RESUMO
The presence of additional N-glycans in the Fab region of IgG has shown to dramatically modify the properties and functionality of these molecules including changes in antibody affinity and stability. However, the underlying molecular mechanism responsible for the presence or absence of these glycans remains unknown. Polypeptide N-linked glycosylation is catalyzed in the lumen of the endoplasmic reticulum by the oligosaccharyltransferase complex. Mammalian cells can express two isoforms of the oligosaccharyltransferase catalytic subunit (STT3-A and STT3-B), which are endowed with distinct enzymatic properties. In this work we employed a murine hybridoma cell culture to study whether the expression of STT3 isoforms could be modulated by progesterone, thus altering the pattern of IgG N-glycosylation. We found that progesterone induces a switch of STT3 isoform expression, increasing IgG N-glycosylation. These effects were dependent on the progesterone-induced blocking factor (PIBF), whose concentration was modulated by progesterone. PIBF was previously found to be an immunomodulatory molecule relevant for the maintenance of pregnancy. We concluded that the STT3-B/STT3-A ratio modulates the N-glycosylation level of IgG, in agreement with previous data showing that full N-glycosylation of polypeptides requires cooperation between both catalytic isoforms. This work provides the first evidence that STT3 isoforms can be hormonally modulated, with marked consequences on IgG N-glycosylation.
Assuntos
Hexosiltransferases/metabolismo , Imunoglobulina G/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Gravidez/metabolismo , Progesterona/farmacologia , Isoformas de Proteínas/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Dinitrofenóis/imunologia , Regulação da Expressão Gênica/imunologia , Glicosilação/efeitos dos fármacos , Hibridomas , Imunomodulação , Camundongos , Proteínas da Gravidez/imunologiaRESUMO
Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis.
Assuntos
Hipersensibilidade Tardia/prevenção & controle , Tolerância Imunológica/imunologia , Proteínas/imunologia , Administração Oral , Animais , Antígenos/administração & dosagem , Efeito Espectador , Carragenina/imunologia , Dinitrofenóis/imunologia , Eosinofilia/imunologia , Eosinofilia/prevenção & controle , Feminino , Hipersensibilidade Tardia/imunologia , Imunidade Celular , Imunidade nas Mucosas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ovalbumina/imunologia , Peritonite/imunologia , Peritonite/prevenção & controle , Proteínas/administração & dosagemRESUMO
In this study, we investigated the influence of intracellular cyclic adenosine monophosphate (cAMP) changes on the rat mast cell hyporesponsiveness following immunological and non-immunological stimuli. Compared with mast cells from normal rats, those recovered from 21-day diabetic animals showed a significant augmentation in the intracellular levels of cAMP, in directly correlated with secretion of lower amounts of histamine after stimulation with antigen, bradykinin and compound 48/80 in vitro. Incubation of normal mast cells with selective inhibitors of phosphodiesterase type 4 (PDE 4) rolipram, NCS 613 and RP 73401, or the cell permeable analogue N6-2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db cAMP), led to a decrease of histamine secretion in vitro. However, the effectiveness of either NCS 613 or db cAMP in inhibiting antigen-induced degranulation is comparable in both normal and diabetic mast cells. We suggest that (a) there is a close correlation between higher levels of intracellular cAMP and hyporesponsiveness of diabetic mast cells, phenomena probably associated with a reduction in the expression and/or activity of PDE 4 and that (b) the mechanism of cAMP-mediated down-regulation of mast cell function is saturated in diabetic rats.
Assuntos
AMP Cíclico/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Mastócitos/fisiologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Antígenos/farmacologia , Bradicinina/farmacologia , Bucladesina/farmacologia , Degranulação Celular/efeitos dos fármacos , Separação Celular , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Diabetes Mellitus Experimental/metabolismo , Dinitrofenóis/imunologia , Relação Dose-Resposta a Droga , Imunoglobulina E/imunologia , Masculino , Mastócitos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Pleura/citologia , Pleura/efeitos dos fármacos , Ratos , Rolipram/farmacologia , Estimulação Química , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The aim of this study was to investigate the role of immunoglobulin E (IgE) in the late phase reaction (LPR) of murine experimental asthma. Our model consisted of an implant of DNP-conjugated, heat-coagulated hen's egg white (DNP-EWI), followed 14 days later by an intratracheal challenge with aggregated DNP-ovalbumin. Airway inflammation was analyzed 48 h after challenge and compared with a similarly immunized group of mice with highly suppressed humoral response due to anti-micro and anti-delta antibody treatment. Total number of cells in the bronchoalveolar lavage (BAL) (with predominance of eosinophils) and EPO activity in the lung homogenate were increased in the DNP-EWI-immunized group compared with immunosuppressed or nonimmunized mice. However, the cellular infiltration and EPO activity observed in the immunosuppressed group were still significantly above those obtained in the nonimmunized group, indicating that inhibition of antibody production did not completely prevent the inflammatory manifestations in BAL and lung. Airway hyperresponsiveness to methacoline was obtained in DNP-EWI-immunized mice, but the respiratory mechanical parameters returned to normal levels in the immunosuppressed group. When these mice were reconstituted with monoclonal anti-DNP antibodies, only IgE, but not IgG1, restored lung inflammation and decreased the conductance of the respiratory system, therefore, increasing hyperresponsiveness. These results indicate that antibodies are not essential for induction of LPR in the lung. However, IgE enhances pulmonary inflammation and hyperresponsiveness.
