RESUMO
We investigated the effect of proteolytic enzyme treatment on the course of passive Heymann nephritis (PHN). PHN was induced by intravenous injection of Heymann antibody into Sprague Dawley rats. Protease-treated rats received intraperitoneal chymopapain and subtilisin. In rats given subnephritogenic doses of Heymann antibody (5 or 10 mg, insufficient to cause proteinuria), glomerular immune deposits were assessed by immunofluorescence and electron microscopy. In rats given 5 mg Heymann antibody and treated with protease in the heterologous phase of the disease (days 1-7), fewer animals were positive for rabbit IgG and rat IgG, as determined by immunofluorescence on day 12, compared with controls (p less than 0.01). Rats given 10 mg Heymann antibody and treated on days 1-5 were less frequently positive for rabbit IgG on day 5 than controls (p less than 0.05). When treatment was given on days 6-12 (autologous phase), fewer rats had glomerular rabbit and rat IgG compared with controls (p less than 0.025). Protease treatment of rats given nephritogenic doses of Heymann antibody (greater than or equal to 40 mg, causing proteinuria) did not result in significant differences in immunofluorescence deposits. However, protease treatment significantly reduced the number of electron dense deposits at all doses of antibody (p less than 0.01). Furthermore, rats given 60 mg Heymann antibody followed by enzyme treatment in the heterologous phase (days 1-7) or throughout the autologous phase (days 6-18) had significantly reduced protein excretion during the autologous phase compared with control rats (p less than 0.05). After onset of significant proteinuria on day 15 in rats given 40 mg Heymann antibody and treated from day 15 until day 25, there was significantly less (p less than 0.05) proteinuria on days 21-22 and 24-25 than in control rats; thus, enzymes could reverse proteinuria. In normal rats, administration of proteases did not have significant effects on urinary protein excretion, serum creatinine, or renal morphology, nor did protease affect anti-rabbit IgG antibody production in rats injected with Heymann antibody. The overall results indicate that proteolytic enzyme treatment can prevent or remove glomerular immune deposits and can prevent or reverse proteinuria.
Assuntos
Glomerulonefrite/terapia , Glomérulos Renais/imunologia , Peptídeo Hidrolases/uso terapêutico , Proteinúria/terapia , Animais , Creatinina/sangue , Imunofluorescência , Masculino , Microscopia Eletrônica , Coelhos , Ratos , Ratos EndogâmicosRESUMO
Antibody reagents were made specific for each of the two forms of human SC, FSC and BSC, which is an integral part of the sIgA molecule. With the fluorescent antibody method the cytological and histological localization of FSC, BSC, and alpha-chains has been studied in various human mucous membranes. SC was present in columnar epithelial cells of the intestines and in the cells of serous acini of bronchial and salivary glands. In contrast, SC was not found in intestinal goblet cells or cells of mucous acini of bronchial and salivary glands. In the columnar epithelial cells of the small and large bowel, FSC was present most prominently in the Golgi zone, and much less prominently in the apical cytoplasm. On the other hand, BSC and alpha-chains were located only in the apical cytoplasm in an overlapping manner. The results favor a model in which sIgA is assembled inside epithelial cells from SC, which was synthesized in the same cell, and IgA, which entered the epithelial cell after synthesis in and secretion by a plasma cell.
Assuntos
Imunoglobulina A/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Animais , Especificidade de Anticorpos , Brônquios/citologia , Brônquios/imunologia , Colo/citologia , Colo/imunologia , Colo/metabolismo , Reações Cruzadas , Células Epiteliais , Epitélio/imunologia , Fluoresceínas , Imunofluorescência , Técnicas Histológicas , Humanos , Imunodifusão , Mucosa Intestinal/citologia , Intestino Grosso/citologia , Intestino Grosso/imunologia , Intestino Grosso/metabolismo , Intestino Delgado/citologia , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Modelos Biológicos , Plasmócitos/imunologia , Coelhos/imunologia , Glândulas Salivares/citologia , Glândulas Salivares/imunologiaRESUMO
Guinea pig 7S immunoglobulins (gamma(1) and gamma(2)) consist of two groups of molecules (K and L) bearing different types of L chains (kappa and lambda). Approximately one-third of the molecules in normal guinea pig gamma(2)-globulin bear the lambda- type of L chains, and all or most of the others bear kappa-chains. kappa- and lambda-chains have similar molecular weights.
