RESUMEN
Sjögren syndrome (SS), a chronic autoimmune disorder causing dry mouth, adversely affects the overall oral health in patients. Activation of innate immune responses and excessive production of type I interferons (IFNs) play a critical role in the pathogenesis of this disorder. Recognition of nucleic acids by cytosolic nucleic acid sensors is a major trigger for the induction of type I IFNs. Upon activation, cytosolic DNA sensors can interact with the stimulator of interferon genes (STING) protein, and activation of STING causes increased expression of type I IFNs. The role of STING activation in SS is not known. In this study, to investigate whether the cytosolic DNA sensing pathway influences SS development, female C57BL/6 mice were injected with a STING agonist, dimethylxanthenone-4-acetic acid (DMXAA). Salivary glands (SGs) were studied for gene expression and inflammatory cell infiltration. SG function was evaluated by measuring pilocarpine-induced salivation. Sera were analyzed for cytokines and autoantibodies. Primary SG cells were used to study the expression and activation of STING. Our data show that systemic DMXAA treatment rapidly induced the expression of Ifnb1, Il6, and Tnfa in the SGs, and these cytokines were also elevated in circulation. In contrast, increased Ifng gene expression was dominantly detected in the SGs. The type I innate lymphoid cells present within the SGs were the major source of IFN-γ, and their numbers increased significantly within 3 d of treatment. STING expression in SGs was mainly observed in ductal and interstitial cells. In primary SG cells, DMXAA activated STING and induced IFN-ß production. The DMXAA-treated mice developed autoantibodies, sialoadenitis, and glandular hypofunction. Our study demonstrates that activation of the STING pathway holds the potential to initiate SS. Thus, apart from viral infections, conditions that cause cellular perturbations and accumulation of host DNA within the cytosol should also be considered as possible triggers for SS.
Asunto(s)
Proteínas de la Membrana/genética , Síndrome de Sjögren/genética , Animales , Autoanticuerpos/sangre , Citocinas/sangre , Citosol/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Inmunidad Innata , Interferón gamma/genética , Interferones/inmunología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Saliva/química , Transducción de Señal , Síndrome de Sjögren/inmunología , XantonasRESUMEN
OBJECTIVES: Adjuvant-induced innate immune responses have been suspected to play a role in the initiation of certain autoimmune disorders. This study investigates the role of alum, an aluminum-based adjuvant in the induction of Sjögren's syndrome-like disorder in mice. METHODS: Inbred, female New Zealand Mixed (NZM) 2758 strain of mice were injected with alum. Control mice were treated similarly with PBS. The mice were monitored for salivary gland dysfunction by measuring pilocarpine-induced salivation. Presence of lymphocytic infiltrates within the submandibular glands was studied by histopathology. Autoantibodies to Ro and La proteins were analysed by ELISA and the presence of anti-nuclear antibodies (ANA) was analysed by indirect immunofluorescence. RESULTS: By eight weeks after treatment, the saliva production in the alum-treated mice was significantly decreased in comparison to the PBS-treated mice. This functional loss persisted till the termination of experiments at 20 wks. The incidence and severity of sialoadenitis was significantly higher in the alum-treated mice. Although there were no differences in the levels of anti-Ro/La autoantibodies in sera of alum and PBS-treated groups, the alum group showed higher ANA reactivity. CONCLUSIONS: In the NZM2758 mice, alum induces a Sjögren's syndrome-like disorder that is characterised by chronic salivary gland dysfunction and the presence of lymphocytic infiltrates within the salivary glands. Thus, the potential of aluminum-based adjuvants for induction of autoimmunity should be closely monitored in individuals genetically susceptible to developing autoimmune disorders.
