RESUMEN
A novel type of bioactive transforming growth factor beta 2 (TGF beta 2)-related immunosuppressive activity, lower in molecular mass (20-23 kDa) than a conventional TGF beta 2 standard (25 kDa), has been shown to be released by non-T non-B suppressor cells of murine decidua into fetal bovine serum (FBS)-containing tissue culture medium during a 48-h incubation at 37 degrees C. Substitution of a serum-free medium has allowed direct PAGE-Western blotting and the demonstration in supernatant (prior to incubation at 37 degrees C) of a TGF beta 2-immunoreactive doublet at a higher molecular mass (26 and 27 kDa) than the standard but with apparent immunosuppressive activity. When decidua were incubated in serum-free medium at 37 degrees C, immunosuppressive activity in the supernatant increased to peak at 18 h in association with the appearance of the previously described lower molecular mass species of molecule. The doublet did not appear to be the result of glycosylation of a conventional 25-kDa TGF beta 2 and could be converted into the lower molecular mass form by incubation for 18 h at 37 degrees C, with pH 4.5 but not with pH 7.5 buffer; this incubation in the absence of decidual cells was not accompanied by increased immunosuppressive activity. This transformation could be blocked by a brief heating of the supernatant to 80 degrees C for 10 min to destroy enzymes present in supernatant prior to mixing with the acidic buffer, but biologic activity neutralizable by anti-TGF beta 2 was retained.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Decidua/metabolismo , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Animales , Western Blotting , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Femenino , Sangre Fetal , Glicosilación , Calor , Concentración de Iones de Hidrógeno , Cinética , Ratones , Ratones Endogámicos C3H , Peso Molecular , Embarazo , Procolágeno/biosíntesisRESUMEN
OBJECTIVE: To examine the effect of glycosylated recombinant human tumor necrosis factor binding protein-1 (r-hTNF binding protein-1), the extracellular domain of the tumor necrosis factor receptor p55 produced in mammalian cells, in a rabbit model of circulatory shock due to Escherichia coli. DESIGN: Prospective, randomized, controlled trial. SETTING: University hospital research laboratory. SUBJECTS: Eighteen female, New Zealand white rabbits. INTERVENTIONS: Anesthetized rabbits, infused with E. coli (10(9) organisms/kg), were pretreated with either r-hTNF binding protein-1 or saline. Mean arterial pressure, central venous pressure, cardiac output, and heart rate were recorded every 20 mins for 1 hr before, and for 4 hrs after, the infusion of E. coli. Blood samples were obtained at 1-hr intervals for platelet count and white blood cell count, r-hTNF binding protein-1, and tumor necrosis factor (TNF) measurements. MEASUREMENTS AND MAIN RESULTS: Administration of r-hTNF binding protein-1 resulted in improvement of mean arterial pressure, cardiac output, and systemic vascular resistance, as compared with the vehicle-treated group (p < .05). Treatment with r-hTNF binding protein-1 was associated with 100% survival, as compared with 55.6% of the saline-treated rabbits (p < .05). Approximately 85% of r-hTNF binding protein-1 was cleared from the circulation 1 hr after the bolus injection (from 171 +/- 27 micrograms/mL at time = 0, to 27 +/- 4 micrograms/mL at 60 mins, decreasing to 6 +/- 2 micrograms/mL for the next 3 hrs). The r-hTNF binding protein-1-treated rabbits had lower serum TNF bioactivity during the first 2 hrs (p < .01). The decreased bioactivity of TNF was confirmed by a specific radioimmunoassay for rabbit TNF. However, at 4 hrs, the vehicle-treated rabbits had lower serum bioactive TNF concentrations (p < .05). The decrease in TNF concentrations in the r-hTNF binding protein-1-treated rabbits resulted from decreased production and, in part, from carry-over of r-hTNF binding protein-1 into the bioassay. CONCLUSIONS: Treatment with r-hTNF binding protein-1 improved hemodynamic variables and survival of E. coli-challenged rabbits. Administration of r-hTNF binding protein-1 suppressed bioactivity of TNF in the circulation of these rabbits, and the production of TNF as well.