Assuntos
Imunoglobulina G , Peptídeos , Animais , Cobaias , Imunodifusão , ImunoeletroforeseRESUMO
The ability of passively administered antibody to suppress the immune response against homologous antigenic determinants while concomitantly enhancing the response against other unrelated determinants of the same antigen molecule has been established in two distinct antigen-antibody systems: (a) guinea pig gamma(2)-immunoglobulin + passive anti-F(ab')(2) antibody, where suppression of anti-F(ab')(2) antibody synthesis is accompanied by enhancement of the anti-Fc response; and (b) human secretory IgA + passive anti-serum IgA antibody, where suppression of antibody production against the alpha and L chains accompanies augmentation of the response to the secretory component. The mechanisms of the suppressive and enhancing effects are probably unrelated for the following reasons: (a) Enhancement of the response to certain determinants may be obtained without discernible suppression of the response to the homologous determinants; and (b) the F(ab')(2) fragments of passive antibody can mediate immune suppression but were not observed to enhance the response against the unrelated determinants of the same antigen molecule. Also, the timing for achieving maximum suppression or enhancement of antibody formation is not the same; enhancement was obtained only at a later time. Both the enhancement and suppressive effects were obtained with the purified gammaG fraction of antisera. This finding rules out an exclusive role of gammaM antibody in the enhancement phenomenon.
Assuntos
Anticorpos/análise , Formação de Anticorpos , Imunidade Materno-Adquirida , Animais , Especificidade de Anticorpos , Antígenos , Galinhas , Colostro/imunologia , Epitopos , Adjuvante de Freund , Cobaias , Humanos , Soros Imunes , Imunoquímica , Imunoglobulina A , Imunoglobulina G , Terapia de Imunossupressão , Isótopos de Iodo , Mieloma Múltiplo/imunologia , Proteínas de Neoplasias , Ovalbumina , Testes de Precipitina , CoelhosRESUMO
Activation of the complement system by IgA was investigated with immune complexes containing a mouse IgA myeloma protein with specificity for phosphorylcholine linked to bovine serum albumin (PC-BSA). These IgA anti-PC-BSA immune complexes activated the alternative complement pathway in mouse and guinea pig serum, while human complement was not affected. The activation proceeded with consumption of C3 but little or no consumption of C5. C3 did not bind to the IgA immune complexes during complement activation although it did bind covalently to IgG immune complexes. It is suggested that IgA immune complexes do not supply a suitable surface for C3 binding and effective alternative pathway convertase assembly; therefore, cleavage is limited and occurs primarily in the fluid phase. Without C3 binding, C5 cleavage does not occur nor can the alternative pathway activation proceed to the amplification step.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Ativação do Complemento , Via Alternativa do Complemento , Imunoglobulina A/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação de Anticorpos , Cálcio/metabolismo , Complemento C3/metabolismo , Relação Dose-Resposta Imunológica , Cobaias , Magnésio/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/imunologia , Coelhos , Soroalbumina Bovina/imunologia , Temperatura , Fatores de TempoRESUMO
The fate of mesenteric lymph node lymphoblasts labeled with either [125I]iododeoxyuridine or [3H]thymidine can be studied after intravenous transfer into syngeneic mice both by measurement of radioactivity in various organs and by combined immunofluorescence and autoradiography of recipient tissues. Many of the lymphoblasts home to the lamina propria of the small intestine within hours of transfer; of these, many visibly secrete IgA. To determine whether the cells that will ultimately secrete IgA are already committed to IgA synthesis before their arrival in the gut, mesenteric lymph node cell populations were treated with various class-specific antisera to mouse immunoglobulins before transfer. Treatment with antiserum to IgA, plus complement, reduced the fraction of injected label recovered from the recipients' intestines, and also reduced the proportion of donor (labeled) cells containing IgA. We conclude that mesenteric lymph nodes are probably the principal source of IgA-secreting plasma cells in the lamina propria of the gut, and that the cells become committed to IgA synthesis and develop cell surface IgA before emigrating. This IgA is apparently synthesized by the cells that bear it since it is not removed by extensive rinsing at 37 degrees C, a maneuver that elutes passively adsorbed immunoglobulin.
