RESUMEN
We present investigations about the mechanism of action of a previously reported 4-anilino-2-trichloromethylquinazoline antiplasmodial hit-compound (Hit A), which did not share a common mechanism of action with established commercial antimalarials and presented a stage-specific effect on the erythrocytic cycle of P. falciparum at 8 < t < 16 h. The target of Hit A was searched by immobilising the molecule on a solid support via a linker and performing affinity chromatography on a plasmodial lysate. Several anchoring positions of the linker (6,7 and 3') and PEG-type linkers were assessed, to obtain a linked-hit molecule displaying in vitro antiplasmodial activity similar to that of unmodified Hit A. This allowed us to identify the PfPYK-1 kinase and the PfRab6 GTP-ase as potential targets of Hit A.
Asunto(s)
Antimaláricos , Malaria Falciparum , Humanos , Antimaláricos/química , Plasmodium falciparum , Relación Estructura-Actividad , Malaria Falciparum/tratamiento farmacológico , EritrocitosRESUMEN
Pre-B cell lines proliferating for several months on stromal cells in the presence of interleukin 7 (IL-7) were established from fetal liver of (NZB x NZW)F1 mice. They express the B lineage-specific markers PB76, B220, and VpreB, but do not express surface immunoglobulin (sIg). Upon removal of IL-7 from the culture, they differentiate to sIg+ B cells that can then be stimulated by lipopolysaccharide to become IgM-secreting cells. Transfer of these pre-B cell lines into SCID mice leads to hypergammaglobulinemia of IgM (600-900 micrograms/ml), IgG2a (1-3 mg/ml), and IgG3 (300-500 micrograms/ml) for the next 3-5 mo. The spleen appears populated with (NZB x NZW)F1-derived pre-B cells, few B cells, and many IgM and/or IgG-producing plasma cells. In contrast, SCID mice populated with pre-B cell lines of normal (C57BL/6 x DBA/2)F1 mouse fetal liver develop normal levels of serum IgM (approximately 100-300 micrograms/ml), almost no detectable levels of IgG, and no plasma cell hyperplasia. The (NZB x NZW)F1 pre-B cell-populated SCID mice contain elevated serum titers of IgG antinuclear autoantibodies, but no retroviral gp70-specific nor erythrocyte-specific autoantibodies. Up to 20% of the SCID mice develop proteinuria as a consequence of IgG deposits in the kidney glomeruli during a 7-mo period of observation. All signs of autoimmune disease seen in these mice are independent of the sex of the SCID host. This experimental system provides a distinction between the disease-determining (NZB x NZW)F1 genes, which are expressed in the B lymphocyte lineage and cause the development of the disease, from those expressed in other cell lineages which only modulate its progression.
Asunto(s)
Enfermedades Autoinmunes/etiología , Linfocitos B/inmunología , Células Madre Hematopoyéticas/inmunología , Activación de Linfocitos , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antivirales/análisis , Línea Celular , Eritrocitos/inmunología , Femenino , Hipergammaglobulinemia/etiología , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Inmunoterapia Adoptiva , Masculino , Ratones , Ratones Endogámicos , Ratones SCIDRESUMEN
Interleukin-4 (IL-4) provides support for humoral immune responses through upregulation of T helper (Th) type 2 cell differentiation, but it is not known whether IL-4 promotes antibody-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). Here, we show that the constitutive expression of an IL-4 transgene by B cells completely prevents the development of lethal lupus-like glomerulonephritis in the (NZW x C57BL/6.Yaa)F1 murine model of SLE. This was associated with marked changes in the serum levels of IgG subclasses, rather than in the total levels of anti-DNA antibodies, with a lack of IgG3, a decrease of IgG2a, and an increase in IgG1 subclasses, and by a strong reduction in the serum levels of gp70-anti-gp70 immune complexes. This effect of the transgene appears to result from a modulation of the Th1 versus Th2 autoimmune response, since the protected mice displayed comparably modified IgG2a and IgG3 antibody response against exogenous T cell-dependent antigen, but not against T cell-independent antigens. Thus, IL-4 prevents the development of this lupus-like autoimmune disease, most likely by downregulating the appearance of Th1-mediated IgG subclasses of autoantibodies such as the IgG3 autoantibodies which have been shown to be especially nephritogenic.