Asunto(s)
Proteínas Portadoras/uso terapéutico , Infecciones por Escherichia coli/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral , Choque Séptico/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Evaluación Preclínica de Medicamentos , Infecciones por Escherichia coli/sangre , Femenino , Estudios Prospectivos , Conejos , Distribución Aleatoria , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/uso terapéutico , Choque Séptico/sangre , Choque Séptico/microbiología , Análisis de Supervivencia , Receptores Señuelo del Factor de Necrosis TumoralRESUMEN
Hematopoietic lineage-restricted stem cell growth has been shown to be significantly inhibited by the addition of exogenous transforming growth factor-beta (TGF-beta) to Dexter-type long-term murine bone-marrow cultures. In order to examine whether TGF-beta produced by these cells has a role in hematopoietic growth regulation, Dexter cultures have been treated with either 1D11.16, a monoclonal antibody that neutralizes the biological activity of TGF-beta types 1, 2, and 3, or with a control antibody. The composition and cellularity of the nonadherent cell populations in these cultures were assessed weekly. Treatment with anti-TGF-beta antibody resulted in a five- to 20-fold increase in nonadherent cells in the cultures when compared to either the control or untreated cultures by week 4. The majority of these cells were granulocyte/macrophage-lineage cells as assessed by histologic and flow-cytometric analysis. There was also a significant increase of megakaryocytes in cultures treated with anti-TGF-beta antibody. Stem-cell analysis, using a colony-forming unit-spleen (CFU-S) assay that combined both the adherent and nonadherent populations from either 4- or 6-week cultures, showed that there are an equivalent number of hematopoietic stem cells per 10(6) cells regardless of antibody treatment. Therefore, cultures treated with anti-TGF-beta antibody contained at least three times as many stem cells as the control cultures. Finally, kinetics studies show that the presence of anti-TGF-beta antibody is required from the onset of culture to produce these effects. These results suggest that TGF-beta is involved in normal growth regulation of bone-marrow hematopoietic cells. By addition of a neutralizing antibody, the normal TGF-beta negative growth signal is disrupted, allowing for expanded growth of several cell populations.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales , Adhesión Celular , División Celular , Ensayo de Unidades Formadoras de Colonias , Granulocitos/citología , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Factores de TiempoRESUMEN
Interleukin 10 (IL-10) suppressed TGF-beta synthesis in mouse bone marrow cultures. Coincidingly, IL-10 down-regulated the production of bone proteins including alkaline phosphatase (ALP), collagen and osteocalcin, and the formation of mineralized extracellular matrix. The mAb 1D11.16 which neutralizes TGF-beta 1 and TGF-beta 2, induced suppressive effects comparable to IL-10 when administered before the increase of cell proliferation in the culture. It appears that mainly TGF-beta 1 plays a role in this system since (a) TGF-beta 2 levels were undetectable in supernatants from osteogenic cultures, (b) no effect was observed when the anti-TGF-beta 2 neutralizing mAb 4C7.11 was added and (c) the suppressive effect of IL-10 could be reversed by adding exogenous TGF-beta 1. It is unlikely that TGF-beta 1 modulates osteogenic differentiation by changing the proliferative potential of marrow cells since 1D11.16 did not affect [3H]thymidine ([3H]TdR) incorporation or the number of fibroblast colony forming cells (CFU-F) which harbor the osteoprogenitor cell population. Furthermore, 1D11.16 did not alter [3H]TdR uptake by the cloned osteoprogenitor cell lines MN7 and MC3T3. Light and scanning electron microscopy showed that IL-10 and 1D11.16 induced comparable morphological changes in the marrow cultures. Control cultures contained flat adherent cells embedded in a mineralized matrix. In contrast, IL-10 and 1D11.16 treated cultures were characterized by round non-adherent cells and the absence of a mineralized matrix. In this study, the mechanism by which IL-10 suppresses the osteogenic differentiation of mouse bone marrow was identified as inhibition of TGF-beta 1 production which is essential for osteogenic commitment of bone marrow cells.
