Histocompatibility Antigens Class II/metabolism , L Cells , Lymphocyte Activation/immunology , Mice , Molecular Sequence Data , Precipitin Tests , Recombinant Proteins/biosynthesis , Transfection
11.
Int Immunol ; 4(10): 1113-21, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1336977

ABSTRACT

MHC class II molecules are involved in the presentation of both exogenous and endogenous antigens to CD4 T cells. Using the trans-membrane hemagglutinin (HA) from measles virus and the secreted hen egg lysozyme (HEL) as antigen models, we have compared the efficiency of MHC class II presentation by naive antigen presenting cells (APCs) pulsed with exogenous antigen with that of their transfected counterparts synthesizing endogenous antigen. B cells expressing even a very low amount of trans-membrane HA were found to present endogenous HA to I-Ed restricted T cell hybridomas with a high efficiency whereas their naive counterparts required to be pulsed with a comparatively high amount of exogenous HA. Similarly, MHC class II presentation of endogenous secreted HEL was found to be much more efficient when compared with that of exogenous HEL. Biochemical studies did not reveal any enhanced intracellular degradation of endogenous HEL. As expected, HEL was released in the surrounding medium within < 1 h. MHC class II presentation of endogenous HEL could not be explained by re-uptake by bystander APCs of HEL secreted in the surrounding medium. No sensitization of naive APCs could be observed either when co-cultured with HEL secreting cells or when cultured for 10 days with a sub-threshold amount of exogenous HEL. At the cell surface, I-Ed molecules immunoprecipitated from HEL secreting cells were found to be slightly enriched in SDS-resistant forms. These data raised the question of how peptides derived from endogenous transmembrane and secreted antigens can so efficiently reach an MHC class II loading compartment.


Subject(s)
Antigen-Presenting Cells/immunology , Autoantigens/immunology , Histocompatibility Antigens Class II/immunology , Isoantigens/immunology , Mice/immunology , T-Lymphocytes/immunology , Animals , Hemagglutinins, Viral/immunology , Hybridomas/immunology , Membrane Proteins/immunology , Muramidase/immunology , Muramidase/metabolism , Receptors, Antigen, T-Cell, alpha-beta/immunology , Recombinant Fusion Proteins/immunology , Transfection
12.
J Allergy Clin Immunol ; 87(1 Pt 1): 58-69, 1991 Jan.
Article in English | MEDLINE | ID: mdl-1825101

ABSTRACT

Interleukin-4 (IL-4) has been demonstrated to induce IgE synthesis by peripheral blood mononuclear cells (PBMNCs) of healthy donors. In this study, we demonstrated that IL-4 also can enhance spontaneous IgE synthesis by PBMNCs of allergic/atopic patients and patients with Buckley's or hyper-IgE syndrome. Spontaneous IgE production by PBMNCs of these patients was suppressed by interferon (IFN)gamma or IFN-alpha in a dose-dependent fashion. Despite high serum IgE levels, no IL-4 could be detected in the serum of the patients, but indirect evidence was obtained indicating that enhanced IL-4 production in vivo may be associated with the high serum-IgE levels in these patients. Spontaneous IL-4 production was measured in PBMNC cultures of 4/21 patients tested. Furthermore, spontaneous IgE synthesis by PBMNCs of three other patients of seven tested in vitro was partly blocked by anti-IL-4 antiserum. In addition, levels of soluble CD23 (which is specifically induced by IL-4) were strongly elevated in sera of patients. Finally, activation of PBMNCs of the patients resulted in levels of IL-4 and of IFN-gamma synthesis that were higher and lower, respectively, than levels produced by PBMNCs of healthy control donors tested in parallel. Collectively, our data indicate that spontaneous IgE synthesis in vitro can be modulated by IL-4, IL-5, IFN-gamma, and IFN-alpha. In addition, our data suggest that enhanced IL-4 and reduced IFN-gamma production is associated with the elevated serum IgE levels observed in these patients.


