RESUMEN
We have performed immunohistochemical or ultrastructural analyses of living mouse small intestines using Epon blocks prepared by 'in vivo cryotechnique' (IVCT). By electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5-10 µm away from cryogen-contact surface tissues. Their microvilli contained dynamically waving actin filaments, and highly electron-dense organelles, such as mitochondria, were seen under the widely organized terminal web. By quick-freezing of fresh resected tissues (FT-QF), many erythrocytes were congested within blood vessels due to loss of blood pressure. By immersion-fixation (IM-DH) and perfusion-fixation (PF-DH), small vacuoles were often seen in the cytoplasm of epithelial cells, and their intercellular spaces were also dilated. Moreover, actin filament bundles were irregular in cross sections of microvilli, compared with those with IVCT. Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval and immunostained for immunoglobulin A (IgA), Ig kappa light chain (Igκ), J-chain and albumin. By cryotechniques, IgA immunoreactivity was detected as tiny dot-like patterns in cytoplasm of some epithelial cells. Both J-chain and Igκ immunoreactivities were detected in the same local areas as those of IgA. By FT-QF, however, the IgA immunoreactivity was more weakly detected, compared with that with IVCT. In thick sections prepared by IM-DH and PF-DH, it was rarely observed in both plasma and epithelial cells. Another albumin was diffusely immunolocalized in extracellular matrices of mucous membranes and also within blood vessels. Thus, IVCT was useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.
Asunto(s)
Criopreservación/métodos , Inmunohistoquímica/métodos , Mucosa Intestinal/ultraestructura , Intestino Delgado/ultraestructura , Microscopía Electrónica/métodos , Citoesqueleto de Actina/fisiología , Albúminas/fisiología , Animales , Proteínas Bacterianas/análisis , Vasos Sanguíneos/fisiología , Células Epiteliales/fisiología , Eritrocitos/fisiología , Etanol/análogos & derivados , Etanol/farmacología , Inmunoglobulina A/inmunología , Inmunoglobulina A/fisiología , Cadenas J de Inmunoglobulina/inmunología , Cadenas J de Inmunoglobulina/fisiología , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/fisiología , Mucosa Intestinal/citología , Intestino Delgado/citología , Ratones , Ratones Endogámicos C57BL , Microvellosidades/fisiología , Microvellosidades/ultraestructura , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Coloración y Etiquetado/métodos , Fijación del Tejido , Conservación de Tejido/métodosRESUMEN
Monoclonal gammopathy is increasingly recognized as a common cause of membranoproliferative glomerulonephritis (MPGN); however, establishing this diagnosis can be challenging. We report the case of a 58-year-old asymptomatic woman who presented with proteinuria with protein excretion of 5,000mg/d, microscopic hematuria, and normal kidney function. Kidney biopsy was consistent with MPGN pattern of injury. Immunofluorescence studies were positive for nonspecific segmental immunoglobulin M (IgM) and C3 staining. Electron microscopy showed subendothelial, subepithelial, and mesangial electron-dense deposits. The workup excluded an infectious or autoimmune disease, but IgG κ monoclonal protein was detected in serum at a concentration of 0.4mg/dL. Because there was a mismatch between the serum monoclonal protein (IgG κ) and immunofluorescence staining pattern (nonspecific IgM, no light chain restriction), laser microdissection and mass spectrometry were performed on the kidney biopsy tissue. This identified the deposits as monoclonal IgG κ, thereby leading to the diagnosis of monoclonal gammopathy-associated MPGN. Our case emphasizes the importance of searching for an underlying cause of MPGN, reviews the technique of laser microdissection-mass spectrometry, and highlights its application as a pathology tool for the evaluation of monoclonal gammopathy-related glomerulonephritis.