Asunto(s)
Células de la Médula Ósea , Interleucina-10/fisiología , Osteogénesis/fisiología , Factor de Crecimiento Transformador beta/biosíntesis , Fosfatasa Alcalina/metabolismo , Animales , Médula Ósea/ultraestructura , Diferenciación Celular , División Celular , Células Cultivadas , Colágeno/metabolismo , Regulación hacia Abajo , Matriz Extracelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Osteocalcina/metabolismoRESUMEN
Addition of transforming growth factor-beta 1 (TGF-beta 1) to in vitro cultures of murine B cells activated with bacterial LPS selectively stimulates IgG2b and IgA class switching and decreases cellular proliferation. To assess a possible role for endogenous TGF-beta in modulating the Ig isotypes produced by LPS-activated cells, we utilized a neutralizing anti-TGF-beta mAb to abrogate endogenous TGF-beta activity. Anti-TGF-beta antibody, over a range of relatively low cell densities, strikingly inhibited both IgG3 and IgG2b production in response to LPS, with little or no change in the concentrations of secreted IgM. This effect of anti-TGF-beta antibody was specific, since it did not occur with an isotype-matched control mAb and was completely reversed with exogenous TGF-beta 1. Optimal IgG3 secretion occurred at concentrations of TGF-beta that were approximately eightfold lower than that necessary for maximal synthesis of IgG2b. Neutralization of endogenous TGF-beta in LPS-activated cultures was associated with an approximately twofold increase in proliferation and viable cell yields, a modest decrease in the percentage of membrane (m)IgG2b+ cells, and a modest increase in the percentage of mIgG3+ cells. This latter finding indicated that TGF-beta was not required for IgG3 class switching, but for maturation of mIgG3+ cells into Ig secretors. Highly purified B cells, obtained by electronic cell sorting, released active TGF-beta in response to LPS and showed a similar marked reduction in LPS-mediated IgG3 and IgG2b secretion in the presence of anti-TGF-beta antibody. Abrogation of endogenous TGF-beta activity in LPS-activated cultures also resulted in a striking reduction in IFN-gamma-mediated IgG2a production, and a more modest decrease in the synthesis of IgG1 and IgE in the presence of IL-4. These data indicate that relatively low concentrations of TGF-beta are essential for stimulating optimal IgG secretion by LPS-activated B cells, in an Ig isotype-nonspecific manner, and may regulate these responses in an autocrine fashion.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina G/biosíntesis , Lipopolisacáridos/farmacología , Activación de Linfocitos , Factor de Crecimiento Transformador beta/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/metabolismo , Células Cultivadas , Femenino , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/clasificación , Interferón gamma/farmacología , Interleucina-4/farmacología , Ratones , Ratones Endogámicos DBARESUMEN
We have investigated the distribution of TGF beta using antibodies specific for its intracellular and extracellular forms in full-thickness biopsies of patients with SSc, primary Raynaud's phenomenon (PRP), systemic lupus erythematosus (SLE), and from normal subjects. Nine of 11 SSc biopsies demonstrated intracellular TGF beta in endothelial cells while only 6 exhibited extracellular TGF beta. Endothelial cells in skin biopsies of all PRP patients displayed both intracellular and extracellular TGF beta. All other control biopsies were negative. In patients with PRP, some positively staining fibroblasts were found scattered throughout the dermis. Lastly, extracellular TGF beta was localized in the papillary dermis of PRP and SSc biopsies and in all the dermal layers of SLE patients. No significant staining of TGF beta was observed in the endothelial cells, fibroblasts, or in the extracellular matrix of the majority of biopsies from normal subjects. These data suggest that TGF beta may be one of the cytokines involved in the early stages of pathogenesis of SSc, and that endothelial cells in SSc and PRP may be a source and/or a target of TGF beta.