Asunto(s)
Aluminio , Autoinmunidad/efectos de los fármacos , Glándulas Salivales , Síndrome de Sjögren , Adyuvantes Inmunológicos/efectos adversos , Aluminio/efectos adversos , Aluminio/inmunología , Animales , Autoanticuerpos/sangre , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos , Monitoreo Fisiológico/métodos , Agonistas Muscarínicos/farmacología , Pilocarpina/farmacología , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/inmunología , Glándulas Salivales/patología , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/inducido químicamente , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/fisiopatologíaRESUMEN
In Sjögren's Syndrome (SS), inherent glandular defects, autoimmunity, and mononuclear cell infiltration within the salivary glands cause reduced salivation leading to xerostomia. Excessive production of type I interferons (IFN), triggered by environmental and genetic factors, is considered pathogenic in this disorder. However, whether type I IFN production is causative or an outcome of the disease process is not known. To address this question, we introduced a deficiency of interferon alpha receptor 1 (Ifnar1) into B6.Aec1Aec2 mice, which are known to have the genetic loci necessary for developing a SS-like disorder. This new mouse strain, B6.Aec1Aec2Ifnar1 (-/-), lacking type I IFN-mediated signaling, was characterized for pilocarpine-induced salivation, the presence of serum autoantibodies, sialoadenitis, and dacryoadenitis. Compared with the B6.Aec1Aec2Ifnar1 (+/+) (wild-type) mice, the B6.Aec1Aec2Ifnar1 (-/-) (knockout) mice had significantly lower mononuclear cell infiltration in the salivary and lacrimal glands. The knockout mice were completely protected from salivary gland dysfunction. Surprisingly, they had a robust autoantibody response comparable with that of the wild-type mice. These findings demonstrate that, in the absence of type I IFN-mediated signaling, systemic autoantibody responses can be dissociated from glandular pathology. Our study suggests that, in genetically susceptible individuals, the type I IFN pathway can instigate certain features of SS.
Asunto(s)
Receptor de Interferón alfa y beta/metabolismo , Salivación/fisiología , Sialadenitis/inmunología , Síndrome de Sjögren/inmunología , Animales , Autoanticuerpos/sangre , Dacriocistitis/genética , Dacriocistitis/inmunología , Dacriocistitis/metabolismo , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Ratones , Ratones Endogámicos , Ratones Noqueados , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Salivación/genética , Salivación/inmunología , Sialadenitis/genética , Sialadenitis/metabolismo , Transducción de Señal/genética , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/fisiopatologíaRESUMEN
OBJECTIVE: Sjögren's syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS-like disease. METHODS: Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll-like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland-infiltrating cells were characterized by flow cytometry. RESULTS: Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)-treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. CONCLUSIONS: Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS-like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.
Asunto(s)
Inmunidad Innata/inmunología , Sialadenitis/inmunología , Síndrome de Sjögren/inmunología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/patología , Quimiocina CCL11/análisis , Quimiocina CCL11/efectos de los fármacos , Quimiocina CCL2/análisis , Quimiocina CCL2/efectos de los fármacos , Quimiocina CCL3/análisis , Quimiocina CCL3/efectos de los fármacos , Quimiocina CCL4/análisis , Quimiocina CCL4/efectos de los fármacos , Quimiocina CCL7/análisis , Quimiocina CCL7/efectos de los fármacos , Quimiocina CXCL10/análisis , Quimiocina CXCL10/efectos de los fármacos , Quimiocina CXCL13/análisis , Quimiocina CXCL13/efectos de los fármacos , Quimiocinas CC/análisis , Quimiocinas CC/efectos de los fármacos , Quimiocinas CXC/análisis , Quimiocinas CXC/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/patología , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Inmunidad Innata/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos NZB , Proteínas Quimioatrayentes de Monocitos/análisis , Proteínas Quimioatrayentes de Monocitos/efectos de los fármacos , Poli I-C/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Sialadenitis/patología , Síndrome de Sjögren/patología , Enfermedades de la Glándula Submandibular/inmunología , Enfermedades de la Glándula Submandibular/patología , Receptor Toll-Like 3/agonistasRESUMEN
Histopathology of the kidney and clinical presentation are critical factors in the diagnosis of immune-mediated glomerulonephritis (GN). The histological manifestations of glomerular injury are shared by multiple underlying mechanisms. Work from our laboratory and from other investigators shows that antinuclear, antihistone or anti-dsDNA antibodies are neither required nor sufficient for development of lupus GN. In addition, antibody to dsDNA can be generated by mechanisms other than loss of tolerance to chromatin. Genetic analyses demonstrate that although there is some interaction between autoantibody production and renal disease, the phenotypes are regulated by distinct genetic intervals. Furthermore, renal failure is not an essential outcome of the immune-complex deposition and proliferative lupus GN. These data are also supported by published studies from systemic lupus erythematosus (SLE) patients. The immune regulation of lupus GN is distinct from other organ-specific diseases and not influenced by CD25(+) or NK1.1(+) regulatory T cells. Thus, fatal GN may depend upon a kidney-reactive T-cell response that, in turn, may be regulated by gender and intrinsic end-organ factors. The data discussed in this review call for a re-evaluation of the current paradigms for pathogenesis of SLE. An interactive model separating autoimmunity from end-organ susceptibility for the pathogenesis of SLE is proposed.