Asunto(s)
Glomerulonefritis Membranoproliferativa/diagnóstico , Glomerulonefritis Membranoproliferativa/metabolismo , Espectrometría de Masas/métodos , Microdisección/métodos , Femenino , Humanos , Cadenas kappa de Inmunoglobulina/fisiología , Persona de Mediana EdadRESUMEN
BACKGROUND: Fanconi syndrome (FS) is a rare renal disorder featuring proximal tubule dysfunction that may occur following tubular reabsorption of a monoclonal light chain (LC), in patients with multiple myeloma. FS may precede the recognition of multiple myeloma by several years. In most cases, crystalline inclusions of monoclonal κ LCs are observed within the lysosomes of proximal tubular cells (PTCs) and probably participate in their functional alteration. METHODS: To investigate the mechanism implicated in proximal tubule dysfunction, we compared the effects of κ LC-CHEB obtained from a patient with myeloma-associated FS to those of control κ LC-BON obtained from a patient without evidence of FS, on the viability and proliferation of two different PTC lines. RESULTS: Our data suggest that the tubular atrophy in myeloma-associated FS does not result from increased apoptosis of PTCs, but from their impaired capacity to proliferate and renew. Indeed, in vitro incubation of cultured PTCs with physiological amounts of the nephrotoxic κ LC-CHEB was sufficient to cause a depression in DNA synthesis and in cell proliferation. This effect was observed neither with control κ LC-BON nor in the absence of κ LC. CONCLUSIONS: The reduced turnover of PTCs may affect tubular repair and regeneration. In addition, the reduced proliferation of myeloma cells producing the same monoclonal κ LC might explain the frequent association of FS with smoldering multiple myeloma.
Asunto(s)
Proliferación Celular , Síndrome de Fanconi/fisiopatología , Cadenas kappa de Inmunoglobulina/fisiología , Túbulos Renales Proximales/patología , Atrofia , Células Cultivadas , Síndrome de Fanconi/etiología , HumanosRESUMEN
Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal V(H) genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3' enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show in this study that STAT5 can repress κE3' enhancer activity. We find that STAT5 binds to a site that overlaps the κE3' PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3' enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3' enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates κE3' enhancer function.
Asunto(s)
Diferenciación Celular/inmunología , Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos/inmunología , Cadenas kappa de Inmunoglobulina/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Unión Competitiva/genética , Unión Competitiva/inmunología , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Cadenas kappa de Inmunoglobulina/metabolismo , Cadenas kappa de Inmunoglobulina/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Células 3T3 NIH , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas/fisiología , Factor de Transcripción STAT5/fisiología , Transactivadores/fisiologíaRESUMEN
Traditionally, mast cells were regarded as key cells orchestrating type I hypersensitivity responses. However, it is now recognized that mast cells are widely involved in nonallergic (non-IgE) chronic diseases. Also, in inflammatory bowel disease (IBD), a disease not associated with increased IgE concentrations, clear signs of activation of mast cells have been found. In this study, we investigated if Ig-free L chain-induced hypersensitivity-like responses through activation of mast cells could contribute to the pathophysiology of IBD. As a mast cell-dependent model for IBD, mice were skin-sensitized with dinitrofluorobenzene followed by intrarectal application of the hapten. In this murine IBD model, F991 prevented mast cell activation and also abrogated the development of diarrhea, cellular infiltration, and colonic lymphoid follicle hyperplasia. Furthermore, passive immunization with Ag-specific Ig-free L chains (IgLCs) and subsequent rectal hapten challenge elicited local mast cell activation and increased vascular permeability in the colon of mice. Clinical support is provided by the observation that serum concentrations of IgLCs of patients suffering from Crohn's disease are greatly increased. Moreover, increased presence of IgLCs was evident in tissue specimens from colon and ileum tissue of patients with IBD. Our data suggest that IgLCs may play a role in the pathogenesis of IBD, which provides novel therapeutic means to prevent or ameliorate the adverse gastrointestinal manifestations of IBD.
