RESUMEN
In a search for genes expressed by dendritic cells (DC), we have cloned cDNAs encoding different forms of an asialoglycoprotein receptor (ASGPR). The DC-ASGPR represents long and short isoforms of human macrophage lectin, a Ca(2+)-dependent type II transmembrane lectin displaying considerable homology with the H1 and H2 subunits of the hepatic ASGPR. Immunoprecipitation from DC using an anti-DC-ASGPR mAb yielded a major 40-kDa protein with an isoelectric point of 8.2. DC-ASGPR mRNA was observed predominantly in immune tissues. Both isoforms were detected in DC and granulocytes, but not in T, B, or NK cells, or monocytes. DC-ASGPR species were restricted to the CD14-derived DC obtained from CD34(+) progenitors, while absent from the CD1a-derived subset. Accordingly, both monocyte-derived DC and tonsillar interstitial-type DC expressed DC-ASGPR protein, while Langerhans-type cells did not. Furthermore, DC-ASGPR is a feature of immaturity, as expression was lost upon CD40 activation. In agreement with the presence of tyrosine-based and dileucine motifs in the intracytoplasmic domain, mAb against DC-ASGPR was rapidly internalized by DC at 37 degrees C. Finally, intracellular DC-ASGPR was localized to early endosomes, suggesting that the receptor recycles to the cell surface following internalization of ligand. Our findings identify DC-ASGPR/human macrophage lectin as a feature of immature DC, and as another lectin important for the specialized Ag-capture function of DC.
Asunto(s)
Células Dendríticas/inmunología , Endocitosis , Lectinas Tipo C , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Secuencia de Aminoácidos , Animales , Receptor de Asialoglicoproteína , Antígenos CD40/metabolismo , Células Cultivadas , Clonación Molecular , Endosomas/química , Granulocitos/inmunología , Humanos , Lectinas/genética , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Monocitos/inmunología , Filogenia , ARN Mensajero/biosíntesis , Ratas , Receptores de Superficie Celular/genética , Homología de Secuencia de Aminoácido , Células Madre/inmunologíaRESUMEN
We have characterized a cytokine produced by Th2 cells, designated as IL-25. Infusion of mice with IL-25 induced IL-4, IL-5, and IL-13 gene expression. The induction of these cytokines resulted in Th2-like responses marked by increased serum IgE, IgG(1), and IgA levels, blood eosinophilia, and pathological changes in the lungs and digestive tract that included eosinophilic infiltrates, increased mucus production, and epithelial cell hyperplasia/hypertrophy. In addition, our studies show that IL-25 induces Th2-type cytokine production by accessory cells that are MHC class II(high), CD11c(dull), and lineage(-). These results suggest that IL-25, derived from Th2 T cells, is capable of amplifying allergic type inflammatory responses by its actions on other cell types.
Asunto(s)
Eosinofilia/inducido químicamente , Enfermedades Gastrointestinales/inducido químicamente , Regulación de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/aislamiento & purificación , Hipergammaglobulinemia/inducido químicamente , Interleucina-13/biosíntesis , Interleucina-4/biosíntesis , Interleucina-5/biosíntesis , Interleucinas , Subgrupos de Linfocitos T/efectos de los fármacos , Células Th2/metabolismo , Secuencia de Aminoácidos , Animales , Linaje de la Célula , Células Cultivadas , Clonación Molecular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Eosinofilia/inmunología , Eosinofilia/patología , Mucosa Gástrica/patología , Enfermedades Gastrointestinales/inmunología , Enfermedades Gastrointestinales/patología , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Sustancias de Crecimiento/toxicidad , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Hiperplasia , Hipertrofia , Integrina alfaXbeta2/análisis , Interleucina-13/genética , Interleucina-17 , Interleucina-4/genética , Interleucina-5/genética , Mucosa Intestinal/patología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Nucleares , Eosinofilia Pulmonar/inducido químicamente , Eosinofilia Pulmonar/inmunología , Eosinofilia Pulmonar/patología , ARN Mensajero/biosíntesis , Receptores de Interleucina-4/deficiencia , Receptores de Interleucina-4/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Subgrupos de Linfocitos T/metabolismo , Células Th2/químicaRESUMEN
Human legumain was characterized following overexpression in a murine cell line as the C-terminal Ig-fusion protein. Upon acid treatment, the prolegumain autoproteolyzed distal to two aspartic acid residues to yield a highly active form. The ability of mature legumain to cleave after aspartic acid residues was confirmed with a small peptide substrate. Substitution of alanine for the putative catalytic cysteine, or for either of two strictly conserved histidine residues, partly or wholly eliminated autoactivation but not the ability of wild-type legumain to correctly process the variants to the properly sized proteins.