Assuntos
Linfócitos B/imunologia , Imunoglobulina A/biossíntese , Intestino Delgado/imunologia , Animais , Anticorpos Anti-Idiotípicos , Diferenciação Celular , Feminino , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmócitos/citologia , Plasmócitos/imunologia , Receptores de Antígenos de Linfócitos B/análise , TemperaturaRESUMO
To test the hypothesis that IgA nephropathy can result from a mucosal immune response, mice were orally immunized with one of three protein antigens for 14 wk. Such mice exhibited an essentially pure mucosal antibody response characterized by specific IgA-producing plasma cells in exocrine sites and specific IgA antibodies in serum. Furthermore, 73% of immunized mice had IgA and 88% had immunogen deposited in the glomerular mesangium, and 64% of immunized mice examined ultrastructurally had electron-dense mesangial deposits. All three were present concurrently in 57% of the immunized mice. No differences in regard to IgG or IgM were observed between immunized and control mice for any of these parameters. Mucosal immunization therefore can result in a specific immune response that leads to mesangial deposition of immune complexes containing IgA antibody. In its fundamental features the experimental renal lesion resembles that seen in the human disease IgA nephropathy.
Assuntos
Antígenos/administração & dosagem , Glomerulonefrite/imunologia , Imunização , Imunoglobulina A , Administração Oral , Animais , Células Produtoras de Anticorpos/imunologia , Brônquios/imunologia , Feminino , Imunoglobulina A/análise , Imunoglobulina A/biossíntese , Intestinos/imunologia , Glomérulos Renais/imunologia , Glomérulos Renais/ultraestrutura , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Ovalbumina/imunologiaRESUMO
The mechanisms by which immune complexes deposit in the glomerular basement membrane have been the subject of much debate, with the relative importance of direct deposition of circulating immune complexes (IC) vs. formation of IC in situ from the binding of circulating antibody to structural or exogenous planted antigen being at issue. In order to determine whether intact IC can deposit as such, covalently linked IC were prepared by a two-step reaction involving the bifunctional reagent toluene-2,4-diisocyanate (TDI), the antigen bovine gamma globulin (BGG), and rabbit anti-BGG antibody. Antigen and antibody were covalently cross-linked, with little self-linkage of antigen or antibody, and IC were purified by gel filtration. The net charge of the complexes was varied by chemical means, either before or after IC formation. When cationic IC were injected intravenously into mice, there was codeposition of antigen and antibody diffusely in the glomerular basement membrane (GBM), and deposits were observed ultrastructurally in the laminae rarae, interna and externa, and the lamina densa. Thus, under conditions of restricted appropriate charge, intact IC can cross the glomerular basement membrane and deposit in subepithelial sites without being excluded by size alone.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Glomérulos Renais/imunologia , Animais , Membrana Basal/imunologia , Bovinos , Endotélio/imunologia , Epitélio/imunologia , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Distribuição Tecidual , gama-GlobulinasRESUMO
Lymphoblasts from the mesenteric lymph nodes (MN) of mice home to the mammary glands of syngeneic recipients late in pregnancy and during lactation, and within hours of transfer most can be shown to contain IgA. Homing does not occur in virgins, in early pregnancy, or after weaning. Homing MN lymphoblasts are sensitive to antiserum to IgA plus complement, but not to other class-specific antisera. Thus, lymphoblasts in MN with the potential to home to the mammary gland are already committed to IgA synthesis and bear surface IgA before reaching their destination. These results explain observations, made by others, of specific IgA antibodies and IgA plasma cells in milk and colostrum after oral immunization. Under natural conditions it is likely that IgA precursor cells, after stimulation in the gut-associated lymphoid tissue by intestinal antigens, migrate to the mammary gland where they secrete antibodies which constitute an important defense mechanism of the newborn. In the absence of lactation, these cells probably form part of the normal traffic to the lamina propria of the small intestine.
Assuntos
Imunoglobulina A Secretora , Imunoglobulina A , Glândulas Mamárias Animais/imunologia , Plasmócitos/imunologia , Prenhez , Animais , Especificidade de Anticorpos , Movimento Celular , Feminino , Intestinos/imunologia , Intestinos/microbiologia , Lactação , Linfonodos/imunologia , Linfonodos/transplante , Masculino , Glândulas Mamárias Animais/citologia , Camundongos , Leite/imunologia , Plasmócitos/metabolismo , Plasmócitos/fisiologia , Gravidez , Transplante HomólogoRESUMO
An Ig molecule containing L chains and H chains similar to human delta-chains has been detected on the surface of radioiodinated murine lymphoid cells. Newborn mice have only IgM on their splenocytes. Between 10 and 15 days, the IgD-like molecule appears and increases in amount until 3 mo of age, when it is the predominant cell surface Ig in terms of radioactivity. IgD is found only in peripheral lymphoid tissues and is present in larger amounts on peripheral lymph node cells (approximately 85% of surface Ig) than on splenocytes (approximately 50%). IgD is also present in comparable amounts on cells from both nu/nu and germfree mice, indicating that its expression may be independent of both thymic influence and antigenic stimulation. These studies suggest that there is a switch from cell surface IgM to IgD that occurs during differentiation of virgin B lymphocytes in the spleen.