Subject(s)
Hypergammaglobulinemia/metabolism , Immunoglobulin E/biosynthesis , Interferon-gamma/pharmacology , Interleukin-4/pharmacology , T-Lymphocytes/metabolism , Antigens, Differentiation, B-Lymphocyte/biosynthesis , Dose-Response Relationship, Drug , Gene Expression Regulation , Humans , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Immunoglobulin M/analysis , In Vitro Techniques , Interferon Type I/pharmacology , Interleukin-2/analysis , Interleukin-5/pharmacology , Receptors, Fc/biosynthesis , Receptors, IgE
13.
J Immunol ; 144(12): 4651-6, 1990 Jun 15.
Article in English | MEDLINE | ID: mdl-1972164

ABSTRACT

Lymphokine secretion profiles were studied of human allergen-specific CD4+ T lymphocyte clones (TLC). To this aim, panels of house dust mite Dermatophagoides pteronyssinus (Dp)-specific TLC were generated from two atopic Dp-allergic patients, suffering from severe atopic dermatitis (AD1) and allergic asthma (AD2), respectively, and from a non-atopic individual (NAD). From AD1 additional TLC were cloned specific for tetanus toxoid or Candida albicans, both Ag that were not relevant for the atopic state of this patient. Secretion of IL-2, IL-4, and IFN-gamma was determined after specific stimulation of these TLC, using autologous monocytes as APC. With respect to the production of IL-4 and IFN-gamma, clearly distinct profiles were observed. All Dp-specific TLC from both atopic donors produced IL-4 but not IFN-gamma, whereas the Dp-specific TLC from NAD, as well as the tetanus toxoid- and C. albicans-specific TLC from AD1, all produced IFN-gamma but not or small quantities of IL-4. Most TLC from all panels produced IL-2. These lymphokine profiles were consistent for at least 3 days and were neither dependent on the dose of allergen nor on the atopic or nonatopic state of the donor of APC. The functional consequence of these restricted lymphokine profiles was stressed by the observation that, whereas Dp-specific TLC from AD1 and AD2 supported in vitro IgE production, this support could be abrogated by a Dp-specific TLC from NAD. The present results suggest that CD4+ T lymphocytes that produce IL-4, but not IFN-gamma, occur in high frequencies in the allergen-specific T cell repertoires of atopic donors, which may have important implications for the pathomechanism of atopic disease.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Hypersensitivity/immunology , Lymphokines/biosynthesis , Allergens/immunology , Animals , Antigen-Presenting Cells/physiology , Candida albicans/immunology , Clone Cells , Humans , Immunoglobulin E/biosynthesis , Interferon-gamma/biosynthesis , Interleukin-2/biosynthesis , Interleukin-4/biosynthesis , Lymphocyte Activation , Mites/immunology , Tetanus Toxoid/immunology
14.
Eur J Immunol ; 20(2): 243-51, 1990 Feb.
Article in English | MEDLINE | ID: mdl-2138081

ABSTRACT

In the present study culture conditions resulting in optimal IgE synthesis by mononuclear cells (MNC) isolated from peripheral blood, tonsils or spleens from healthy nonallergic donors were investigated. The highest rate of IgE synthesis was obtained in a two-step culture system in which the MNC were preincubated with interleukin 4 (IL4; 200 U/ml) for 48 h, washed and subsequently incubated with IL4 (200 U/ml) for 9 days. Despite these culture conditions, IL4-induced IgE synthesis varied considerably (1-150 ng/ml) and MNC from 16/70 donors failed to produce IgE. Kinetic studies indicated that IL4 was required at the onset of the incubation phase. IgE synthesis was reduced by greater than 95% when addition of IL4 in the incubation period was delayed 24 h or more. IL4-induced IgE synthesis was blocked by interferon-gamma (IFN-gamma). This inhibition is most effective when IFN-gamma was added in the 48-h preincubation step or during the first 48 h of the incubation period. Interestingly, IL4 was found to block spontaneous and lectin- or factor-induced IFN-gamma production by MNC, purified CD3+, CD4+ or CD8+ Tcells. This down-regulatory effect of IL4 on IFN-gamma production occurred at the mRNA transcription level. Furthermore, it is shown that IL4 induced the release of soluble CD23 and that recombinant soluble CD23 enhanced IL4-induced IgE synthesis, but only when IL4 was present at suboptimal concentrations. Collectively, our data indicate that IL4 and IFN-gamma regulate the level of IgE synthesis by influencing each other's activities reciprocally during the first 3 days of the culture.