Asunto(s)
Enfermedad de Raynaud/metabolismo , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Anciano , Espacio Extracelular/metabolismo , Femenino , Histocitoquímica , Humanos , Persona de Mediana Edad , Enfermedad de Raynaud/patología , Esclerodermia Sistémica/patologíaRESUMEN
Bacterial lipopolysaccharide (LPS) has been reported to induce immunoglobulin (Ig)G2b class switching, yet we observed strain differences in IgG2b secretion in response to this mitogen. Specifically, BALB/c B cells, unlike those from DBA/2, synthesized relatively low amounts of IgG2b relative to IgG3, IgG1, or IgM. This report demonstrates that transforming growth factor (TGF) beta 1, previously shown to induce IgA class switching, selectively stimulates IgG2b secretion by BALB/c resting B cells activated with LPS. This activity was specifically reversed with a neutralizing anti-TGF-beta 1 antibody. The ability of TGF-beta 1 to act directly on highly purified membrane (m)IgM+ mIgG2b- cells to stimulate IgG2b production, stimulate an increase in IgG2b-secreting cells, and selectively increase the steady-state levels of germline gamma 2b RNA, suggests that it promotes IgG2b class switching. In this regard, addition of anti-TGF-beta antibody to cultures of DBA/2-derived resting B cells activated by LPS, alone, led to selective reduction in IgG2b secretion, indicating that endogenous TGF-beta 1 accounts for the high IgG2b secretory response observed in that strain. Finally, TGF-beta 1 failed to stimulate IgG2b secretion by B cells activated with dextran-conjugated anti-IgD antibody. We propose that TGF-beta 1 is a switch factor for the murine IgG2b subclass for appropriately activated B cells. In combination with other data, this would show that all six non-IgM, non-IgD isotypes in the mouse can be selectively induced by specific cytokines.
Asunto(s)
Linfocitos B/metabolismo , Inmunoglobulina G/metabolismo , Lipopolisacáridos/farmacología , Factor de Crecimiento Transformador beta/fisiología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Células Cultivadas , Femenino , Inmunoglobulina G/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBARESUMEN
Six monoclonal antibodies (mAb) are described that bound to the bovine bone glycoprotein, osteoglycin. The protein osteoglycin was originally called Osteoinductive Factor (OIF). The antibodies were characterized with respect to their reaction patterns in Western blots, indirect immunoprecipitation, and binding epitope. The antibodies bound to one of two sequential determinants, either residues 62-76 or 95-105, in the C-terminal region of mature osteoglycin. One mAb, 2C11, was found to be useful for affinity purification of osteoglycin. Another mAb, 3B2, was able to immunohistochemically stain osteoblasts, osteocytes, chondrocytes, occasional osteoclasts and nail bed epithelial cells in rat neonatal forelimb. The mAbs will provide an essential tool for the further characterization of this unique glycoprotein.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Huesos/química , Glicoproteínas/análisis , Glicoproteínas/inmunología , Animales , Western Blotting , Huesos/citología , Huesos/inmunología , Bovinos , Ensayo de Inmunoadsorción Enzimática , Epítopos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Osteoblastos/química , Osteoblastos/citología , Osteoblastos/inmunología , Osteocitos/química , Osteocitos/citología , Osteocitos/inmunología , Pruebas de PrecipitinaRESUMEN
Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Sinovitis/prevención & control , Factor de Crecimiento Transformador beta/fisiología , Enfermedad Aguda , Animales , Resorción Ósea , Enfermedad Crónica , Femenino , Ratas , Ratas Endogámicas Lew , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
OBJECTIVE: To demonstrate expression of transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) by cultured rheumatoid arthritis (RA) synovial cells and to investigate their role as synovial cell mitogens. METHODS: Polypeptide growth factors were detected and identified by immunocytochemical staining and Western blot analysis. Messenger RNA (mRNA) transcripts encoding TGF beta and bFGF were identified by polymerase chain reaction analysis. The influence of neutralizing growth factor monoclonal antibodies (MAb) on RA synovial cell growth was investigated. TGF beta bioactivity was determined by Mv1Lu assay. RESULTS: Lysates of RA, as compared with normal, synovial cells contained greater amounts of TGF beta and bFGF. Western blot analysis