Asunto(s)
Ácido Aspártico , Cisteína Endopeptidasas/metabolismo , Proteínas de Plantas , Secuencia de Aminoácidos , Animales , Western Blotting , Dominio Catalítico , Línea Celular , Cisteína/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/aislamiento & purificación , Activación Enzimática , Etiquetas de Secuencia Expresada , Histidina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
We report that Th2 cell cultures generated using T cells or splenocytes from IL-13-deficient mice produce significantly reduced levels of IL-4, IL-5, and IL-10 compared with wild-type. In contrast, IL-4 and IL-5 production by mast cells stimulated in vitro with PMA, ionomycin, or IgE cross-linking are unaffected. In vitro Th2 cell differentiation cannot be rescued by the addition of exogenous factors, but in vivo antigen challenge and administration of IL-13 can increase Th2-like cytokine responses as can infection with the parasitic nematode Nippostrongylus brasiliensis. IL-13-deficient mice also have lower basal levels of serum IgE and biased antigen-specific immunoglobulin responses. Thus, IL-13 is an important regulator of Th2 commitment and may therefore play a central role in atopy and infectious diseases.
Asunto(s)
Interleucina-13/deficiencia , Células Th2/citología , Animales , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/citología , Diferenciación Celular , Resistencia a Medicamentos/genética , Marcación de Gen/métodos , Inmunización , Inmunoglobulina E/sangre , Interleucina-13/genética , Interleucina-13/fisiología , Interleucina-4/biosíntesis , Interleucina-5/biosíntesis , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Insercional , Infecciones por Nematodos/inmunología , Neomicina/farmacología , Fosfoglicerato Quinasa/genética , Receptores de IgE/biosíntesis , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Bazo/citología , Células Th2/efectos de los fármacos , Células Th2/metabolismoRESUMEN
We describe a new cystatin in both mice and humans, which we termed leukocystatin. This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions. As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs. In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns. The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAGTM fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins. The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system.