Assuntos
Membrana Celular/imunologia , Imunoglobulina D/análise , Linfócitos/imunologia , Fatores Etários , Animais , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Medula Óssea/imunologia , Precipitação Química , Eletroforese em Gel de Poliacrilamida , Vida Livre de Germes , Soros Imunes , Imunoglobulina M/análise , Imunoglobulinas , Técnicas Imunológicas , Linfonodos/imunologia , Ativação Linfocitária , Linfócitos/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos , Mieloma Múltiplo/imunologia , Baço/imunologia , Timo/imunologiaRESUMO
Immunoglobulin A (IgA) glycosylation, recognized as an important pathogenic factor in IgA nephropathy (IgAN), is apparently controlled by the polarity of T helper (Th) cytokine responses. To examine the role of cytokine polarity in IgAN, inbred mice were immunized by intraperitoneal priming with inactivated Sendai virus (SeV) emulsified in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), which promote Th1- or Th2-immune response, respectively, and then boosted identically twice orally with aqueous suspensions of inactivated virus. Next, some mice were challenged intranasally with infectious SeV. Mice primed with CFA or IFA had equal reductions in nasal viral titre relative to non-immune controls, and equally increased serum levels of SeV-specific IgA antibody. Mice primed with CFA showed higher SeV-specific IgG than those with IFA. Splenocytes from mice primed with IFA produced copious amounts of interleukin (IL)-4 and IL-5, but little interferon-gamma and IL-2; those primed with CFA had reciprocal cytokine recall responses. Total serum IgA and especially SeV-specific IgA from mice primed with IFA showed a selective defect in sialylation and galactosylation. Although the frequency and intensity of glomerular deposits and haematuria did not differ, glomerulonephritis in mice primed with IFA and challenged with infectious virus was more severe than in those given CFA, as judged by serum creatinine level. We conclude that the polarity of T cell cytokines controls the pattern of IgA glycosylation and exerts direct or indirect effects on functional glomerular responses to immune complex deposition.
Assuntos
Citocinas/imunologia , Glomerulonefrite por IGA/imunologia , Imunoglobulina A/imunologia , Vírus Sendai/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adjuvantes Imunológicos , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Adjuvante de Freund , Glicosilação , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Líquido da Lavagem Nasal/imunologia , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
The effect of the charge of circulating immune complexes on glomerular localization was studied in a model of passive serum sickness. Preformed immune complexes of heterogeneous or restricted charge, prepared in vitro from isoelectrically focused or chemically modified proteins, were injected intravenously into mice. The distribution of immune complexes in the kidney was compared by immunofluorescence and electron microscopy. Cationic but not anionic or electrophoretically heterogeneous immune complexes gave rise to diffuse localization in the glomerular basement membrane. The binding in subepithelial and subendothelial sites correlated with the known distribution of structural anionic sites. The observations suggest that electrostatic interactions between fixed anionic sites and immune complexes may be an important factor in glomerular trapping. Alternative mechanisms based on initial localization of excess free cationic antigen cannot be completely excluded and are also considered.
Assuntos
Complexo Antígeno-Anticorpo , Membrana Basal/imunologia , Doenças do Complexo Imune/fisiopatologia , Glomérulos Renais/imunologia , Animais , Modelos Animais de Doenças , Ponto Isoelétrico , Glomérulos Renais/ultraestrutura , Camundongos , Microscopia EletrônicaRESUMO
The therapeutic effects of saccharolytic and proteolytic enzymes were investigated in models of IgA nephropathy. Mesangial glomerulonephritis was induced in mice by intravenous injection of preformed soluble immune complexes of dextran sulfate and either IgA (J 558) or IgM (MOPC 104 E) anti-dextran MAb (passive model) or by immunization with DEAE dextran (active model). In the passive model, only 30-40% of dextranase-treated mice given IgA or IgM immune complexes had mesangial Ig or dextran deposits, compared with 100% of saline-treated controls (P less than 0.01). There was no significant difference in mice given only protease. In the active model, dextranase and protease separately each reduced glomerular dextran and C3 deposits, and hematuria (P less than 0.01). Dextranase also reduced the glomerular IgA deposits (20 vs. 100% of saline-treated mice) and the frequency and severity of mesangial matrix expansion (both P less than 0.02), but did not reduce the modest IgG or IgM codeposits. Protease reduced IgG and IgM deposits, proteinuria and mesangial hypercellularity compared with saline (P less than 0.02), but did not diminish IgA, and had no effect on mesangial matrix expansion. The combination of dextranase plus protease attenuated all components of glomerular injury as judged by clinical and pathological parameters, but inactivated dextranase plus inactivated protease had no effect on any parameter. We conclude that enzymatic digestion of antigen and antibody can reduce immune deposits, mesangial proliferation, proteinuria, and hematuria in experimental glomerulonephritis.