identified a single TGF beta band (MW approximately 25 kd) in each of the cell lysates examined. Western blot analysis using MAb DE6 identified a doublet of bFGF bands (MW approximately 18.0 kd) in normal synovial cell lysates and 4 bFGF bands (MW approximately 18.0, 22.0, 22.6, and 25.2 kd) in RA synovial cell lysates. RA and normal synovial cells expressed mRNA transcripts encoding TGF beta 1 but not TGF beta 2, and FGF-2 (basic FGF). Additional mRNA transcripts encoding FGF-5 and FGF-7 were expressed by RA, but not normal, synovial cells in culture. In contrast to MAb 1D11.16, which caused a dose-dependent decrease in RA synovial cell growth, MAb DG2 (up to 100 micrograms/ml) had no effect on cell growth. CONCLUSION: RA and normal synovial cells cultured in serum-free medium express TGF beta 1 and native bFGF. However, only RA synovial cells in culture express higher molecular weight isoforms of bFGF. TGF beta 1 appears to regulate synovial cell growth in vitro through an external autocrine loop. Despite expression of high-affinity bFGF receptors on cultured synovial cells, the mechanisms by which bFGF modulates synovial cell growth are unknown.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Membrana Sinovial/patología , Factor de Crecimiento Transformador beta/metabolismo , Artritis Reumatoide/patología , Secuencia de Bases , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Humanos , Inmunohistoquímica , Sondas Moleculares/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Membrana Sinovial/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Transforming growth factor beta (TGF-beta) 1 and 2 have both become increasingly important in cutaneous biology, but their expression and distribution in human skin are not entirely clear. In this report, normal forearm skin from four volunteers was investigated for TGF-beta 1 and beta 2 immunostaining with antibodies that detect preferentially either cell- or matrix-associated forms of these peptides. Marked cell-associated TGF-beta 1 was found in the dermis, particularly around blood vessels and ducts; cellular TGF-beta 2 immunostaining was less prominent, and was predominantly around blood vessels. Neither TGF-beta 1 nor -beta 2 could be detected in the epidermis or epithelial structures, and the dermal matrix contained minimally detectable amounts of the two isoforms. In all cases, dermal matrix and cells contained greater amounts of TGF-beta 1 than TGF-beta 2. Previous studies have shown that both TGF-beta 1 and -beta 2 can induce dramatic increases in extracellular matrix, and both peptides have been implicated in the pathogenesis of fibrosis. We therefore investigated TGF-beta 1 and -beta 2 immunostaining in involved forearm skin of four patients with systemic sclerosis. Compared to normal skin, fibrotic specimens showed increased amounts of matrix and epidermal TGF-beta 1, but not TGF-beta 2. We conclude that TGF-beta 1 and -beta 2 expression in human skin is differentially regulated, and that their distribution is varied and complex.
Asunto(s)
Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/metabolismo , Distribución Tisular , Factor de Crecimiento Transformador beta/genéticaRESUMEN
A unique form of transforming growth factor-beta (TGF-beta), TGF-beta 2.3 heterodimer, has been purified from bovine bone extract. TGF-beta 2.3 migrated as a single 25-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas under reducing conditions it migrated as a 12.5 kDa band. The TGF-beta 2.3 reacted positively with anti-TGF-beta 2 and anti-TGF-beta 3 antibodies on immunoblots. Equal levels of TGF-beta 2 and TGF-beta 3 sequences were detected by N-terminal sequencing. TGF-beta 2.3 eluted as a single sharp peak by reverse-phase high performance liquid chromatography. However, prior reduction of the protein with dithiothreitol resulted in the protein eluting in two peaks, one containing predominantly TGF-beta 3 and the other containing predominantly TGF-beta 2. TGF-beta 2.3 inhibited proliferation of mink lung epithelial cells and promoted the formation of colonies of normal rat kidney fibroblasts in culture with specific biological activity similar to those of TGF-beta 1 and TGF-beta 2. These results demonstrate that the protein is TGF-beta 2.3 heterodimer, consisting of one polypeptide chain each of TGF-beta 2 and TGF-beta 3 linked by one or more disulfide bonds. In addition, TGF-beta 1.2 heterodimer, previously found only in porcine platelets, has also been purified from bovine bone extract.