Asunto(s)
Colitis/inmunología , Colitis/metabolismo , Cadenas kappa de Inmunoglobulina/fisiología , Cadenas lambda de Inmunoglobulina/fisiología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Adulto , Animales , Colitis/patología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunización Pasiva , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/sangre , Enfermedades Inflamatorias del Intestino/patología , Masculino , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Regulación hacia Arriba/inmunología , Adulto JovenRESUMEN
Random V(D)J junctions ensure that the diversity of the Ig primary repertoire is adapted to the vast heterogeneity of Ags. In two-thirds of cases, recombination between variable segments induces a frameshift in the open reading frame and generates a premature termination codon. In B cells harboring biallelic V(D)J rearrangement of Ig genes, transcription is known to occur on both the functional and nonfunctional alleles, generating considerable amounts of primary transcripts with out-of-frame V regions. In this study, we analyzed in cell lines and primary B cells the RNA surveillance of nonfunctional Igkappa transcripts arising from nonproductive rearrangement. We demonstrated that splicing inhibition, nonsense-mediated decay and nonsense-altered splicing each have an individual partial effect that together associate into an efficient surveillance machinery, downregulating nonfunctional Igkappa mRNA. Moreover, we provide evidence that the RNA surveillance efficiency increases throughout B cell development. Whereas splicing inhibition remains constant in most cell lines, differences in nonsense-mediated decay and nonsense-altered splicing are responsible for the higher RNA surveillance observed in plasma cells. Altogether, these data show that nonfunctionally rearranged alleles are subjected to active transcription but that multiple RNA surveillance mechanisms eradicate up to 90% of out-of-frame Igkappa mRNA.
Asunto(s)
Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B/inmunología , Cadenas kappa de Inmunoglobulina/genética , Empalme del ARN/inmunología , ARN Mensajero/antagonistas & inhibidores , Recombinación Genética/inmunología , Transcripción Genética/inmunología , Alelos , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Codón sin Sentido/antagonistas & inhibidores , Codón sin Sentido/genética , Codón sin Sentido/fisiología , Codón de Terminación/antagonistas & inhibidores , Codón de Terminación/genética , Codón de Terminación/fisiología , Mutación del Sistema de Lectura/inmunología , Cadenas kappa de Inmunoglobulina/fisiología , Ratones , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , ARN Mensajero/fisiología , Células Madre/citología , Células Madre/inmunología , Células Madre/metabolismoRESUMEN
OBJECTIVE: Autoimmune diseases predominantly affect women, suggesting that female sex hormones may play a role in the pathogenesis of such diseases. We have previously shown that persistent mild-to-moderate elevations in serum prolactin levels induce a break in self tolerance in mice with a BALB/c genetic background. The aim of this study was to evaluate the effects of hyperprolactinemia on the mechanisms of B cell tolerance induction. METHODS: Effects of prolactin on splenic B cell subsets were studied in female BALB/c mice. B cell receptor (BCR)-mediated apoptosis and proliferation of transitional B cells were analyzed by flow cytometry. Expression of apoptotic genes was examined by microarrays and real-time polymerase chain reaction analysis. B cells coexpressing kappa/lambda light chains were assessed by flow cytometry and immunohistochemistry. Activation status of transitional type 3 (T3) B cells was evaluated by BCR-induced calcium influx studies. RESULTS: BCR-mediated apoptosis of the T1 B cell subset, a major checkpoint for negative selection of autoreactive specificities, was decreased in prolactin-treated mice. Microarray studies indicated that this event may be mediated by the prolactin-induced up-regulation of the antiapoptotic gene interferon-gamma receptor type II and down-regulation of the proapoptotic gene Trp63. Prolactin treatment also altered the amount of receptor editing, as indicated by the increased number of transitional B cells coexpressing kappa/lambda light chains. Additionally, hyperprolactinemia modified the level of B cell anergy by increasing the degree of BCR-induced calcium influx in the T3 B cells. CONCLUSION: Persistently elevated serum prolactin levels interfere with B cell tolerance induction by impairing BCR-mediated clonal deletion, deregulating receptor editing, and decreasing the threshold for activation of anergic B cells, thereby promoting autoreactivity.