Asunto(s)
Linfocitos B/inmunología , Interleucina-4/biosíntesis , Nefritis Lúpica/genética , Nefritis Lúpica/prevención & control , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/sangre , Formación de Anticuerpos , Diferenciación Celular , Elementos de Facilitación Genéticos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunoglobulina G/sangre , Cadenas Pesadas de Inmunoglobulina/genética , Interleucina-4/genética , Nefritis Lúpica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Regiones Promotoras Genéticas , Linfocitos T/inmunología , Células Th2/citología , Células Th2/inmunologíaRESUMEN
We have previously shown that long-term in vitro proliferating fetal liver pre-B cell lines derived from autoimmune-prone (NZB x NZW)F1 (BW) mice, but not normal (B6 x DBA2)F1 mice, can differentiate in severe combined immunodeficient (SCID) mice to produce elevated levels of serum immunoglobulin (Ig) M and IgG, and high titers of antinuclear antibodies The contribution of parental NZB and NZW strains to B cell abnormalities of BW hybrid mice was investigated here by preparing pre-B cells and transferring them into immunodeficient SCID- and RAG-2-targeted mice. We show that transfer of NZB pre-B cells led to a marked IgM hypergammaglobulinemia and to the production of limited amounts of IgG2a. On the other hand, the transfer of NZW pre-B cell lines led to moderately elevated IgM levels and marked hypergammaglobulinemia of IgG2a. High IgM and low IgG anti-DNA titers are found in the recipients of NZB pre-B cells, whereas those receiving NZW pre-B cells contained lower levels of IgM and high titers of IgG anti-DNA. In marked contrast, essentially identical titers of antibodies directed against a non-self-antigen, DNP, are found in all group of pre-B cell recipients. Thus, B-lineage cells of both NZB and NZW parental strains manifest abnormalities associated with the development of this lupus-like disease. Therefore, the present study strongly suggests a complex inheritance of B cell abnormalities in autoimmune-prone (NZB x NZW)F1 mice and emphasizes the critical importance of intrinsic B cell defects in the development of murine systemic lupus erythematosus.
Asunto(s)
Linfocitos B/fisiología , Lupus Eritematoso Sistémico/sangre , Animales , Anticuerpos Antinucleares/análisis , Linfocitos B/química , Hipergammaglobulinemia/complicaciones , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Hígado/citología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Ratones SCIDRESUMEN
To assess the potency of low-affinity anti-red blood cell (RBC) autoantibodies in the induction of anemia, we generated an immunoglobulin (Ig)G2a class-switch variant of a 4C8 IgM anti-mouse RBC autoantibody, and compared its pathogenic potential with that of its IgM isotype and a high-affinity 34-3C IgG2a autoantibody. The RBC-binding activity of the 4C8 IgG2a variant was barely detectable, at least 1,000 times lower than that of its IgM isotype, having a high-binding avidity, and that of the 34-3C IgG2a monoclonal antibody (mAb). This low-affinity feature of the 4C8 mAb was consistent with the lack of detection of opsonized RBCs in the circulating blood from the 4C8 IgG2a-injected mice. However, the 4C8 IgG2a variant was highly pathogenic, as potent as its IgM isotype and the 34-3C IgG2a mAb, due to its capacity to interact with Fc receptors involved in erythrophagocytosis. In addition, our results indicated that the pentameric form of the low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity. Demonstration of the remarkably high pathogenic potency of low-affinity autoantibodies, if combined with appropriate heavy chain effector functions, highlights the critical role of the Ig heavy chain constant regions, but the relatively minor role of autoantigen-binding affinities, in autoimmune hemolytic anemia.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Autoanticuerpos/sangre , Eritrocitos/inmunología , Anemia Hemolítica Autoinmune/sangre , Animales , Anticuerpos Monoclonales , Línea Celular , Citometría de Flujo , Variación Genética , Hemólisis , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Inmunoglobulina M/sangre , Inmunoglobulina M/genética , Región de Cambio de la Inmunoglobulina , Hígado/inmunología , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Receptores Fc/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Using three different Fcgamma receptor (FcgammaR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcgammaR, FcgammaRI, and FcgammaRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcgammaRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcgammaRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcgammaRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, approximately 20-100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcgammaRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcgammaRIII was revealed by the use of two different IgG2a anti-red blood cell autoantibodies, which displayed a striking preferential utilization of FcgammaRIII, compared with the high-affinity FcgammaRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcgammaRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity.