Subject(s)
Antigens, CD/physiology , Antigens, Differentiation, B-Lymphocyte/physiology , Immunoglobulin E/biosynthesis , Interferon-gamma/pharmacology , Interleukin-4/pharmacology , Receptors, Fc/physiology , Cells, Cultured , Humans , In Vitro Techniques , Interferon-gamma/biosynthesis , Receptors, IgE , Recombinant Proteins , Solubility , T-Lymphocytes/metabolism , Time Factors
15.
J Immunol ; 144(3): 875-82, 1990 Feb 01.
Article in English | MEDLINE | ID: mdl-1967277

ABSTRACT

We had previously shown that the signal of activation delivered via CD2 varies according to the mitogenic pair of CD2 mAb used. We had selected two typical mAb pairs, D66 + T11(1) and GT2 + T11(1), the former delivering the "richest" signal, the latter the poorest. Here we analyzed the cytolytic activities generated within PBL-stimulated by these two pairs. When purified CD2+,3- cells were cultured with either one of these two pairs, no significant lymphokine-activated killer (LAK) activity--namely the activity exerted on NK-resistant malignant cell lines or fresh tumor cells--was detected, thereby demonstrating the inability of CD2 mAb pairs to directly trigger the LAK precursors. By contrast, when purified CD2+,3+ cells were cultured, only D66 + T11(1) was able to trigger a potent CTL activity, as judged by targeting their activity, at the effector phase, with a bridging CD3 mAb on a FcR+ target cell or by using heteroaggregates on FcR- malignant cells. When whole PBL were used, a similar and moderate LAK activity was generated after culture with either one of the 2 CD2 mAb pairs. This, in fact, masked quite different events. The amounts of endogeneous IL-2 released in PBL cultures with GT2 + T11(1) was rather low, although it was sufficiently high in PBL cultures with D66 + T11(1) to generate a potent LAK activity. Yet, PBL stimulated with D66 + T11(1) released concomitantly a high amount of IL-4 which inhibited the development of the LAK activity, as demonstrated by unmasking this activity with an anti-IL4 antiserum and which did not inhibit the T CTL activity; this IL-4 secretion was not seen with GT2 + T11(1). Therefore, stimulation by these two typical CD2 mAb pairs induce a striking different pattern of IL synthesis.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/physiology , Cytotoxicity, Immunologic , Killer Cells, Lymphokine-Activated/immunology , Lymphocyte Activation , Receptors, Immunologic/physiology , T-Lymphocytes, Cytotoxic/immunology , Antigen-Antibody Reactions , Antigens, CD/physiology , Antigens, Differentiation, T-Lymphocyte/analysis , CD2 Antigens , CD3 Complex , Cells, Cultured , Humans , In Vitro Techniques , Interleukin-2/pharmacology , Interleukin-4/biosynthesis , Receptors, Antigen, T-Cell/analysis
16.
J Cell Biochem ; 39(3): 253-64, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2496134