Asunto(s)
Autoinmunidad/inmunología , Nefritis Lúpica/inmunología , Animales , Anticuerpos Antinucleares/genética , Anticuerpos Antinucleares/inmunología , Autoinmunidad/genética , ADN/genética , ADN/inmunología , Femenino , Humanos , Nefritis Lúpica/genética , Nefritis Lúpica/patología , Masculino , Ratones , Ratones EndogámicosRESUMEN
Research on murine autoimmune ovarian disease (AOD) models suggests that the following sequence of events operate in prevention and induction of AOD. Potentially pathogenic T cells for oocyte antigens that exist in normal mice are kept in check by regulatory CD25(+) T cells. Oocyte-specific pathogenic T cells are activated when the regulation is lost, as after day 3 thymectomy, or when T cells are stimulated through molecular mimicry. Activated, proinflammatory T cells induce interstitial ovarian inflammation without disruption in ovarian function. Activated T cells also help B cells that respond to endogenous oocyte antigens, to produce oocyte autoantibodies of diversified specificities. Autoantibodies, nonpathogenic in themselves, retarget T cell-mediated inflammation to ovarian follicles resulting in ovarian atrophy and ovarian failure. Future studies should determine the applicability of these findings to human ovarian autoimmunity.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Enfermedades del Ovario/inmunología , Enfermedades del Ovario/prevención & control , Receptores de Superficie Celular , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas del Huevo/inmunología , Femenino , Humanos , Tolerancia Inmunológica , Glicoproteínas de Membrana/inmunología , Linfocitos T/inmunología , Glicoproteínas de la Zona PelúcidaRESUMEN
The detection of noninfectious ovarian inflammation (oophoritis) and serum ovarian autoantibodies in a patient with premature ovarian failure is indicative of an autoimmune etiology. The mechanisms of autoimmune ovarian injury leading to loss of function are currently unknown. In this study we investigated the impact of oophoritis on ovarian function based on two murine autoimmune ovarian disease (AOD) models. AOD can be induced by thymectomy at Day 3 after birth (d3tx). D3tx mice develop ovarian inflammation and atrophy with loss of oocytes. In these mice, ovarian atrophy and not oophoritis correlated with abnormal estrous cyclicity. The second AOD model is induced by active immunization of adult mice with a murine ZP3 peptide (pZP3) in adjuvant. After active immunization, the zona pellucida antibody titer, not oophoritis, correlated with reduced fertility. To investigate the effect of oophoritis in the absence of antibody response or ovarian atrophy, pZP3-specific T cells were passively transferred into naive syngeneic mice. This recruited cytokine-producing cells into the ovaries so that elevated cytokine production and its effect on ovarian function could be examined. Recipients of pZP3-specific T cells developed severe granulomatous oophoritis, and the diseased ovaries had elevated ovarian mRNA levels of interferon-gamma, interleukin-1beta, and tumor necrosis factor alpha. Despite these changes, fertility rates and gonadotropin-induced follicular development remained essentially normal. Therefore, normal ovarian function is compatible with severe ovarian inflammation mediated by autoreactive T cells.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Ooforitis/inmunología , Ovario/inmunología , Ovario/fisiopatología , Receptores de Superficie Celular , Células TH1/inmunología , Animales , Autoanticuerpos/sangre , Enfermedades Autoinmunes/fisiopatología , Citocinas/biosíntesis , Proteínas del Huevo/inmunología , Estro , Femenino , Infertilidad Femenina/inmunología , Interferón gamma/genética , Interleucina-1/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ooforitis/fisiopatología , ARN Mensajero/metabolismo , Timectomía , Factor de Necrosis Tumoral alfa/genética , Zona Pelúcida/inmunología , Glicoproteínas de la Zona PelúcidaRESUMEN