Asunto(s)
Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Células Madre Hematopoyéticas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biomarcadores de Tumor , Pollos , Cistatinas/química , Cistatinas/genética , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/genética , Humanos , Ratones , Datos de Secuencia Molecular , Pliegue de Proteína , ARN Mensajero/metabolismoRESUMEN
Functional receptors for interleukin (IL)-4 and IL-13 on endothelial cells consist of the 130-kDa IL-4 receptor alpha-chain (IL-4Ralpha) and a 65-75-kDa IL-13 binding subunit that are expressed in a ratio of about 1:3, respectively. The restricted number of IL-4Ralpha limits subunit heterodimerization and in turn receptor-mediated signaling. We report here, the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of the receptor subunits for IL-4 and IL-13. By flow cytofluorometry and receptor-binding analysis of iodinated IL-4 and IL-13, stimulation with TNF-alpha-induced a 2-3-fold increase of the IL-4Ralpha expression. The up-regulation was also confirmed at the transcriptional level by reverse transcription-polymerase chain reaction. Radioligand cross-linking experiments revealed no change in the subunit composition of the TNF-alpha-induced receptor complex. Nevertheless, TNF-alpha stimulation led to increased activation of the IL-4-specific signal transducers and activators of transcription protein (Stat6) by IL-4 and IL-13. Thus, TNF-alpha corrects the subunit imbalance of the endothelial IL-4.IL-13 receptor complex thereby increasing receptor heterodimerization and in turn the signaling capability by IL-4 and IL-13.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/metabolismo , Activación Enzimática , Femenino , Humanos , Modelos Moleculares , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interleucina-4 , Factor de Transcripción STAT6 , Espectrometría de Fluorescencia , Regulación hacia ArribaRESUMEN
The effect of cytokine treatment on the in vivo maturation and Ig isotype switching of human B cells was studied in a modified SCID-hu mouse model. SCID mice, subcutaneously cotransplanted with small fragments of fetal human thymus and bone (SCID-hu BM/T mice) generated all human leukocyte lineages including T and B lymphocytes, macrophages, and granulocytes. All SCID-hu BM/T mice spontaneously produced human IgM and IgG, whereas IgE and IgA were detected in 37 and 80% of the mice, respectively, indicating that productive human T-B cell interactions resulting in Ig isotype switching occur in these mice. Administration of IL-4 to SCID-hu BM/T mice enhanced human B cell maturation, as judged by the increase in the percentages of CD45+, CD19+ bone marrow B cells expressing CD20, CD23, CD40, sIgM, and sIgD. Furthermore, these cells were also functionally more mature because they spontaneously produced human IgG/IgG4 in vitro and could be induced to secrete human IgE by addition of anti-CD40 mAb alone. In contrast, B cells isolated from PBS-treated mice only produced significant Ig levels after stimulation with anti-CD40 mAb in the presence of exogenous IL-4. IL-4 administration also induced human IgE synthesis in 44% of the mice, which had no serum IgE before treatment. More importantly, ongoing human IgE synthesis in SCID-hu BM/T mice was suppressed by > 90% following administration of an IL-4 mutant protein, which acts as an IL-4 and IL-13 receptor antagonist. These results suggest that IL-4/IL-13 receptor antagonists have potential clinical utility in treating human atopic diseases associated with enhanced IgE production.
Asunto(s)
Antígenos CD/química , Linfocitos B/inmunología , Inmunoglobulina E/biosíntesis , Inmunosupresores/farmacología , Interleucina-4/farmacología , Activación de Linfocitos , Receptores de Interleucina/antagonistas & inhibidores , Receptores de Interleucina/química , Animales , Linfocitos B/metabolismo , Movimiento Celular/inmunología , Humanos , Inmunoglobulina E/efectos de los fármacos , Inmunoglobulinas/sangre , Inmunofenotipificación , Subunidad alfa1 del Receptor de Interleucina-13 , Ratones , Ratones SCID , Ratones Transgénicos , Receptores de Interleucina-13 , Receptores de Interleucina-4 , Timo/trasplante , Quimera por TrasplanteRESUMEN
Studies on the binding of IL-2 to its receptor (IL-2R) have generally been limited to receptors expressed on cell surfaces. This has hampered detailed kinetic and mechanistic studies at the molecular level. We have prepared the soluble extracellular domains of all three receptor subunits (called alpha, beta and gamma) by recombinant techniques and have used these to perform detailed kinetic studies of their binding properties using the technique of surface plasmon resonance. We describe a novel approach whereby the receptors are assembled on an antibody surface, being held by an epitope engineered into the C-terminus of each of these domains. Thus the receptors are oriented naturally leading to homogeneous ligand binding kinetics. We have characterized the interactions of the heteromeric complexes of these subunits with mouse and human IL-2 and their analogs, as well as the recently discovered cytokine, IL-15. We have also studied the extracellular domains of the mouse receptor subunits for the first time and have used these as well as mouse-human hybrid receptors to probe the mechanism of assembly of these complexes. We show that no additional proteins are required to reproduce the properties of these complexes in vitro. In addition, kinetic studies with site-specific analogs of IL-2 and the mouse-human receptor hybrids clearly indicate that the extracellular domains of alpha and beta can together readily bind ligand with kinetic properties distinct from those of the constituent subunits. In contrast, a complex containing ligand and the extracellular domains of beta and gamma was comparatively difficult to assemble and required prolonged exposure to IL-2. Our method enabled us to calculate the stoichiometry of these complexes and to determine that anchoring these subunits is necessary to efficiently drive complex formation. The kinetic and equilibrium differences between the mouse and human receptor complexes, and between IL-2 and IL-15 binding to these receptors clarify the roles of the alpha and gamma subunits in the differential response of cells to different cytokines that may be present simultaneously in the environment.