ABSTRACT

IL-4 specifically induced IgE production by peripheral blood lymphocytes or by tonsil or spleen cells from healthy donors. IL-4-induced IgE synthesis was dependent on CD4+ T cells and monocytes and was blocked by IFN-gamma, IFN-alpha, and prostaglandin E-2 (PGE-2). These substances also inhibited IL-4-induced CD23 expression and subsequent release of soluble CD23 (s-CD23). In addition, IgE production was blocked by F(ab')2 fragments of an mAb against CD23. In contrast, IL-5 enhanced IL-4-induced IgE production, provided IL-4 was added at nonsaturating concentrations. This increase in IgE production correlated quantitatively with an enhanced release of s-CD23. Collectively, these results indicate that there is a correlation between s-CD23 release and IgE production. However, s-CD23 fractionated from supernatants of the lymphoblastoid cell line RPMI-8866 was ineffective in inducing IgE production in the absence of IL-4, but acted synergistically with suboptimal concentrations of IL-4. In addition, it is demonstrated that alloreactive T-cell clones produced varying concentrations of IL-4, IL-2, or IFN-gamma upon stimulation. Only supernatants of 2/4 of these T-cell clones induced a low degree of IgE synthesis, but in the presence of anti-IFN-gamma antibodies, all four supernatants induced a strong induction of IgE production. This IgE synthesis was blocked specifically by anti-IL-4 antibodies, indicating that IL-4 is the sole inducer of IgE synthesis. Our findings demonstrate that IL-4-induced IgE production involves complex interactions of T cells, B cells, and monocytes and is positively modulated by IL-5 and s-CD23 but down-regulated by IFN-gamma, IFN-alpha, and PGE-2, respectively.


Subject(s)
Immunoglobulin E/biosynthesis , Interferon-gamma/immunology , Interleukins/immunology , Antibodies, Monoclonal/immunology , Dinoprostone/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-4 , Interleukin-5 , Interleukins/analysis , Isoantigens/immunology
17.
J Immunol Methods ; 117(1): 67-81, 1989 Feb 08.
Article in English | MEDLINE | ID: mdl-2643668

ABSTRACT

A rabbit antiserum to partly purified recombinant E. coli-expressed human interleukin-4 (IL-4) has been produced which neutralizes the T cell growth factor, B cell growth factor, and Fc epsilon R2/CD23 inducing activities of IL-4. The antiserum demonstrated sufficient avidity to immunoprecipitate labelled COS7-expressed recombinant human IL-4. In contrast, rabbits immunized with conjugates of various synthetic IL-4 oligopeptides produced antisera which recognized IL-4 in both enzyme-linked immunosorbent assay (ELISA) and Western blotting formats, but failed to immunoprecipitate IL-4 from solution, or to neutralize bioactivity. Two rat monoclonal antibodies, 11B4, 22C10 were produced from a rat immunized with purified COS7 cell-expressed IL-4. These IgG2a antibodies recognized both E. coli-expressed and mammalian cell-expressed (COS7 and L cell) recombinant human IL-4 in solution (immunoprecipitation), as well as on solid phase (indirect ELISA and dot-blotting). The 11B4 antibody inhibited IL-4 bioactivity at an IC50 which was 25-50-fold in molar excess of factor. Both antibodies also recognized IL-4 bound to an immobilized rabbit IgG fraction of anti-IL-4. The 11B4 antibody was used to develop an immunoenzymetric assay capable of detecting less than 100 pg of analyte/ml. Supernatants from PBL, activated under varying conditions were tested for IL-4 levels. PHA and ConA were found to induce a relatively low degree of IL-4 production by these PBL. An approximately ten-fold greater level of IL-4 production was observed when they were stimulated with A23187 in combination with PMA. Various patient sera and cell line supernatants were also tested. These IL-4 immunoreagents are important tools for further studies of IL-4 immunobiology.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Immune Sera , Immunoenzyme Techniques , Interleukins/immunology , Neutralization Tests , Amino Acid Sequence , Animals , Antibodies, Monoclonal/analysis , Humans , Immune Sera/analysis , Immune Sera/pharmacology , Interleukin-4 , Interleukins/chemical synthesis , Interleukins/isolation & purification , Male , Molecular Sequence Data , Rabbits , Rats , Rats, Inbred Lew , Recombinant Proteins/chemical synthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification
18.
Autoimmunity ; 4(4): 267-76, 1989.
Article in English | MEDLINE | ID: mdl-2518830