Immunization with zona pellucida 3 (ZP3) glycoprotein induces infertility in primates and is a target antigen for a contraceptive vaccine. However, loss of ovarian function is a long-term side effect. A possible mechanism is autoimmune ovarian disease induced by ZP3-specific autoreactive T cells, demonstrated in mice immunized with a murine ZP3 peptide in complete Freund's adjuvant. Indeed, a murine contraceptive vaccine that elicits antibodies to zona pellucida (ZP) without concomitant pathogenic T-cell activation has been achieved by a chimeric peptide (CP) consisting of a native ZP3 B-cell epitope and a foreign helper T-cell peptide. Herein, we evaluate the CP strategy in primate for human ZP3 (hZP3) vaccine development. A CP was constructed that consisted of a known helper T-cell epitope from the malarial circumsporozoite protein and a native B-cell epitope of hZP3. The human CP elicited antibodies to ZP3 in macaques without a measurable T-cell response to the hZP3 peptide. The serum antibodies reacted with macaque and human ZP and significantly inhibited human sperm binding to oocytes in vitro. Moreover, the CP elicited antibodies to human ZP in mice that lack murine major histocompatibility complex (MHC) class II molecules but express transgenic human HLA-DR3, -DQ6, or DQ8 molecules. Therefore, this study 1) provides evidence to support the feasibility of the CP strategy in hZP3 vaccine development and 2) describes a novel approach for evaluating the influence of polymorphic human MHC on vaccine immunogenicity without human immunization.
Asunto(s)
Anticonceptivos , Vacunas/inmunología , Zona Pelúcida/inmunología , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Activación de Linfocitos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Interacciones Espermatozoide-Óvulo/fisiologíaRESUMEN
Zona pellucida (ZP) glycoproteins possess sperm receptor-binding activities. Antibodies against ZP can block sperm-egg interaction and thereby prevent fertilization. The feasibility of developing a safe contraceptive vaccine based on the ZP has been hampered by the finding that active immunization with autologous or heterologous ZP proteins results in infertility that is associated with ovarian dysfunction. A mouse model was used to investigate mechanisms of the ovarian pathology that is induced by active immunization with a 13mer peptide derived from mouse ZP3 (mZP3(330-342)). This peptide includes one native B-cell epitope and two nested T-cell epitopes. Ovarian pathology could be transferred into naive recipients by CD4+ T cells, but not by antibodies, from immunized mice, suggesting the importance of T cells in the mechanism of ovarian pathogenesis. Moreover, immune responses, as well as disease induction, were restricted to H-2a,k,u,s,axb haplotypes. On the basis of this mouse model, a strategy to generate a contraceptive anti-ZP antibody response without a pathogenic T-cell response, irrespective of H-2 haplotype, is described. The B-cell epitope was modified by amino acid substitution to eliminate the overlapping oophoritogenic T-cell epitope, and was linked to a promiscuous foreign T-cell epitope, bovine RNase94-104. The resultant chimaeric peptide (CP2) induced anti-ZP antibodies in 100% of the eight strains of inbred mice with different H-2 haplotypes without significant disease induction. An antifertility trial in B6AF1 female mice immunized with CP2 showed that the anti-ZP antibody was associated with a reduction in fertility. This infertility was reversed with a decline in anti-ZP antibody titre. Preliminary data show that this strategy of vaccine design may also be applied to primates.