Asunto(s)
Huesos/química , Factor de Crecimiento Transformador beta/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Western Blotting , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Visón , Datos de Secuencia Molecular , Ratas , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Three isoforms of the transforming growth factor beta (TGF-beta) family, TGF-beta 1, TGF-beta 2, and TGF-beta 3, bind specifically and with high affinity to several cell surface components known as type I, type II, and type III proteins. The type I and II proteins may serve as biological receptors, whereas the type III protein does not appear to be associated with TGF-beta-mediated cell responses, and its function remains unknown. Binding data on confluent monolayers of rat skeletal myoblasts of the L6 cell line reveals two novel patterns of TGF-beta 1 binding. Saturation of the type I receptor with native TGF-beta 2 induces a 7-fold increase in binding of radiolabeled TGF-beta 1 at the type II protein. No induction of type II receptor binding was observed on subconfluent cells indicating a density-dependent phenomenon. The data suggest that the type I and type II proteins may interact during ligand binding in a manner which may be indicative of a regulatory role that is activated by the phase of cell growth or differentiation. A second observation is the binding of TGF-beta to a glycoprotein of 180 kDa and referred to here as the "type VI" binding protein. This protein is not related to previously described TGF-beta binding proteins, and its distribution appears universal among cell types. The level of TGF-beta 1 binding to this protein is dependent on the presence of TGF-beta 2. It is not known whether this protein transmits biological information or whether it serves as an accessory protein of a TGF-beta receptor complex.
Asunto(s)
Proteínas de la Membrana/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Marcadores de Afinidad , Animales , Autorradiografía , Unión Competitiva , Bovinos , Células Cultivadas , Pollos , Cricetinae , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Glicosilación , Humanos , Hidrólisis , Riñón/citología , Riñón/metabolismo , Músculo Liso/citología , Músculo Liso/metabolismo , Músculos/citología , Músculos/metabolismo , RatasRESUMEN
Mononuclear cells (MNC) stimulated either with lipopolysaccharide (LPS) or with surface-adsorbed IgG elaborated significant amounts of tumor necrosis factor (TNF) bioactivity, as well as immunoenzymatically detectable TNF-alpha and interleukin-1 beta. (IL1-beta). In contrast, IgG-stimulated cells released little IL1 bioactivity, but released an IL1 inhibitor, as determined by the thymocyte costimulatory assay (LAF assay). This inhibition was not due to an inhibitory effect of cyclooxygenase products, e.g. prostaglandin-E2 in the LAF assay. In contrast, antibodies against transforming growth factor type beta (TGF-beta), which is an important inhibitor of the LAF assay, augmented the LAF activity of supernatants from LPS-stimulated and IgG-stimulated MNC. Anti-TGF-beta-modulated LAF inhibition was enhanced by acid treatment of supernatants from mononuclear cells, but not of those from purified monocytes. Antibody blocking experiments point for the first time to a TGF-beta species other than type 1 as a monocyte-derived TGF-beta activity. Thus, TGF-beta released in active form from monocytes may be the more important antagonist of IL1 than cyclooxygenase-derived mediators. It implies that the LAF assay, in the absence of anti-TGF-beta antibodies, is an inadequate indicator of IL1 activity.