Asunto(s)
Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Anemia Hemolítica Autoinmune/etiología , Anemia Hemolítica Autoinmune/genética , Anemia Hemolítica Autoinmune/inmunología , Animales , Autoanticuerpos/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Eritrocitos/inmunología , Variación Genética , Isotipos de Inmunoglobulinas/genética , Isotipos de Inmunoglobulinas/metabolismo , Región de Cambio de la Inmunoglobulina/genética , Técnicas In Vitro , Hierro/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
During B cell development, there is an ordered expression of heavy chain variable region (VH) genes during ontogeny such that JH proximal VH genes are rearranged and expressed before the more JH distal VH genes. Thus, the relative chromosomal position of VH genes is biologically significant. We have previously employed deletion mapping to order the nine described murine VH gene families as follows: 3609-J558-(J606/VGAM3-8/S107)-3660-(X24/Q52/7183 ). (Families within parentheses were not mapped relative to each other.) In this report we continue this analysis by mapping two recently described heavy chain variable region gene families (VH10 and VH11). VH10 is located at the JH proximal end of the major cluster of J558 VH gene segments. VH11 (a very small family) is intermingled with the 3660 family. Although in general VH genes are thought to be clustered, we and others have reported some interspersion between families. To further address this issue, we have analyzed 80 recombinant phage clones containing J558 VH gene segments for the presence of other VH family genes. Our data indicate that the J558 and 3609 VH families are extensively intermingled as has recently been described for the most JH proximal Q52 and 7183 families.
Asunto(s)
Linfocitos B/fisiología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Animales , Bacteriófago lambda , Southern Blotting , Línea Celular , Mapeo Cromosómico , Análisis Mutacional de ADN , Sondas de ADN , Desoxirribonucleasa EcoRI , Vectores Genéticos , Hibridomas , Hígado , Ratones , Ratones Endogámicos BALB C , Familia de MultigenesRESUMEN
We have analyzed at the clonal level (limiting dilution assay) the repertoire of lipopolysaccharide (LPS)-responsive murine B cells committed to the production of autoantibodies characteristic of systemic lupus erythematous (SLE), i.e. anti-single-stranded DNA (ssDNA), anti-double-stranded DNA, anti-Sm and rheumatoid factors (RF). Our results demonstrated that: (1) the frequency of precursor B cells producing each lupus autoantibody (approximately 1 in every 100-400 LPS-responding B cell) was similar in two non-autoimmune (C57BL/6 and BALB/c) and four SLE-prone (NZB, (NZB x NZW)F1, MRL/MpJ and BXSB/MpJ) mice despite the marked differences in autoimmune responses in the different SLE-prone mice, and (2) the relative frequency of autoantibody-secreting precursor B cells was constant throughout life, and equally distributed among activated and resting B-cell populations and among B cells from the peritoneal cavity and spleen. The lack of association of anti-ssDNA secretion with anti-Sm or RF secretion in cultures set up with a smaller number of B cells ruled out the possibility that the similar frequency of different autoantibody-secreting cell precursors is due to the poly-specificity of IgM autoantibodies. Notably, the frequencies of autoantibody-secreting precursor cells were significantly lower, approximately 4 and 10 times, than those of anti-tetanus toxoid and anti-dinitrophenyl antibody-producing precursor B cells, respectively. The similar frequency of precursor B cells producing four different lupus autoantibodies on the one hand and the considerable variation in each autoimmune response among SLE-prone mice on the other, support the hypothesis that specific stimulatory mechanisms may govern each autoimmune response in different SLE strains of mice.