Assuntos
Dextranase/uso terapêutico , Glomerulonefrite por IGA/terapia , Peptídeo Hidrolases/uso terapêutico , Animais , Complexo Antígeno-Anticorpo/metabolismo , Complemento C3/metabolismo , Imunofluorescência , Mesângio Glomerular/patologia , Glomerulonefrite por IGA/patologia , Hematúria/terapia , Imunização Passiva , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Glomérulos Renais/imunologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Proteinúria/terapiaRESUMO
We sought to determine whether systemic administration of proteases ameliorates membranous nephritis induced in rats by immunization and challenge with cationic bovine gamma globulin, and whether targeting of protease to glomerular capillaries increases efficacy. Proteases substituted with biotin were targeted via the cationic protein avidin A, which by virtue of its charge has affinity for the glomerular basement membrane. Despite identical pretreatment proteinuria, rats given untargeted protease (biotin-conjugated without avidin, or unconjugated plus avidin) had significantly less proteinuria than saline-treated controls and nephrotic rats given avidin plus biotin-conjugated (targeted) protease had even less proteinuria and reduced glomerular rat IgG and C3. Among more severely nephrotic rats, targeted protease was again more effective than untargeted protease at reducing proteinuria, and also decreased the size of electron-dense glomerular deposits, hypercholesterolemia, and creatininemia. Inactivated targeted proteases had no effect on proteinuria, hypercholesterolemia, or azotemia. Finally, active targeted protease did not affect proteinuria in the nonimmune mediated nephrosis induced by puromycin aminonucleoside. We conclude that systemic protease can specifically diminish glomerular immune deposits, proteinuria, hyperlipidemia, and creatininemia associated with experimental immune complex glomerulonephritis but not toxic nephrosis, and that targeted protease is more effective than untargeted protease.
Assuntos
Endopeptidases/uso terapêutico , Glomerulonefrite/tratamento farmacológico , Albuminúria/tratamento farmacológico , Animais , Ensaio de Imunoadsorção Enzimática , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Imunoglobulina G/análise , Masculino , Nefrose/tratamento farmacológico , Proteinúria/tratamento farmacológico , Ratos , Ratos Endogâmicos , gama-Globulinas/imunologiaRESUMO
Complement-receptor lymphocytes have generally been considered to be a subpopulation of bone-marrow derived (B) lymphocytes. However, the present studies show that essentially all cells with integral surface immunoglobulin from normal human peripheral blood bear receptors for the third component of complement. Moreover, after removal of phagocytes, all cells with complement receptors bear surface Ig. Thus, circulating B cells and complement-receptor lymphocytes are the same population.
Assuntos
Proteínas do Sistema Complemento , Linfócitos/imunologia , Receptores de Antígenos de Linfócitos B , Adulto , Animais , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Membrana Celular/imunologia , Complemento C3 , Humanos , Masculino , Fagócitos/imunologiaAssuntos
Membrana Celular/imunologia , Imunoglobulina D/isolamento & purificação , Imunoglobulinas/isolamento & purificação , Linfócitos/imunologia , Animais , Eletroforese em Gel de Poliacrilamida , Cabras/imunologia , Soros Imunes , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos BALB C , Coelhos/imunologia , Baço/citologia , Trítio , Tirosina/metabolismoRESUMO
We have shown that in mice cholera toxin can be an effective adjuvant for gastrointestinal immune responses against a virus. The adjuvant properties can be increased and even dissociated from the toxic properties if virus and toxoid are covalently linked. Finally, oral immunization with these preparations of cholera toxin/toxoid and Sendai virus can be used to prime for respiratory immune responses to Sendai virus in which protection from infection correlates with IgA in the upper and with IgG in the lower respiratory tract.