Asunto(s)
Interleucina-2/metabolismo , Interleucinas/metabolismo , Receptores de Interleucina-2/metabolismo , Secuencia de Aminoácidos , Animales , Biopolímeros , Epítopos/inmunología , Espacio Extracelular/química , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Humanos , Interleucina-15 , Interleucina-2/farmacología , Cinética , Ligandos , Ratones , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Relación Estructura-ActividadRESUMEN
The lymphocyte cell-surface Ag CD38 catabolizes NAD to adenosine 5' diphosphoribose (ADPR) and cyclic ADPR (cADPR). We show here that the soluble extracellular domain of CD38 (sCD38) mediates ADP ribosylation of several proteins. This was demonstrated by mass spectrometric analyses which revealed the addition of mass in units of 541.1 Da to these proteins, presumably corresponding to the covalent attachment of one or more ADPR moieties. Separate experiments showed that the same proteins became specifically radiolabeled following incubation with [32P]NAD plus sCD38. Additionally, it is shown that sCD38 can autoribosylate. Moreover, sCD38-mediated protein ribosylation was found to occur specifically at cysteine residues, since it was effectively blocked by addition of L-cysteine but not by other amino acids, and CD38-mediated protein ribosylation could be reversed by the addition of HgCl2, which specifically cleaves thiol-glycosidic bonds. ADPR purified from the reaction of sCD38 with NAD could itself be covalently transferred to target proteins at rates similar to the sCD38-mediated reaction, indicating that the ribosylation proceeds via the generation of this reactive intermediate. In vitro mutagenesis of a catalytic Glu residue that is conserved in numerous ADP-ribosyl transferases revealed that this amino acid is also important for catalysis in CD38. These data suggest that CD38 has the potential to cause ribosylation of experimental proteins, and raises the possibility that its specific ribosylation of a currently unidentified lymphocyte protein may contribute to its array of immunoregulatory activities.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación/fisiología , N-Glicosil Hidrolasas/metabolismo , N-Glicosil Hidrolasas/fisiología , Poli(ADP-Ribosa) Polimerasas/fisiología , Proteínas/metabolismo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adenosina Difosfato Ribosa/fisiología , Animales , Antígenos de Diferenciación/genética , Cisteína/metabolismo , Glicoproteínas de Membrana , Ratones , Mutagénesis Sitio-Dirigida/genética , N-Glicosil Hidrolasas/genética , Proteínas Recombinantes/metabolismoRESUMEN
Interleukin (IL)-13 elicits a subset of the biological activities of the related IL-4. The basis of this functional similarity is that their specific cell-surface receptors (called IL-13R and IL-4R) are distinct, yet are complex and share a common subunit(s). The IL-4R primary binding subunit (called IL-4R alpha) does not by itself bind IL-13. We show that the ability of IL-13 to partially compete for IL-4 binding to some human cell types depended on co-expression of IL-4R and IL-13R. However, IL-13 binding was always associated with IL-4 binding. Hyper-expression of IL-4R alpha on cells expressing both IL-4R and IL-13R decreased their binding affinity for IL-4, abrogated the ability of IL-13 to compete for IL-4 binding, and yet had no effect on IL-13R properties. Anti-human IL-4R alpha monoclonal antibodies which blocked the biological function and binding of IL-4 also blocked the function and binding of IL-13. These data show that IL-4R alpha is a secondary component of IL-13R.