Asunto(s)
Autoanticuerpos/análisis , Linfocitos B/inmunología , Modelos Animales de Enfermedad , Lupus Eritematoso Sistémico/inmunología , Ratones Endogámicos NZB/inmunología , Ratones Endogámicos/inmunología , Ratones Mutantes/inmunología , Animales , Animales Recién Nacidos/inmunología , Autoanticuerpos/inmunología , Diferenciación Celular , Células Clonales/inmunología , Femenino , Lipopolisacáridos/farmacología , Lupus Eritematoso Sistémico/genética , Activación de Linfocitos/efectos de los fármacos , Ratones , Modelos Biológicos , Cavidad Peritoneal/patología , Ratas , Ratas Endogámicas/inmunología , Bazo/patologíaRESUMEN
A cDNA clone encoding the variable region of the heavy chain of a BALB/c antibromelinized mouse red blood cell (BrMRBC) monoclonal antibody has been characterized. The nucleic acid sequence indicates that the variable region of the heavy chain is likely encoded by variable-VCP12, diversity-DSP 2 (5, 7 or 8) and joining-JH1 germ-line genes. An identical combination of V genes was observed for six other CBA/J and NZB anti-BrMRBC hybridomas. The BALB/c VCP12 nucleotide sequence is less than 80% homologous to members of the 10 known VH families. Moreover, the genomic restriction fragments detected under moderate stringency conditions with the radiolabeled cDNA probe did not correspond to those obtained with probes of the most homologous VH families (7183 and X24). The results indicate that the VCP12 gene defines a new VH family which we propose to designate VH11. The observation of 1, 2 or 3 genomic restriction fragments at the most, with mice bearing the Igh-Vd or j, Igh-Va or b or Igh-Vc haplotypes, suggests the existence of a few VCP12-related genes.
Asunto(s)
Autoanticuerpos/genética , Eritrocitos/inmunología , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Animales , Anticuerpos Monoclonales/genética , Secuencia de Bases , Southern Blotting , Bromelaínas , Clonación Molecular , ADN/genética , Ratones , Ratones Endogámicos BALB C/genética , Datos de Secuencia Molecular , Familia de Multigenes , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
An IgG3 monoclonal antibody, 6-19, derived from unmanipulated MRL/MpJ-lpr/lpr mice, exhibiting cryoglobulin and anti-IgG2a rheumatoid factor activities, induces skin leukocytoclastic vasculitis and glomerulonephritis when injected into normal mice. To determine the role of the gamma 3 heavy chain constant region in the generation of cryoglobulins and associated tissue lesions, we have established an IgG1 class switch variant, clone SS2F8, from the 6-19 hybridoma by sequential sublining. Here we report that the SS2F8 monoclonal antibody, which loses the cryoglobulin activity but retains the rheumatoid factor activity, fails to generate skin and glomerular lesions. The lack of pathogenicity of the IgG1 SS2F8 switch variant is not due to mutations in variable regions, since nucleotide sequence analysis shows no differences between both clones. In addition, we have observed that the IgG1 SS2F8 switch variant exhibits < 10% of the rheumatoid factor activity, as compared with the IgG3 6-19 monoclonal antibody, suggesting that the self-associating property of the gamma 3 isotype promotes antibody-binding activity. The present study indicates that the cryoglobulin activity associated with the gamma 3 isotype is critically involved in the pathogenicity of 6-19 anti-IgG2a rheumatoid factor monoclonal antibody and highlights the pathogenic relevance of autoantibodies of the IgG3 subclass in murine systemic lupus erythematosus.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Glomerulonefritis/inmunología , Regiones Constantes de Inmunoglobulina/inmunología , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Factor Reumatoide/inmunología , Piel/inmunología , Vasculitis Leucocitoclástica Cutánea/inmunología , Animales , Anticuerpos Monoclonales/toxicidad , Secuencia de Bases , Clonación Molecular , Cruzamientos Genéticos , Crioglobulinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Glomerulonefritis/patología , Regiones Constantes de Inmunoglobulina/toxicidad , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/toxicidad , Inmunoglobulina G/clasificación , Inmunoglobulina G/toxicidad , Cadenas Pesadas de Inmunoglobulina/toxicidad , Riñón/inmunología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Piel/patología , Vasculitis Leucocitoclástica Cutánea/patologíaRESUMEN
Murine IgG3 anti-IgG2a rheumatoid factor (RF) monoclonal antibodies (mAb) with cryoglobulin activity, are able to induce, in normal mice, skin leukocytoclastic vasculitis and lupus-like glomerulonephritis resembling 'wire-loop' lesions (subendothelial immune deposits). The development of glomerular, but not skin, lesions in immunoglobulin-deficient mice (lacking the corresponding IgG2a autoantigen) receiving IgG3 RF cryoglobulins indicates that the RF activity of IgG3 monoclonal cryoglobulins and subsequent formation of IgG3-IgG2a immune complexes play a critical role in the development of skin vasculitis. In contrast, nephritogenic activity is solely contributed by IgG3-associated cryoglobulin activity. Polymorphonuclear leukocyte (PMN) infiltration is one of the major pathologic changes observed in both types of lesions. Treatment with mAbs against the adhesion molecules leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) (both known for their involvement in PMN-endothelial cell interaction) inhibits the development of skin vascular lesions. However, it has no effect on the generation of glomerulonephritis. Apparently, adhesion molecule requirements for PMN interaction with glomerular capillary endothelial cells are different from those for PMN infiltration of the skin. However, the PMN depletion experiment has clearly shown that PMNs play an active role in the development of 'wire-loop' glomerular lesions. In the absence of the glomerular PMN infiltration, IgG3 RF cryoglobulins induce a different type of glomerular lesion, characterized by voluminous intracapillary thrombi and mesangial deposits, yet lacking subendothelial deposits. This is consistent with the fact that the latter lesions can be induced by certain IgG3 mAbs, which are unable to provoke glomerular PMN infiltration. Finally, the activation of the complement system does not appear to play a major role in either skin or glomerular lesions induced by IgG3 RF cryoglobulins.