Asunto(s)
Inmunoglobulina G/farmacología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Dinoprostona/farmacología , Dinoprostona/fisiología , Humanos , Indometacina/farmacología , Interleucina-1/biosíntesis , Interleucina-1/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos C3H , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
A polyclonal antibody (CL-B1/29) raised against a synthetic peptide with an amino acid sequence identical to the first 29 N-terminal residues of bovine bone-derived transforming growth factor-beta 2 (TGF-beta 2) was characterized and used for immunolocalization of TGF-beta 2 in adult mice. Reduced staining of immunoblots and tissue after absorption of the antiserum with the immunizing peptide or with TGF-beta 2 but not with purified TGF-beta 1 demonstrated that the reagent is specific for TGF-beta 2, with little or no crossreactivity with TGF-beta 1. The immunolocalization of TGF-beta 2 was investigated in formalin-fixed, paraffin-embedded cultured cells and murine tissue. Specimens pre-digested with testicular hyaluronidase demonstrated immunostaining predominantly of extracellular connective tissue matrix, whereas specimens pre-digested with pronase E demonstrated primarily cytoplasmic staining. Immunoreactivity was widely distributed in connective tissue, muscle, adsorptive and secretory epithelia, especially of endocrine tissue, and neural tissue of adult mice.
Asunto(s)
Anticuerpos , Factor de Crecimiento Transformador beta/análisis , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Tejido Conectivo/química , Glándulas Endocrinas/química , Epitelio/química , Matriz Extracelular/química , Hialuronoglucosaminidasa , Ratones , Músculos/química , Sistema Nervioso/química , Pronasa , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
A unique protein that promotes ectopic osteoinduction in the rat has been isolated and characterized. Osteoinductive factor (OIF) was extracted from the organic matrix of bovine bone with 4 M guanidine HCl and purified by gel filtration, ion-exchange chromatography, affinity chromatography, and reversed phase high performance liquid chromatography. OIF is a glycoprotein with an apparent molecular mass of 22-28 kDa based on sodium dodecyl sulfate gel electrophoresis. Enzymatic or chemical deglycosylation of OIF reduces its mass to about 12 kDa with apparent loss of activity. OIF activity in the model used is substantially increased by addition of transforming growth factor (TGF)-beta 1 or TGF-beta 2, suggesting an important role for TGF-beta 1 and -2 in bone regeneration and repair. The N-terminal sequence of OIF has no homology to other reported proteins.
Asunto(s)
Desarrollo Óseo , Huesos/fisiología , Glicoproteínas/aislamiento & purificación , Sustancias de Crecimiento/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Desarrollo Óseo/efectos de los fármacos , Huesos/efectos de los fármacos , Bovinos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , Peso Molecular , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas , Factores de Crecimiento Transformadores/farmacologíaRESUMEN
Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.
Asunto(s)
Anticuerpos Monoclonales , Reacciones Antígeno-Anticuerpo , Pruebas de Neutralización , Factores de Crecimiento Transformadores/inmunología , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/fisiología , Afinidad de Anticuerpos , Unión Competitiva , Western Blotting , Cromatografía de Afinidad , Reactivos de Enlaces Cruzados , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Precipitina , Receptores de Superficie Celular/análisis , Receptores de Factores de Crecimiento Transformadores beta , Factores de Crecimiento Transformadores/aislamiento & purificación , Factores de Crecimiento Transformadores/metabolismoRESUMEN
Long-term cultured bone marrow cells were characterized with respect to a number of B and pre-B cell markers. Cells expressing ThB, B-220, and IgM were found within cultures set up according to the procedure of Whitlock and Witte. This culture system was modified by placing sorted pre-B cells (ThB+, IgM-) from bone marrow in culture with previously-established bone marrow adherent layers. These cultures commenced growth without the lag associated with the Whitlock cultures. These cultured nonadherent cells show a high frequency of IgM+ cells, but do not express either IgD or Ia, and we refer to them as immature B cells. Cells with a similar phenotype (IgM+, Ia-, IgD-) are found within the spleens of young but not adult mice. The phorbol ester PMA induces expression of IgD on the cultured immature B cells, but has no effect on Ia expression. This suggests that the processing of H chain RNA transcripts may be affected by protein kinase C. These results demonstrate that the appearance of IgM, IgD, and Ia are independently controlled in long-term cultured B-lineage cells.