Assuntos
Vírus da Parainfluenza 1 Humana/imunologia , Vacinas Virais/administração & dosagem , Administração Oral , Animais , Anticorpos Antivirais/biossíntese , Toxina da Cólera/administração & dosagem , Imunização , Camundongos , Infecções por Paramyxoviridae/prevenção & controleRESUMO
Microbial IgA proteases cleave human serum IgA1 immunoglobulin, but human secretory IgA is resistant to hydrolysis. We have found this resistance to be due to an inhibition of protease activity that is mediated by the Fab region of secretory IgA. The IgA proteases of the genus Neisseria are more sensitive to inhibition than is the protease of Streptococcus sanguis. There is also a serum inhibitor of Neisseria proteases that co-chromatographs with IgG. Monoclonal (myeloma) human IgG proteins and plasma protease inhibitors such as alpha-1-antitrypsin and alpha-2-macroglobulin do not inhibit. Human sera do not contain inhibitor to S. sanguis protease activity. We conclude that microbial IgA proteases are subject to inhibition by IgA in secretions and IgG in serum, and this activity is most consistent with being an anti-enzyme antibody. The insensitivity of S. sanguis IgA protease to inhibition is unexplained but provides further evidence that the IgA proteases are structurally diverse.
Assuntos
Imunoglobulina A Secretora/metabolismo , Neisseria gonorrhoeae/enzimologia , Neisseria meningitidis/enzimologia , Inibidores de Proteases , Serina Endopeptidases , Streptococcus sanguis/enzimologia , Sangue , Cromatografia DEAE-Celulose , Cromatografia em Gel , Colostro/imunologia , HumanosRESUMO
Disulfide bonds are a major force in stabilizing the three-dimensional structure of immunoglobulins. To determine the pattern of interchain disulfide bonding between the four H chains, four L chains and single J chain of rat dimeric IgA (dIgA), we analyzed dIgA from the LO DNP-64 hybridoma by diagonal SDS-PAGE. Bands corresponding to one, two, three and four H chains, one and two L chains and the free J chain were observed under non-reducing conditions, suggesting that the interchain disulfide bonds in rat dIgA are unstable under denaturing conditions. Similar patterns of disulfide bonding were observed in three other hybridoma or myeloma dIgAs from LOU/CN rats. In contrast, when dIgA pretreated with iodoacetamide (IA) was analyzed by the same technique, only bands corresponding to four H chains, one and two L chains and the free J chain were observed, suggesting that blocking free sulfhydryl groups stabilizes the inter-H chain disulfide bonds. Reaction of dimeric LO DNP-64 dIgA with 5,5'-dithiobis-(2-nitrobenzoic acid) or with 14C-IA demonstrated that this dIgA contains an average of 4 moles of free sulfhydryl groups per mole of protein under non-denaturing conditions and 9 moles of free sulfhydryl groups under denaturing conditions. Taken together, the results suggest that interchain disulfide bonds in rat dIgA are unstable, presumably due to the influence of nearby free sulfhydryl groups, and that non-covalent forces are critical for stabilizing the dIgA complex. The results also indicate that J chain is entirely non-covalently associated with the H chains, an apparently unique feature of rat dIgA. A model for interchain disulfide bonding in rat dIgA is proposed.
Assuntos
Imunoglobulina A/química , Cadeias Pesadas de Imunoglobulinas/química , Região de Junção de Imunoglobulinas/química , Animais , Dissulfetos , Eletroforese em Gel Bidimensional , Imunoglobulina A/isolamento & purificação , Modelos Químicos , RatosRESUMO
The protective efficacy of anti-Sendai virus IgA was compared to that of IgG after topical application of monoclonal antibodies (MAb) to the respiratory tract of mice. BALB/c mice were passively intranasally immunized with 50 microliters ascites containing equivalent ELISA titers of MAb 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Lung viral titers were determined by plaque assay 3 days following challenge. In most instances IgA MAb afforded equivalent protection to IgG MAb in that there was no significant difference in virus recovery from the lungs of animals treated with either IgA or IgG MAb, including subclasses of IgG. When IgA MAb was fractionated into monomers and oligomers, there was no inherent advantage to the oligomeric form with respect to passive protection against viral challenge. The data indicate that IgA and IgG antibodies are equally efficacious in protecting the airways from viral infection. The experiments suggest that the advantage of IgA for protecting mucosal surfaces, such as the respiratory tract, relates to the presence of a specialized mechanism for transporting oligomeric IgA across epithelial surfaces. The results also support the rationale for active mucosal immunization protocols designed to generate an IgA response.