Asunto(s)
Linfocitos B/fisiología , Hiperprolactinemia/fisiopatología , Tolerancia Inmunológica/fisiología , Prolactina/fisiología , Animales , Apoptosis/fisiología , Linfocitos B/patología , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Hiperprolactinemia/patología , Cadenas kappa de Inmunoglobulina/fisiología , Cadenas lambda de Inmunoglobulina/fisiología , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas/fisiología , Receptores de Interferón/fisiología , Transactivadores/fisiología , Receptor de Interferón gammaRESUMEN
The truncated/V(H)-less mouse H chain Dmu forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dmu-lambda1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dmu's unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent lambda1 L chains could associate with both full-length H chains and Dmu, whereas secretion-incompetent lambda1 L chains could only do so with full-length H chains. In contrast, Dmu could not form receptors with a panel of kappa L chains irrespective of their secretion properties. This was due to an incompatibility of Dmu with the kappa-joining and constant regions. Finally, the Dmu-lambda1 receptor was less active than the full-length mouse mu-lambda1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dmu H chain alleles to the repertoire.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Diferenciación Celular/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B , Cadenas Pesadas de Inmunoglobulina/fisiología , Inmunoglobulina de Cadenas Ligeras Subrogadas/fisiología , Cadenas kappa de Inmunoglobulina/fisiología , Cadenas lambda de Inmunoglobulina/fisiología , Células Madre/inmunología , Alelos , Animales , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Línea Celular , Línea Celular Transformada , Células Cultivadas , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina de Cadenas Ligeras Subrogadas/biosíntesis , Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/genética , Ratones , Ratones Noqueados , Mutagénesis , Receptores de Antígenos de Linfocitos B/biosíntesis , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/fisiología , Células Madre/metabolismoRESUMEN
Acquired Fanconi syndrome (FS) is a complication of monoclonal gammopathies featuring a generalized dysfunction of the proximal tubule of the kidney, due to the storage within proximal tubular cells of a monoclonal immunoglobulin light chain. We engineered transgenic mice in which the endogenous mouse Jkappa cluster was replaced by a human VkappaJkappa rearranged gene cloned from a patient with smoldering myeloma-associated FS. The V region belonged to the VkappaI subgroup and was related to the O2-O12 germ-line gene, a V segment previously found associated with FS and light-chain crystallization in several patients with myeloma. Association of the human VkappaI domain with a mouse kappa constant domain in transgenic animals yielded a nephrotoxicity pattern similar to that observed in patients, strongly suggesting that the whole pathogenic effect of FS light chains can be ascribed to a peculiar structure of the V domain. Morphologic alterations of the kidney tubular cells, which contained rhomboid-shape crystals, were observed in mice, together with alterations of the proximal tubule reabsorption function. Moreover, the number of renal crystalline inclusions was dramatically reduced after conditional deletion of the human VkappaI transgene, showing that proximal tubular lesions are reversible upon suppression of the nephrotoxic light chain secretion.
Asunto(s)
Síndrome de Fanconi/etiología , Región Variable de Inmunoglobulina/fisiología , Cadenas kappa de Inmunoglobulina/fisiología , Enfermedades Renales/patología , Animales , Anticuerpos Monoclonales , Síndrome de Fanconi/patología , Humanos , Enfermedades Renales/etiología , Túbulos Renales/patología , Ratones , Ratones TransgénicosRESUMEN
Guillain-Barré syndrome is associated with antecedent Campylobacter jejuni infection. Only a minority of the infected individuals, however, develops the disease, implying a role for genetic factors in conferring susceptibility. To determine the role of immunoglobulin KM genes (genetic markers of the constant region of kappa chains) in the etiology of this syndrome, we genotyped 83 patients and 196 healthy controls from Norway for KM1 and KM3 alleles by polymerase chain reaction-restriction fragment length polymorphism. The frequency of KM3 homozygotes was significantly increased in patients compared with controls (86.7% vs. 74%, P=0.01, odds ratio=2.3). Conversely, the frequency of KM1/KM3 heterozygotes was significantly decreased in patients compared with controls (13.3% vs. 26%, P=0.01, odds ratio=0.4). These results suggest that KM genes may be relevant to the etiology of Guillain-Barré syndrome.