Asunto(s)
Crioglobulinemia/inmunología , Neutrófilos/fisiología , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales , Complejo Antígeno-Anticuerpo , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Hibridomas/inmunología , Inmunoglobulina G/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Leucocitos/patología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Endogámicos BALB C , Neutrófilos/patología , Ratas , Factor Reumatoide/inmunología , Piel/irrigación sanguínea , Vasculitis/inmunología , Vasculitis/patologíaRESUMEN
The variable (V) region sequences of six immunoglobulin M (IgM, kappa) monoclonal autoantibodies that recognize bromelinized isologous red blood cells, obtained by fusions of peritoneal cells from NZB or CBA/J nonimmunized mice with BALB/c myeloma cells, were determined by direct mRNA sequencing. The V regions of the light chains (VL) are almost identical with one another, as are the V regions of the heavy chains (VH), which, however, differ by six linked-base substitutions, depending on the strain of mice producing the autoantibodies. Such variations may reflect allelic differences. The VH segments determined have no obvious correspondence to any VH genes identified so far. They may belong to the small VH group 4, where 73% homology, at the most, can be calculated at the protein level for codons 1 to 94. Alternatively, the VH regions may be members of a new group of VH sequences not previously found. The V kappa regions appear closely homologous to members of the V kappa-9 subgroup of myeloma proteins of unknown antigen-binding specificity. The joining segments, J kappa and JH, used by the autoantibodies investigated, originate from the J kappa 2 and JH1 germ-line gene segments, respectively. The nine base-long diversity segments, D, derive from one member of the germ-line D gene SP2 family.
Asunto(s)
Autoanticuerpos/inmunología , Eritrocitos/inmunología , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Animales , Diversidad de Anticuerpos , Bromelaínas/farmacología , Eritrocitos/efectos de los fármacos , Ratones , ARN Mensajero/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Two out of five murine IgG3 anti-trinitrophenyl (TNP) monoclonal antibodies (mAb) obtained either by immunization with TNP-keyhole limpet hemocyanin (KLH) (CB1, CB5, CB6 and 4H10) or with dinitrophenyl-lipopolysaccharide (9A6), exhibited anti-IgG rheumatoid factor (RF) activity (CB6 and 4H10). The anti-IgG activity of these two anti-TNP RF was specifically inhibited by murine IgG as well as by the hapten TNP. In order to identify the structural basis for the anti-IgG activity, the nucleotide sequences encoding the VH and VL regions were determined. By comparing the V regions of the non-RF and RF anti-TNP mAb, it was found that one anti-TNP RF antibody, CB6, shares virtually identical VL and VH regions with two anti-TNP antibodies, CB1 and CB5, but markedly differs from these in the D region. Furthermore, the light chain framework region 2 (FR2) and FR3 of non-RF mAb, CB1, CB5 and 9A6, have amino acid sequences almost identical to those claimed for anti-IgG1 RF activity (Shlomchik et al., J. Exp. Med. 1986. 164: 407). Our findings suggest, at least in the case of CB6 mAb, the involvement of CDR, but not light chain FR residues, in IgG binding.