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Anticoncepción Inmunológica , Proteínas del Huevo/inmunología , Glicoproteínas de Membrana/inmunología , Enfermedades del Ovario/inmunología , Receptores de Superficie Celular , Vacunas Sintéticas/inmunología , Zona Pelúcida/inmunología , Secuencia de Aminoácidos , Animales , Proteínas del Huevo/genética , Mapeo Epitopo , Femenino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Ovario/inmunología , Linfocitos T/inmunología , Glicoproteínas de la Zona PelúcidaRESUMEN
Female bonnet monkeys Macaca radiata (n = 8, four per group) were immunized with purified 55 kDa glycoprotein from porcine zona pellucida (ZP3) and ZP3 conjugated to the beta subunit of human chorionic gonadotrophin (beta hCG) using adjuvants permissible for human use (alum, muramyl dipeptide and sodium phthalyl derivative of lipopolysaccharide). The animals were monitored for anti-ZP3 antibody titres, biweekly progesterone concentrations, menstrual cyclicity and status of fertility. All the animals generated a good anti-ZP3 antibody response, continued to have ovulatory cycles, remained infertile in the presence of high anti-ZP3 antibody titres and showed no disturbance in cyclicity (except summer amenorrhoea). Examinations by laparoscope showed normal ovaries with developing follicles or corpora lutea on the surface. Fifty per cent of the animals conceived after a decline in antibody titres. Ovaries of animals that failed to regain fertility were examined for changes in morphology at times when anti-ZP3 antibody titres in the circulation were low and following a booster when titres were high. None of the ovaries showed any sign of inflammation or lymphocytic infiltration. Follicles at different stages of development were seen in all of the ovaries. No significant reduction in the number of follicles, except in one monkey (MRA 178), was observed. There was no increase in the numbers of atretic or degenerating follicles. The results showed that ZP3 immunization with permissible adjuvants could be used for immunocontraception without obvious ovarian changes.
Asunto(s)
Gonadotropina Coriónica , Anticoncepción Inmunológica , Proteínas del Huevo/administración & dosificación , Glicoproteínas de Membrana/administración & dosificación , Folículo Ovárico/fisiología , Fragmentos de Péptidos , Progesterona/sangre , Adyuvantes Inmunológicos , Animales , Anticuerpos/análisis , Gonadotropina Coriónica Humana de Subunidad beta , Proteínas del Huevo/inmunología , Estro/efectos de los fármacos , Femenino , Fertilidad/efectos de los fármacos , Inmunización Pasiva , Macaca radiata , Glicoproteínas de Membrana/inmunología , Ovario/anatomía & histologíaRESUMEN
A murine monoclonal antibody (MA-30) recognizing a sequential epitope on porcine zona pellucida-3 beta glycoprotein (ZP3 beta) and capable of inhibiting sperm-egg interaction was previously described. Polyclonal antibodies against a approximately 6 kDa fragment from the tryptic digest of ZP3 beta, reacting with MA-30, can also inhibit porcine gamete interaction in vitro. Partial N-terminal sequencing of the smallest fragment from the ZP3 beta tryptic digest having reactivity with MA-30 shows sequence homology with human ZP3.