Asunto(s)
Enfermedades Autoinmunes/etiología , Síndrome de Guillain-Barré/etiología , Regiones Constantes de Inmunoglobulina/fisiología , Cadenas kappa de Inmunoglobulina/fisiología , Alelos , Antígenos Bacterianos/inmunología , Enfermedades Autoinmunes/epidemiología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Infecciones por Campylobacter/complicaciones , Campylobacter jejuni/inmunología , Enteritis/complicaciones , Gangliósidos/inmunología , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Síndrome de Guillain-Barré/epidemiología , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/inmunología , Humanos , Inmunoglobulina A/inmunología , Regiones Constantes de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Imitación Molecular , Noruega/epidemiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Infecciones del Sistema Respiratorio/complicaciones , RiesgoRESUMEN
Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Proteínas de Choque Térmico , Cadenas Ligeras de Inmunoglobulina/fisiología , Inmunoglobulina M/metabolismo , Complejos Multienzimáticos/metabolismo , Ubiquitina/metabolismo , Animales , Linfocitos B/metabolismo , Linfocitos B/ultraestructura , Transporte Biológico , Brefeldino A/farmacología , Células COS , Proteínas Portadoras/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Permeabilidad de la Membrana Celular , Chaperón BiP del Retículo Endoplásmico , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/fisiología , Cadenas lambda de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/fisiología , Cadenas mu de Inmunoglobulina/metabolismo , Lipopolisacáridos/farmacología , Ratones , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal , Transfección , Vesículas Transportadoras/metabolismoRESUMEN
Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1-B5) that interact with the variable domain of Ig kappa L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1-B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (r(s) = 0.82; p < 0.001) between the maximal percent IL-4 release induced by protein L and that induced by anti-IgE and between intact protein L and the B1-B4 fragment (r(s) = 0.90; p < 0.01). Removal of IgE bound to basophils markedly reduced the IL-4 release induced by anti-IgE, protein L, and B1-B4. Preincubation of basophils with protein L or anti-IgE caused complete cross-desensitization to subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda chains) blocked anti-IgE-induced IL-4 release, but not the releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with kappa L chains of the IgE isotype.
Asunto(s)
Proteínas Bacterianas/fisiología , Basófilos/metabolismo , Proteínas de Unión al ADN/fisiología , Inmunoglobulina E/fisiología , Cadenas kappa de Inmunoglobulina/fisiología , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Receptores de IgE/biosíntesis , Superantígenos/fisiología , Adolescente , Adulto , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Antiidiotipos/fisiología , Basófilos/efectos de los fármacos , Basófilos/inmunología , Basófilos/microbiología , Sitios de Unión de Anticuerpos , Células Cultivadas , Reacciones Cruzadas , Ciclosporina/farmacología , Regulación de la Expresión Génica/inmunología , Liberación de Histamina/efectos de los fármacos , Liberación de Histamina/inmunología , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Interleucina-13/genética , Interleucina-4/genética , Cinética , Persona de Mediana Edad , Proteínas de Mieloma/metabolismo , Peptostreptococcus/inmunología , Peptostreptococcus/patogenicidad , ARN Mensajero/biosíntesis , Secuencias Repetitivas de Aminoácido/inmunologíaRESUMEN
Calcium and phosphate metabolism abnormalities are frequent in myeloma patients and the role of renal lesions in such ionic perturbations may have been overlooked. The authors herein report the complete primary structure of a Bence Jones Vkappal light chain responsible for myeloma-associated proximal tubulopathy with increased phosphaturia. Plasma and serum biochemical evaluations indicated a proximal tubular dysfunction mainly manifested as tubular acidosis and phosphate loss. The study of a kidney biopsy showed interstitial and tubular lesions with numerous myeloma casts and peculiar features of the proximal tubular cells, which carried numerous phagolysosomal inclusions with occasional crystalline periodic striation. The nephrotoxic light chain primary structure was deduced from the bone marrow monoclonal plasma cells RNA. The kappal sequence was highly homologous to kappa chains previously characterized in patients with Fanconi syndrome. It was related to the Vkappal subgroup and was composed of a variable segment encoded by the O8/O18 germline gene rearranged to Jkappa4. The primary sequence presented unusual features restricted to the variable region, including substitutions of residues 28 and 31 in the complementary determining region 1 (CDR1) by amino acids of different charge. An unusual conformation of the kappal domain, likely resulting from somatic hypermutation, could alter the catabolism of the protein after its internalization and result in the tubular cell dysfunction. Comparison with Fanconi syndrome studies suggests that Vkappal Bence Jones proteins may damage proximal tubular cells to an extent varying according to light chain (LC) sequence and structure, either leading to crystal formation and Fanconi syndrome or inducing partial inhibition of proximal tubule function.