Asunto(s)
Autoanticuerpos/inmunología , Región Variable de Inmunoglobulina/genética , Factor Reumatoide/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/genética , Secuencia de Bases , Sitios de Unión de Anticuerpos , Cadenas Pesadas de Inmunoglobulina/fisiología , Cadenas Ligeras de Inmunoglobulina/fisiología , Región Variable de Inmunoglobulina/fisiología , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , ARN Mensajero/genética , Factor Reumatoide/genética , Trinitrobencenos/inmunologíaRESUMEN
Hybridoma collections from spleen or peritoneal cells of newborn or adult individuals were screened by RNA hybridization for expression of the VH11-gene family using a V-region probe VCP12, which encodes anti-BrMRBC antibodies. No VH11 expression was observed in hybridomas derived from newborn spleen cells in either BALB/c, NZB or (CBA/N x BALB/c) F1 mice (0/93). Adult NZB and BALB/c spleen cell collections contained only one hybridoma expressing VH11 (1/242). Interestingly, however, the VH11-positive hybridoma showed no anti-BrMRBC reactivity, while one anti-BrMRBC clone in the same collection expressed a Q52 VH gene. In contrast, hybridomas derived from peritoneal cells showed an absolute correlation between expression of VH11 genes and anti-BrMRBC reactivity (15/32). The high expression in the peritoneal cavity of such cells is likely the result of local positive selection.
Asunto(s)
Eritrocitos/inmunología , Genes de Inmunoglobulinas , Familia de Multigenes , Animales , Animales Recién Nacidos , Sondas de ADN , Regulación de la Expresión Génica , Hibridomas/inmunología , Ratones , Ratones Endogámicos , Cavidad Peritoneal/inmunología , Bazo/inmunologíaRESUMEN
In vivo pathological manifestations of eight monoclonal anti-mouse red blood cell (MRBC) autoantibodies obtained from unmanipulated NZB mice were determined in BALB/c mice. Three (two IgG1 and one IgG2a) of four IgG monoclonal antibodies (mAb) and two of four IgM mAb were able to induce anemia following their i.p. injection. All five pathogenic anti-MRBC mAbs reacted only with MRBC, whereas non-pathogenic anti-MRBC mAbs showed binding to different species of RBC. Competition studies suggested the presence of at least two distinct epitopes recognized by our pathogenic anti-MRBC mAb. Histological examinations revealed that anemia resulted from either marked sequestration of agglutinated MRBC in spleens and livers or erythrophagocytosis, most remarkably by Kupffer cells in livers. This difference was correlated with the ability of each mAb to mediate Fc receptor-dependent phagocytosis by macrophages. The absence of complement-mediated hemolysis in vitro and the development of anemia in C5-deficient or C3-depleted mice indicated a minor role, if any, for complement-mediated lysis in the anemia induced by our anti-MRBC mAb. Our results suggest that (i) at least two different pathogenic epitopes are implicated in autoimmune hemolytic anemia; and (ii) sequestration of agglutinated MRBC in spleens and livers and Fc receptor-dependent phagocytosis, but not complement-mediated hemolysis, are the major mechanisms for the development of autoimmune hemolytic anemia.
Asunto(s)
Anemia Hemolítica Autoinmune/etiología , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Eritrocitos/inmunología , Anemia Hemolítica Autoinmune/patología , Animales , Proteínas del Sistema Complemento/fisiología , Hemaglutinación , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Fagocitosis , Receptores Fc/fisiología , Bazo/patologíaRESUMEN
In order to understand further the processes involved in immunological injury of the kidney, we have prepared monoclonal antibodies against brush border (BB) antigens of rat proximal tubule. The 27 antibodies which constitute the basis of this report have been cloned, characterized immunochemically, and classified in three specificity groups on the basis of tissue reactivity. The first group is made up of six antibodies reacting with antigens simultaneously present on BB and glomerulus: three are directed against a high molecular weight (MW) protein which migrates with an apparent MW of 330,000; two react with a 90,000 MW protein that is present diffusely on renal and intestinal BB as well as on endothelial cells; one recognizes an antigen exclusively present on superficial tubules and glomerular epithelial cells, which could not be chemically characterized. The second group is made up of eight antibodies present on renal and intestinal BB: five react with a 120,000 MW antigen, one with a 300,000 MW antigen. The third group comprises 13 antibodies. Two are directed against antigens present within the cytoplasm or the basolateral membranes of renal tubules. Eleven react with intracellular antigens probably related to the cytoskeleton. Since they have been identified through several fusions, some of the monoclonal antibodies described are probably directed against immunodominant proteins of the BB. They open new possibilities for purifying the corresponding antigens by affinity chromatography as well as for obtaining BB preparations selectively depleted of the strongest immunogens thus favouring antibody production to previously unrecognized antigens.