Asunto(s)
Receptores de Interleucina/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Unión Competitiva , Células Cultivadas , Humanos , Interleucina-13/antagonistas & inhibidores , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13 , Ratones , Ratones Endogámicos BALB C , Receptores de Interleucina-13 , Receptores de Interleucina-4RESUMEN
IL-13 is a relatively novel cytokine produced by activated T cells. IL-13 inhibits the production of proinflammatory cytokines and chemokines by activated monocytes, induces B cell proliferation and differentiation, including IgE production, and the expression of certain adhesion molecules on endothelial cells. All these biological properties of IL-13 are shared with IL-4, but in contrast to IL-4, IL-13 does not act on T cells. In this review, the similarities in structure and biological function of IL-13 and IL-4 and the commonalities of their receptors are summarized. The potential role of IL-13 in allergic responses is briefly discussed.
Asunto(s)
Interleucina-13/inmunología , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Animales , Linfocitos B/inmunología , Humanos , Hipersensibilidad Inmediata/inmunología , Interleucina-13/biosíntesis , Interleucina-4/inmunología , Monocitos/inmunología , Receptores de Interleucina/inmunologíaAsunto(s)
Interleucina-13/fisiología , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Citocinas/biosíntesis , Regulación de la Expresión Génica , VIH/fisiología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Inflamación/fisiopatología , Interleucina-13/química , Interleucina-13/genética , Interleucina-13/farmacología , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/fisiología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Receptores de Interleucina/química , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/genética , Receptores de Interleucina/fisiología , Receptores de Interleucina-13 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Células Th2/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The recently described interleukin-13 (IL-13) has been shown to share many of the effects of IL-4 on normal B cells, including growth-promoting activity and induction of CD23. In this study, we compared the effects of IL-13 and IL-4 on B chronic lymphocytic leukaemias (B-CLL) cells. After anti-CD40 activation, both IL-13 and IL-4 promoted the DNA synthesis of B-CLL cells and increased the recovery of viable cells. The time kinetics of the proliferative response of B-CLL cells to IL-13 or IL-4 were superimposable and showed the long-lasting effect of both cytokines. As on normal B cells, both IL-4 and IL-13 synergized with IL-10 to enhance B-CLL DNA synthesis. Moreover, IL-13, like IL-4, was able to increase CD23 expression on anti-CD40-activated leukaemic B cells. The CD23 up-regulation and the DNA synthesis induced by IL-13 on anti-CD40-activated B-CLL cells, were significantly reduced when B-CLL cells were cultured with anti-IL-4 receptor monoclonal antibody, suggesting a common pathway for IL-13 and IL-4 signalling. However, after cross-linking of surface IgM, IL-4 strongly inhibited the IL-2-induced DNA synthesis of B-CLL cells, whereas IL-13 did not inhibit IL-2-driven proliferation of anti-IgM-activated B-CLL cells. Furthermore, while IL-4 strongly up-regulated the expression of CD23 on anti-IgM-activated leukaemic B cells, IL-13 only marginally increased it. Finally, IL-13, in contrast to IL-4, did not prevent the entry of B-CLL cells into apoptosis. Thus IL-13 and IL-4 display comparable effects on anti-CD40-activated B-CLL cells, which are blocked by anti-IL-4 receptor (IL-4R) monoclonal antibodies. However, IL-13-dependent effects are absent or inefficient in non-activated or anti-IgM-activated B-CLL cells. This suggests that such cells may lack functional IL-13 receptors, though IL-13R and IL-4R on B-CLL cells share a common component.