ABSTRACT

T lymphocytes present in thyroid infiltrates of 6 patients with Hashimoto's thyroiditis (HT) and of 4 patients with Graves' disease (GD) were analyzed at clonal level and their profiles of mitogen-induced lymphokine secretion were characterized. Production of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-gamma (IFN-gamma) was measured in culture supernatants of a total number of 332 T cell clones (TCC) from HT, of 269 TCC from GD infiltrates and of 266 control TCC derived from normal lymphoid tissues. No significant difference was found in the ability to produce IL-2 between TCC from HT or GD infiltrates and control TCC. The proportion of HT- or GD-derived TCC able to produce IL-4 was extremely low (4 and 5%, respectively) in comparison with controls (19%). In contrast, the proportion of interferon-gamma (IFN-gamma)-producing (IFN-P) TCC derived from either HT (87%) or GD (80%) infiltrates was much higher (p less than 0.0005) than that found in controls (59%). In addition, most of IFN-P TCC from either HT or GD usually released higher amounts (p less than 0.002) of IFN-gamma than did control clones. No significant difference was found between GD infiltrates and controls in the proportions of TCC able to secrete TNF-alpha (39% and 47%, respectively), whereas the proportion of TNF-alpha-producing (TNF-P) TCC derived from HT (78%) was significantly higher (p less than 0.0001). In addition, most of both CD8 and CD4 TCC from HT released higher amounts of TNF-alpha than did TNF-P clones from controls or GD. These data suggest that T cells present in autoimmune thyroid infiltrates share a number of functions, such as high production of IFN-gamma, but differ with regard to their ability to secrete TNF-alpha, which is peculiar of most T cells present in the thyroid of HT patients.


Subject(s)
T-Lymphocytes/immunology , Thyroid Gland/immunology , Thyroiditis, Autoimmune/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Antigens, Differentiation, T-Lymphocyte , Autoimmunity , CD4 Antigens , CD8 Antigens , Clone Cells/immunology , Graves Disease/immunology , Graves Disease/pathology , Humans , Interferon-gamma/biosynthesis , Interleukin-2/biosynthesis , T-Lymphocytes/pathology , Thyroid Gland/pathology , Thyroiditis, Autoimmune/pathology
19.
Int Arch Allergy Appl Immunol ; 88(1-2): 203-5, 1989.
Article in English | MEDLINE | ID: mdl-2468614

ABSTRACT

To explore the binding domains of rCIgE(301-376) necessary for inhibition of passive transfer of rye grass and Chinese elm IgE to human basophils, we employed monoclonal antibodies known to bind to IgE(301-336), M-272, and to IgE(367-376), M-27. By preincubating M-272, but not M-27, with rCIgE(301-376), passive transfer of specific IgE to basophils was partially inhibited. This implies that M-272 recognizes a binding site on rCIgE(301-376) or sterically interferes with the rCIgE(301-376) high-affinity binding domains on human basophils.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Antigens, Differentiation, B-Lymphocyte/metabolism , Basophils/immunology , Histamine Release , Immunoglobulin E/immunology , Receptors, Fc/metabolism , Allergens , Antigen-Antibody Reactions , Humans , Immunization, Passive , In Vitro Techniques , Peptide Fragments , Receptors, IgE , Recombinant Proteins/immunology , Structure-Activity Relationship , Trees
20.
J Immunol ; 141(9): 3128-34, 1988 Nov 01.
Article in English | MEDLINE | ID: mdl-2459242

ABSTRACT

We have produced three different mAb specific for human IgE-Fc. Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule. All three mAb were able to induce basophil degranulation as measured by the induction of histamine release. mAb 173 recognizes a thermolabile epitope in the CH4 domain. It does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain. This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides. mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23. These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.


Subject(s)
Antibodies, Anti-Idiotypic/physiology , Antibodies, Monoclonal/physiology , Antigens, Differentiation, B-Lymphocyte/metabolism , Epitopes/immunology , Immunoglobulin E/immunology , Immunoglobulin E/metabolism , Peptide Fragments/immunology , Receptors, Fc/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity , Antigen-Antibody Reactions , Antigens, Differentiation, B-Lymphocyte/immunology , Binding, Competitive , Humans , Immunoglobulin E/physiology , Mice , Mice, Inbred BALB C , Peptide Mapping , Receptors, Fc/immunology , Receptors, IgE , Recombinant Proteins/immunology , Structure-Activity Relationship
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