Asunto(s)
Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoglobulina D/metabolismo , Inmunoglobulina M/metabolismo , Animales , Células de la Médula Ósea , Células Cultivadas , Ratones , Ratones Endogámicos , Ésteres del Forbol/farmacologíaRESUMEN
The addition of anti-immunoglobulin (anti-Ig) to purified rabbit B-cells or concanavalin A (Con A) to purified rabbit T-cells within minutes resulted in the phosphorylation of a number of cytosolic proteins. Two-dimensional (2-D) electrophoresis and autoradiography of 32P-labeled cell sonicates was used to identify proteins whose phosphorylation was enhanced by these mitogens. Two proteins, pp58 and pp90, were phosphorylated 1.5 min after addition of anti-Ig to B-cells. Four other proteins, pp60, pp65, pp67 and pp95, were phosphorylated at later times. Three of these proteins were also phosphorylated after addition of Con A to purified T-cells. These phosphoproteins do not correspond to any previously described cytoplasmic proteins. Although all of these phosphoproteins were present in the cytosolic fraction, pp58 may be associated with the cytoskeleton. Protein pp58 is also distinguished from the rest by its absence from 2-D gels run under non-reducing conditions. Treatment of the B-cells with F(ab')2 fragments of anti-Ig stimulated phosphorylation but Fab' fragments did not--indicating that receptor cross-linking is required to induce phosphorylation. Both pp58 and pp90 contained phosphoserine, but neither phosphothreonine nor phosphotyrosine. Quantitatively the 32P-labeling of pp58 was 2.7-fold over background at 10 min after anti-Ig addition. The identification of these phosphoproteins, which may play a role in activational cascades or in cytoskeletal rearrangements, hopefully will help to clarify the interrelationships between cyclic nucleotide dependent and independent kinases in lymphocyte activation.
Asunto(s)
Linfocitos B/metabolismo , Citosol/metabolismo , Mitógenos/farmacología , Proteínas/metabolismo , Linfocitos T/metabolismo , Aminoácidos/análisis , Animales , Anticuerpos Antiidiotipos/inmunología , Concanavalina A/farmacología , Electroforesis , Femenino , Fragmentos Fab de Inmunoglobulinas/inmunología , Activación de Linfocitos , Masculino , Fosfoproteínas/análisis , Fosforilación , ConejosRESUMEN
Trifluoperazine (TFP), an inhibitor of the calcium-binding protein, calmodulin (CaM), was used to assess the role of calmodulin in the responses of rabbit lymphoid cells to stimulation with mitogen and antigen. After binding goat anti-rabbit Fab antibody, rabbit B cells lose their surface immunoglobulin (Ig) through endocytosis and then reexpress this protein during the next 24 hr. This reexpression was markedly inhibited by TFP. The brief and early addition of TFP markedly inhibited the increased [3H]thymidine (Tdr) uptake by rabbit T cells treated with concanavalin A and B cells exposed to anti-Fab. TFP greatly inhibited the induction by keyhole limpet hemocyanin (KLH) of the in vitro syntheses of antibody, Ig, and protein by KLH-primed lymph node cells (LNC). The earlier the TFP the greater was the inhibition of induction of these syntheses. However, once induced, synthesis and secretion of antibody were not inhibited by TFP. In striking contrast to the inhibition by TFP of the mitogenic and antigenic responses of lymphoid cells was the lack of effect of this drug on resting lymphocytes. Since TFP was not cytotoxic for either resting or mitogen- or antigen-stimulated LNC, it is highly unlikely that the observed inhibitory effects of this drug were due to its cytotoxicity. We postulate that an early signal for the activation of LNC proliferation, differentiation, and the syntheses of antibody, Ig, and protein involves a calcium-CaM-mediated reaction. Based on this work and that of others, the calcium-CaM complex may mediate an interaction between the ligand-occupied surface receptor and the cytoskeleton.