Asunto(s)
Linfocitos B/inmunología , Interleucina-13/inmunología , Interleucina-4/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígenos CD40 , Células Cultivadas , ADN/biosíntesis , Citometría de Flujo , Humanos , Inmunoglobulina M/inmunología , Staphylococcus aureus/inmunología , Regulación hacia ArribaRESUMEN
Interleukin 13 (IL-13) and interleukin 4 (IL-4) are two closely related proteins produced by activated T cells. IL-4 is a well characterized mediator of various aspects of the immune response, including anti-inflammatory effects on monocytes and macrophages, regulation of B cell function, T cell growth, and regulation of adhesion molecule expression on endothelial cells. IL-13, a more recently characterized cytokine, appears to exhibit IL-4-like activities on monocytes, macrophages and human B cells, but has no effect on T cells. While there is a close parallel between IL-4 activities on human and mouse cells, IL-13 activities in these two systems appear to differ substantially with a notable absence of effect on mouse B cells. This review briefly summarizes the current state of knowledge of the interrelated activities of IL-13 and IL-4, explores the basis of these effects at the receptor level and attempts to rationalize the existence of these close relatives via differences in their production by T cells.
Asunto(s)
Interleucina-4/inmunología , Interleucinas/inmunología , Animales , Humanos , Interleucina-13RESUMEN
We have investigated the relevance of interleukin-1 receptor type I (IL-1R tI) in the implantation process in vivo in a murine model. Indirect immunofluorescence experiments demonstrate that IL-1R tI is located in mouse endometrial lumenal epithelium with increased intensity in the periimplantation period, whereas IL-1 beta staining is located in the mouse placenta. PMSG/human CG (hCG)-stimulated and mated 12-week-old B6C3F-1 female mice were randomly allocated to three groups: A, control noninjected; B, buffer-injected animals; and C, animals injected ip with 20 micrograms recombinant human IL-1 receptor antagonist (rhIL-1ra) every 12 h beginning on pregnancy day 3. Injections were continued until day 9, and animals were killed 12 h after the last injection. Pregnancy rates in the three groups were: noninjected, 58.8% (10 of 17); buffer-injected, 73.7% (14 of 19); rhIL-1ra-injected, 6.7% (1 of 15), P = 0.0001155, Fisher exact test. To rule out the possibility that pregnancy failure was due to an embryotoxic effect of rhIL-1ra, 2-cell mouse embryos (n = 276) were flushed from the same group of animals used for in vivo experiments and cultured with increasing concentrations of rhIL-1ra: 0 microgram/ml (n = 91), 1 microgram/ml (n = 36), 50 micrograms/ml (n = 36), 100 micrograms/ml (n = 52), and 200 micrograms/ml (n = 61) rhIL-1ra. The percentages of 2-cell mouse embryos reaching the blastocyst stage after 72 h in culture were 85.7%, 91.6%, 94.4%, 96%, and 85.2%, respectively. We further cultured these blastocysts for 5 days on fibronectin-coated plates with or without 200 micrograms/ml rhIL-1ra. In both groups, hatching, attachment to fibronectin, outgrowth, and migration were documented to be similar. Furthermore, our longitudinal morphological study of embryonic implantation in control and rhIL-1ra-injected mice shows that the blockade of IL-1R tI interferes with the attachment of mouse blastocysts to maternal endometrium in vivo. In summary, we demonstrate that blockade of maternal endometrial IL-1R tI with IL-1ra prevents implantation in the mouse by interfering with embryonic attachment, without adverse effects on blastocyst formation, hatching, fibronectin attachment, outgrowth, and migration in vitro.