Asunto(s)
Anticuerpos Monoclonales/fisiología , Región Variable de Inmunoglobulina/fisiología , Cadenas kappa de Inmunoglobulina/fisiología , Túbulos Renales Proximales/patología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Secuencia de Bases , Síndrome de Fanconi/genética , Síndrome de Fanconi/inmunología , Síndrome de Fanconi/patología , Femenino , Humanos , Región Variable de Inmunoglobulina/sangre , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/sangre , Cadenas kappa de Inmunoglobulina/química , Cadenas kappa de Inmunoglobulina/genética , Túbulos Renales Proximales/inmunología , Datos de Secuencia Molecular , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Células Tumorales CultivadasRESUMEN
The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway.
Asunto(s)
Subgrupos de Linfocitos B/patología , Proteínas del Sistema Complemento/fisiología , Dermatitis por Contacto/inmunología , Inmunoglobulina M/metabolismo , Cadenas kappa de Inmunoglobulina/fisiología , Subgrupos de Linfocitos T/inmunología , Animales , Sitios de Unión de Anticuerpos/genética , Activación de Complemento/genética , Activación de Complemento/inmunología , Dermatitis por Contacto/genética , Dimerización , Oído Externo/inmunología , Edema/genética , Edema/inmunología , Femenino , Haptenos/metabolismo , Cadenas Pesadas de Inmunoglobulina/administración & dosificación , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/farmacología , Inmunoglobulina M/administración & dosificación , Cadenas kappa de Inmunoglobulina/administración & dosificación , Cadenas kappa de Inmunoglobulina/aislamiento & purificación , Cadenas kappa de Inmunoglobulina/farmacología , Inyecciones Intravenosas , Linfopenia/genética , Linfopenia/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Ratones Mutantes , Subunidades de Proteína , Trinitrobencenos/inmunologíaRESUMEN
It is widely appreciated that the isotype of the H chain of the Ab molecule influences its functional properties. We have now investigated the contribution of the isotype of the L chain to the structural and functional properties of the Ab molecule. In these studies, the L chain variable region of a murine anti-dansyl Ab was joined to either human kappa or lambda constant region domains and expressed with mouse-human chimeric H chains of the four human IgG isotypes. The resulting Abs were secreted as fully assembled molecules although, as has been previously observed, IgG4 with either kappa or lambda L chains was also secreted as HL half-molecules. However, the isotype of the L chain can influence the kinetics of intracellular assembly with IgG1lambda, IgG2lambda, and IgG4lambda assembling more slowly than their kappa counterparts. The isotype of the L chain also influenced the susceptibility of the interchain disulfide bonds to attack by reducing agents with variable effects, depending on the isotype of the H chains. For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain influenced the rate of clearance in mice, with IgG2lambda having a shorter in vivo half-life than IgG2kappa. Only slight differences were also observed between lambda and kappa molecules in their kinetics of binding to and dissociation from the hapten dansyl. These studies demonstrate that the isotype of the L chain has only a slight impact on the structural and functional properties of variable region identical Abs.