Asunto(s)
Proteínas del Huevo/inmunología , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular , Interacciones Espermatozoide-Óvulo/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos , Unión Competitiva , Proteínas del Huevo/genética , Femenino , Humanos , Técnicas In Vitro , Masculino , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Porcinos , Glicoproteínas de la Zona PelúcidaRESUMEN
PROBLEM: Immunization with zona pellucida (ZP) leads to block in fertility with variable degree of ovarian dysfunctions. To design an immunocontraceptive vaccine based on synthetic peptides of zona pellucida, it is imperative to identify and define epitopes involved in sperm binding. METHOD: Epitopic domains recognized by monoclonal antibodies (MAbs) specific to either porcine ZP3 alpha or ZP3 beta glycoproteins were delineated in an enzyme-linked immunosorbent assay (ELISA) based on the ability of a MAb in solution to inhibit the binding of biotinylated ZP3 to another MAb coated on a microtitration plate. Immunoblot studies were carried out to determine the nature of reactive determinants. Porcine oocytes preincubated with MAbs were tested for sperm binding in vitro. RESULTS: Out of 23 MAbs generated, 10 had specificity for ZP3 alpha and 13 for ZP3 beta. By using these antibodies, eight epitopic domains on both ZP3 alpha and ZP3 beta were discernible. On ZP3 beta, epitopic domain DI partially overlaps with DII and DV with DVI, whereas on ZP3 alpha domains DI to DV were in close proximity with a partial overlap, suggesting the dominance of this region. All 10 MAbs against ZP3 alpha, and 10 out of 13 against ZP3 beta recognized deglycosylated forms of antigens. Seven antibodies having specificities for ZP3 alpha and ZP3 beta respectively recognized linear epitopes. MA-30, having specificity for ZP3 beta and MA-420 for ZP3 alpha and recognizing linear epitopes significantly inhibit the binding of boar sperm to porcine oocytes in vitro. CONCLUSIONS: Collectively, these studies indicate the value of utilizing MAbs for identifying and characterizing functionally significant ZP determinants. MAbs recognizing sequential epitopes will help in the elucidation of the amino acid sequence of the epitopes, which will subsequently help in design of synthetic immunocontraceptive vaccines.
Asunto(s)
Proteínas del Huevo , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular , Zona Pelúcida/inmunología , Animales , Anticuerpos Monoclonales , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Femenino , Immunoblotting , Masculino , Ratones , Ratones Endogámicos BALB C , Interacciones Espermatozoide-Óvulo/inmunología , Porcinos , Glicoproteínas de la Zona PelúcidaRESUMEN
Seven monoclonal antibodies (MAs) generated against porcine zona pellucida glycoprotein, ZP3 (comprising both ZP3 alpha and ZP3 beta) were characterized for their specificities to ZP3 alpha (MA 7 and MA 28) or ZP3 beta (MA 1, MA 2, MA 10, MA 27 and MA 30) and their relative affinities in competitive ELISA. Among the seven MAs tested, MA 28 showed the highest affinity for ZP3 and ZP3 alpha and MA 30 for ZP3 beta. All the antibodies bound to the zona pellucida in an indirect immunofluorescence assay, but only four (MA 7, MA 28, MA 10 and MA 30) were able to inhibit the binding of boar sperm to the porcine oocyte. Reduction followed by carboxyamidomethylation of the antigen or its chemical deglycosylation reduces reactivity to MA 7 and MA 10, suggesting that these antibodies read conformational or discontinuous determinants. The epitope recognized by MA 28 is sequential or conformational, stabilized by disulfide bonds while MA 30 reads a sequential determinant. ZP3 alpha digested with alpha-chymotrypsin, trypsin and V8 protease, respectively, revealed fragments in the range of 27-20 kDa with MA 28 in immunoblots. Proteolytic digests of ZP3 beta show that MA 30 recognizes approximately 14 kDa fragment of an alpha-chymotrypsin digest and a approximately 6 kDa fragment of a tryptic digest. These studies will help in delineation of smaller determinants of ZP involved in sperm binding.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas del Huevo , Epítopos/inmunología , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular , Interacciones Espermatozoide-Óvulo , Porcinos/inmunología , Zona Pelúcida/inmunología , Animales , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Femenino , Masculino , Glicoproteínas de Membrana/química , Ratones , Ratones Endogámicos BALB C/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Conformación Proteica , Glicoproteínas de la Zona PelúcidaRESUMEN
Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.