Asunto(s)
Blastocisto/fisiología , Implantación del Embrión/efectos de los fármacos , Endometrio/fisiología , Placenta/fisiología , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/farmacología , Animales , Blastocisto/citología , Blastocisto/efectos de los fármacos , Gonadotropina Coriónica/farmacología , Implantación del Embrión/fisiología , Endometrio/citología , Endometrio/efectos de los fármacos , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Gonadotropinas Equinas/farmacología , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Ratones , Ratones Endogámicos , Técnicas de Cultivo de Órganos , Placenta/citología , Placenta/efectos de los fármacos , Embarazo , Proteínas Recombinantes/farmacologíaRESUMEN
Interleukin 13 (IL-13) is a recently described protein secreted by activated T cells which is a potent in vitro modulator of human monocyte and B-cell functions. The data, reviewed here by Gerard Zurawski and Jan de Vries, shows that IL-13 shares biological activities with IL-4, their genes are closely linked in both the human and mouse genomes, and there is sequence homology between IL-13 and IL-4 proteins. Although the cloned IL-4 receptor protein (IL-4R) does not bind IL-13, it appears that the functional IL-4R and IL-13R share a common subunit that is important for signal transduction.
Asunto(s)
Interleucina-4/fisiología , Interleucinas/fisiología , Leucocitos Mononucleares/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Interleucina-13 , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The high affinity receptor for interleukin-2 (IL-2) contains three subunits called IL-2R alpha, beta and gamma. A biological and receptor binding analysis based on 1393 different mutant mouse IL-2 (mIL-2) proteins was used to define the function of each of the 149 residues. By this genetic analysis, 44 residues were assigned important functions, 21 of which were structural. The remaining 23 residues consisted of 19 residues, from three separate regions, that were important for IL-2R alpha interaction; three residues, from two separate regions, that were important for IL-2R beta interaction; and a single residue important for IL-2R gamma interaction. We built a model mIL-2 structure based on the homologous human IL-2 (hIL-2) crystal structure. The roles of the 21 residues presumed to be important for structure were consistent with the model. Despite discontinuity in the primary sequence, the residues specific for each IL-2R subunit interaction were clustered and located to three disparate regions of the tertiary mIL-2 structure. The relative spatial locations of these three surfaces are different from the two receptor binding sites known for the structurally related human growth hormone and the significance of this observation is discussed.
Asunto(s)
Interleucina-2/química , Receptores de Interleucina-2/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Interleukin 4 (IL-4) and IL-13 share many biological functions. Both cytokines promote growth of activated human B cells and induce naive human surface immunoglobulin D+ (sIgD+) B cells to produce IgG4 and IgE. Here we show that a mutant form of human IL-4, in which the tyrosine residue at position 124 is replaced by aspartic acid (hIL-4.Y124D), specifically blocks IL-4 and IL-13-induced proliferation of B cells costimulated by anti-CD40 mAbs in a dose-dependent fashion. A mouse mutant IL-4 protein (mIL-4.Y119D), which antagonizes the biological activity of mouse IL-4, was ineffective. In addition, hIL-4.Y124D, at concentrations of up to 40 nM, did not affect IL-2-induced B cell proliferation. hIL-4.Y124D did not have detectable agonistic activity in these B cell proliferation assays. Interestingly, hIL-4.Y124D also strongly inhibited both IL-4 or IL-13-induced IgG4 and IgE synthesis in cultures of peripheral blood mononuclear cells, or highly purified sIgD+ B cells cultured in the presence of anti-CD40 mAbs. IL-4 and IL-13-induced IgE responses were inhibited > 95% at a approximately 50- or approximately 20-fold excess of hIL-4.Y124D, respectively, despite the fact that the IL-4 mutant protein had a weak agonistic activity. This agonistic activity was 1.6 +/- 1.9% (n = 4) of the maximal IgE responses induced by saturating concentrations of IL-4. Taken together, these data indicate that there are commonalities between the IL-4 and IL-13 receptor. In addition, since hIL-4.Y124D inhibited both IL-4 and IL-13-induced IgE synthesis, it is likely that antagonistic mutant IL-4 proteins may have potential clinical use in the treatment of IgE-mediated allergic diseases.