Asunto(s)
Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Cadenas kappa de Inmunoglobulina/fisiología , Cadenas lambda de Inmunoglobulina/fisiología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Activación de Complemento , Pruebas Inmunológicas de Citotoxicidad , Ditiotreitol/farmacología , Semivida , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/metabolismo , Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/fisiología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Cadenas kappa de Inmunoglobulina/química , Cadenas lambda de Inmunoglobulina/química , Cinética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Receptor editing is a means by which immature bone marrow B cells can become self-tolerant. Rearrangements of heavy (H) and/or light (L) chain genes are induced by encounter with autoantigens to change the specificity from self to nonself. We have developed site-directed transgenic mice (sd-tg) whose transgenes code for the H chain of antibodies that bind DNA. B cells that express the transgenic H chain associate mainly with four of the 93 functional Vkappa genes of the mouse. Numerous aspartate residues that might inhibit DNA binding by the V(H) domain distinguish these L chain Vkappa sequences, but engaging these Vkappa editors often requires multiple rearrangements. Among the edited B cells is a subset of multispecific cells that express multiple receptors. One consequence of multispecificity is partial autoreactivity; these multispecific B cells may contribute to autoimmunity.
Asunto(s)
Anticuerpos Antinucleares/genética , Autoantígenos/inmunología , Autoinmunidad/genética , Subgrupos de Linfocitos B/inmunología , ADN/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Reordenamiento Génico de Cadena Ligera de Linfocito B , Genes de Inmunoglobulinas , Cadenas kappa de Inmunoglobulina/fisiología , Autotolerancia/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Antinucleares/química , Anticuerpos Antinucleares/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Subgrupos de Linfocitos B/metabolismo , Codón/genética , Hibridomas/inmunología , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/química , Cadenas kappa de Inmunoglobulina/genética , Punto Isoeléctrico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Reacción en Cadena de la Polimerasa , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransgenesRESUMEN
We evaluated the effect of eight species of light chains on cultured human kidney proximal tubule cell proliferation. Exposure to light chains for 48 hours caused dose-dependent inhibition in tritium ((3)H)-thymidine incorporation by simian virus 40 immortalized human proximal tubule cells, although the effect was variable among different species of light chains. We studied cytotoxic effects of selected toxic light chains in further detail. Two of these light chains caused significant DNA degradation. A lambda-light chain caused lactate dehydrogenase release from exposed cells at 48 hours, but not at 24 hours. Cytomorphological and electron microscopic examination of cells exposed to light chains for 24 hours showed condensed nuclei, cell detachment, paucity of mitotic activity, and apoptosis, and at 48 hours of exposure, changes consistent with necrosis. Apoptosis assay by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method showed a sixfold increase in the number of apoptotic cells exposed to the same lambda-light chain for 24 hours. Rhodamine-phalloidin staining showed variable but significant disruptions in the actin cytoskeleton. These studies show that some myeloma light chains are toxic to cultured human proximal tubule cells and induce cytoskeletal injury and DNA damage consistent with apoptosis followed by secondary necrosis. Direct proximal tubule cell toxicity may be an important mechanism of renal involvement in multiple myeloma.
Asunto(s)
Muerte Celular/fisiología , Cadenas kappa de Inmunoglobulina/fisiología , Cadenas lambda de Inmunoglobulina/fisiología , Túbulos Renales Proximales/citología , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Apoptosis , División Celular , Células Cultivadas , ADN/biosíntesis , Relación Dosis-Respuesta Inmunológica , Humanos , Túbulos Renales Proximales/patología , L-Lactato Deshidrogenasa/metabolismo , Necrosis , Timidina/metabolismoRESUMEN
The B cell antigen receptor (BCR) is a large complex that consists of a disulfide-linked tetramer of two transmembrane heavy (mu) chains and two light (lambda or kappa) chains in association with a heterodimer of Igalpha and Igbeta. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a transforming protein called K1, which has structural and functional similarity to Igalpha and Igbeta. We demonstrate that K1 downregulates the expression of BCR complexes on the surface. The NH(2)-terminal region of K1 specifically interacts with the mu chains of BCR complexes, and this interaction retains BCR complexes in the endoplasmic reticulum, preventing their intracellular transport to the cell surface. Thus, KSHV K1 resembles Igalpha and Igbeta in its ability to induce signaling and to interact with mu chains of the BCR. However, unlike Igalpha and Igbeta, which interact with mu chains to direct BCR complexes to the cell surface, K1 interacts with mu chains to block the intracellular transport of BCR complexes to the cell surface. These results demonstrate a unique feature of the K1 transforming protein, which may confer virus-infected cells with a long-term survival advantage.