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Antígenos/inmunología , Túbulos Renales Proximales/inmunología , Microvellosidades/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Citoplasma/inmunología , Técnica del Anticuerpo Fluorescente , Glomérulos Renales/inmunología , Ratones , Ratas , Ratas EndogámicasRESUMEN
The differentiation of Ig+ B cells into plaque-forming cells is dependent on antigen and factors produced by T cells and/or macrophages. We describe here the production of T-cell factors termed lymphocyte promotor factors (LPF). A foetal calf serum-specific T-cell line and its clones synthesize LPF, which is defined as factors that polyclonally stimulate normal spenic T cells to differentiate into cytotoxic T lymphocytes (T-LPF) and normal splenic B cells to differentiate into plaque-forming cells into (PFC) (B-LPF) in the apparent absence of specific antigen. The proliferation of and the B-LPF production of all T-cell clones tested were foetal calf serum-specific and I-Ab-restricted. Some of these clones produced only T-LPF, some clones produced only B-LPF, and some clones produced both T-LPF and B-LPF. B-LPF stimulate the polyclonal differentiation of Ig+ B cells into PFC without the apparent need for helper T cells, is different from T-LPF, and induces almost exclusively IgM PFC. The B-LPF described in the present paper are compared with previously described T-cell factors, which stimulate antigen-specific B-cell responses or bystander B-cell responses. The conclusion is that B-LPF are probably different from B-cell growth factors, T-cell replacing factors, allogeneic effector factors, and interleukin 2.
Asunto(s)
Linfocitos B/citología , Linfocitos T/inmunología , Animales , Células Productoras de Anticuerpos , Linfocitos B/inmunología , Diferenciación Celular , Línea Celular , Mapeo Cromosómico , Células Clonales , Eritrocitos/fisiología , Genes , Antígenos H-2/genética , Técnica de Placa Hemolítica , Antígenos de Histocompatibilidad Clase II/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ovinos/inmunología , Bazo/citología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Some further aspects of B-lymphocyte-promotor factor (B-LPF) activity have been studied. This activity was present in the supernatants of certain helper-T-cell lines, and it induced polyclonal activation of Ig+ B cells into Ig-secreting cells. It was found that B-LPF induced polyclonal, terminal B-cell differentiation (1) in T-cell- and macrophage-depleted spleen cell populations, (2) in both Lyb-5- and Lyb-5+ cells as well as in small and blast-like splenic B cells, and (3) in normal rather than memory B cells. B-LPF function was neither restricted to major histocompatibility complex gene products nor to immunoglobulin allotypes. B-LPF-like activity was also produced by some B-cell lymphomas/hybrids and by the P388-D1 macrophage line. B-LPF activity was found in three MW fractions: (I) greater than 180,000 (pI greater than 7.0 and 4.5-5.5); (II) 50,000-70,000 (pI greater than 7.0; 6.0-6.5, and 4.5-5.5); and (III) 10,000-15,000 (pI greater than 7.0). All three MW forms of B-LPF activity carried antiserum 6036-defined and AB-1.9.3 monoclonal antibody-defined determinants, and they reacted with chicken gammaglobulin (CGG)-Sepharose but not with human serum albumin-Sepharose. These data indicate that the three MW forms of B-LPF activity are associated/dissociated forms of a 10,000-15,000 MW form (subunit) rather than three different molecular species with B-LPF activity. A comparative study between antigen-specific helper factors and B-LPF was hampered by the finding that the helper-T-cell hybridomas used (e.g., T85-109-45/1) only produced B-LPF in our hands. Previously, it has been described that these helper-T-cell hybrids produced CGG-specific, I-Ak-restricted helper factors. However, one surprising observation was that B-LPF produced by both T85 hybrid cells and L12 T lymphoma cells was absorbed and could be eluted from CGG-Sepharose columns. The relationship of B-LPF to other nonspecific and apparently specific T-helper-cell products is discussed in particular in the light of the observations that many immunologically active molecules are built up from 10,000-12,000 molecular weight domain-like polypeptide structures.