Asunto(s)
Linfocitos B/fisiología , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Interleucina-4/farmacología , Interleucinas/antagonistas & inhibidores , Receptores de Interleucina/fisiología , Receptores Mitogénicos/fisiología , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Antígenos CD40 , Humanos , Interleucina-13 , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/antagonistas & inhibidores , Interleucina-4/química , Activación de Linfocitos/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Receptores de Interleucina-13 , Receptores de Interleucina-4 , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Recently, we described the cloning and expression of a human cDNA which is the homologue to P600, a gene transcribed by mouse Th2 clones. Based on its activities on human monocytes and B cells this gene was designated IL-13. In the present study we investigated the effects of IL-13 alone or in combination with IL-4, IFN-gamma, or IL-10 on human monocytes. IL-13 induced significant changes in the phenotype of monocytes. Like IL-4, it enhanced the expression of CD11b, CD11c, CD18, CD29, CD49e (VLA-5), class II MHC, CD13, and CD23, whereas it decreased the expression of CD64, CD32, CD16, and CD14 in a dose-dependent manner. IL-13 induced up-regulation of class II MHC Ag and its down-regulatory effects on CD64, CD32, and CD16 expression were prevented by IL-10. IFN-gamma could also partially prevent the IL-13-induced down-regulation of CD64, but not that of CD32 and CD16. However, IL-13 strongly inhibited spontaneous and IL-10- or IFN-gamma-induced ADCC activity of human monocytes toward anti-D coated Rh+ erythrocytes, indicating that the cytotoxic activity of monocytes was inhibited. Furthermore, IL-13 inhibited production of IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, macrophage inflammatory protein-1 alpha, granulocyte/macrophage-CSF, granulocyte-CSF, IFN-alpha, and TNF alpha by monocytes activated with LPS. In contrast, IL-13 enhanced the production of IL-1 ra by these cells. Similar results on cytokine production were observed or have been obtained with IL-4. Thus IL-13 shares most of its activities on human monocytes with IL-4, but no additive or synergistic effects of IL-4 and IL-13 on human monocytes were observed, suggesting that these cytokines may share common receptor components. Taken together, these results indicate that IL-13 has anti-inflammatory and important immunoregulatory activities.
Asunto(s)
Citocinas/biosíntesis , Interferón gamma/farmacología , Interleucina-10/farmacología , Interleucina-4/farmacología , Interleucinas/farmacología , Monocitos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos de Superficie/análisis , Secuencia de Bases , Células Cultivadas , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-13 , Datos de Secuencia Molecular , Monocitos/inmunología , Monocitos/metabolismo , Receptores de IgG/análisis , Sialoglicoproteínas/metabolismoRESUMEN
Interleukin-4 (IL-4) and interleukin-13 (IL-13) are two cytokines that are secreted by activated T cells and have similar effects on monocytes and B cells. We describe a mutant form of human interleukin-4 (hIL-4) that competitively antagonizes both hIL-4 and human interleukin-13 (hIL-13). The amino acid sequences of IL-4 and IL-13 are approximately 30% homologous and circular dichroism (CD) spectroscopy shows that both proteins have a highly alpha-helical structure. IL-13 competitively inhibited binding of hIL-4 to functional human IL-4 receptors (called hIL-4R) expressed on a cell line which responds to both hIL-4 and IL-13. Binding of hIL-4 to an hIL-4 responsive cell line that does not respond to IL-13, and binding of hIL-4 to cloned IL-4R ligand binding protein expressed on heterologous cells, were not inhibited by IL-13. hIL-4 bound with approximately 100-fold lower affinity to the IL-4R ligand binding protein than to functional IL-4R. The mutant hIL-4 antagonist protein bound to both IL-4R types with the lower affinity. The above results demonstrate that IL-4 and IL-13 share a receptor component that is important for signal transduction. In addition, our data establish that IL-4R is a complex of at least two components one of which is a novel affinity converting subunit that is critical for cellular signal transduction.