Asunto(s)
Herpesvirus Humano 8/fisiología , Proteínas de la Membrana/fisiología , Receptores de Antígenos de Linfocitos B/fisiología , Proteínas del Envoltorio Viral/fisiología , Proteínas Virales/fisiología , Sitios de Unión , Línea Celular , Regulación hacia Abajo , Herpesvirus Humano 8/genética , Humanos , Cadenas kappa de Inmunoglobulina/química , Cadenas kappa de Inmunoglobulina/fisiología , Cadenas lambda de Inmunoglobulina/química , Cadenas lambda de Inmunoglobulina/fisiología , Cadenas mu de Inmunoglobulina/química , Cadenas mu de Inmunoglobulina/fisiología , Proteínas de la Membrana/genética , Sistemas de Lectura Abierta , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/genética , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genéticaRESUMEN
T cell tolerance is established and maintained through various mechanisms, the critical component being the persistence of the specific Ag. However, at the molecular level, the nature of the recovering TCR repertoire following breakdown of tolerance is unknown. We address this important question by following kappa light chain constant region (C kappa)-specific CD4+ T cells of kappa light chain knock-out (kappa-/-) mice born to kappa+/- mothers. These cells, which were in contact with maternal kappa+ Igs from early ontogeny until weaning, were strongly tolerized. Tolerance was reversible and waned with the disappearance of peptide C kappa 134-148 presentation in lymphoid organs, including the thymus. Whereas three specific V beta-J beta rearrangements emerged in the peptide C kappa 134-148-specific CD4+ T cell response of all regular kappa-/- mice, soon after breakdown of tolerance only one of these rearrangements was detected. The two others displayed a significant delay in reappearance and were still rare at 26 wk of age, while the control proliferative response had already recovered 3 mo earlier. At 52 wk of age, a complete recovery of the three canonical V beta-J beta rearrangements was observed. Thus, although profoundly perturbed for several months, the T cell repertoire returns to equilibrium, highlighting the resilient nature of this system.
Asunto(s)
Tolerancia Inmunológica , Cadenas kappa de Inmunoglobulina/fisiología , Intercambio Materno-Fetal/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos/genética , Animales Recién Nacidos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Epítopos de Linfocito T/biosíntesis , Epítopos de Linfocito T/genética , Femenino , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Tolerancia Inmunológica/genética , Regiones Constantes de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Activación de Linfocitos/genética , Masculino , Intercambio Materno-Fetal/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , Embarazo , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Abs using the kappaII-A2 V gene segment predominate the human Ab repertoire to the Haemophilus influenzae b (Hib) polysaccharide (PS). All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain complementarity-determining region-3 (CDR-3) having an insertional arginine (Arg) at position 95a, the V-J junction. These findings suggest an essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing chain recombination experiments in which a series of A2 L chains, differing at position 95a, were combined individually with an Fd region known to generate a Hib PS-combining site when paired with an A2-Arg(95a)-Jkappa1 V region. Hib PS binding of the recombinant Fabs was evaluated quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with Jkappa1 gave equivalent and strongest binding to Hib PS. Fabs having A2-Jkappa1 L chains with either tyrosine, glycine, alanine, leucine, serine, or threonine in position 95a, or having an A2-Arg(95a)-Jkappa3 L chain, gave intermediate binding. Fabs having A2-Jkappa1 L chains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the importance of L chain junctional residue, as well as Jkappa usage and CDR-3 length, in determining Hib PS-binding affinity. Contrary to expectation, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.