Assuntos
Formação de Anticorpos , Asma/fisiopatologia , Hiper-Reatividade Brônquica/imunologia , Imunoglobulina E/imunologia , Animais , Asma/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Dinitrofenóis/imunologia , Clara de Ovo , Peroxidase de Eosinófilo , Eosinófilos/enzimologia , Eosinófilos/patologia , Imunoglobulinas/sangue , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Peroxidases/metabolismoRESUMO
OBJECTIVE: To develop mouse monoclonal and rabbit polyclonal antibodies against immunoglobulin of Argentine boa constrictors and to demonstrate the ability of these reagents to detect antibody responses in boa constrictors by use of an ELISA and western blot analysis. ANIMALS: Two 3-year-old Argentine boa constrictors. Procedure-Boa constrictors were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each snake received biweekly inoculations of 250 microg of DNP-BSA (half SC, half IP) for a total of 6 inoculations followed by monthly inoculations for 3 months. Preimmune blood samples were collected. Subsequently, blood was collected immediately prior to each booster inoculation. Anti-DNP antibodies were isolated from immune plasma samples by affinity chromatography. Affinity-purified boa anti-DNP immunoglobulin was used for production of polyclonal and monoclonal antibodies. An ELISA and western blot analysis were used to monitor immune responses, for purification of boa anti-DNP immunoglobulin, and for assessment of polyclonal and monoclonal antibody specificity. RESULTS: A 6-fold increase in optical density (OD405) of immune boa plasma, compared with preimmune plasma, was detected by the polyclonal antibody, and a 12- and 15-fold increase was detected by monoclonal antibodies HL1787 and HL1785, respectively, between weeks 4 and 8. Results of western blot analysis confirmed anti-DNP antibody activity in immunized boa plasma and in affinity column eluates. Polyclonal and monoclonal antibodies detected specific anti-DNP antibody responses in immunized boas. CONCLUSIONS AND CLINICAL RELEVANCE: Polyclonal and monoclonal antibodies recognized boa constrictor immunoglobulin. These antibodies may be useful in serologic tests to determine exposure of snakes to pathogens.
Assuntos
Anticorpos/imunologia , Boidae/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Anticorpos/sangue , Anticorpos/isolamento & purificação , Argentina , Western Blotting , Reações Cruzadas , Dinitrofenóis/imunologia , Feminino , Masculino , Soroalbumina Bovina/imunologia , Especificidade da EspécieRESUMO
The objective of the present study was to investigate whether the injection of a tolerated protein (indirect effects) affects the formation of granulomas around Schistosoma mansoni eggs trapped in the lungs after intravenous (iv) injection into normal (noninfected) C57BL/6 mice (6 animals per group). To induce oral tolerance to chicken egg ovalbumin a 1/5 dilution of egg white in water was offered ad libitum in a drinking bottle for 3 days. Control mice received water. After 7 days, control and experimental animals were injected iv with 2,000 S. mansoni eggs through a tail vein. In some mice of both groups the iv injection of eggs was immediately followed by intraperitoneal (ip) immunization with 10 micro g of dinitrophenylated conjugates of ovalbumin (DNP-Ova) emulsified in complete Freund's adjuvant (CFA) or only CFA; 18 days later, mice were bled and killed by ether inhalation. The lungs were fixed in formalin and embedded in paraffin. Serial sections of 5 m were stained with Giemsa, Gomori's silver reticulin and Sirius red (pH 10.2). Granuloma diameters were measured in histological sections previously stained with Gomori's reticulin. Anti-DNP and anti-soluble egg antigen (SEA) antibodies were analyzed by ELISA. In mice orally tolerant to ovalbumin the concomitant ip injection of DNP-Ova resulted in significantly lower anti-SEA antibodies (ELISA*: 1395 +/- 352 in non-tolerant and 462 +/- 146 in tolerant mice) and affected granuloma formation around eggs, significantly decreasing granuloma size (area: 22,260 +/- 2478 to 12,993 +/- 3242 m ). Active mechanisms triggered by injection of tolerated antigen (ovalbumin) reduce granuloma formation.
Assuntos
Antígenos de Helmintos/imunologia , Dinitrofenóis/imunologia , Granuloma/parasitologia , Haptenos/imunologia , Pneumopatias Parasitárias/imunologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Schistosoma mansoni/imunologia , Administração Oral , Animais , Dinitrofenóis/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Granuloma/imunologia , Granuloma/patologia , Haptenos/administração & dosagem , Tolerância Imunológica , Pneumopatias Parasitárias/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
In this study, we postulated that repeated cycles of IgE passive sensitisation and antigen challenge may play a role in up-regulating eosinophil response in allergic conditions. Antigen-mediated stimulation of the pleural cavity of rats passively sensitised with a single injection of IgE anti-DNP resulted in mast cell degranulation, increase in vascular permeability and mild neutrophilia, but no pleural eosinophilia. In contrast, a second cycle of sensitisation and challenge, performed within 7 days, showed a marked eosinophilia in parallel with a lower plasma leakage and comparable neutrophilia. The eosinophilic phenomenon was not reproduced when (1) IgE sensitisation or antigen challenge was omitted in the first cycle, or (2) the first cycle was replaced by either a histamine and 5-HT dual challenge or a PAF challenge. Furthermore, we found an increase in eotaxin levels in animals subjected to two rather than one cycle of sensitisation and challenge. Treatment with the PAF receptor antagonist BN 52021 or with the lipoxygenase inhibitor zileuton, but not mast cell granule depletion, prevented the allergen-evoked eosinophil accumulation in rechallenged animals. Our results indicate that repeated cycles of IgE-driven inflammation may lead to eosinophil accumulation in a mechanism dependent on eotaxin, PAF and leukotrienes.
Assuntos
Anticorpos Monoclonais/imunologia , Dinitrofenóis/imunologia , Eosinofilia/imunologia , Imunização Passiva , Imunoglobulina E/imunologia , Pleurisia/imunologia , Soroalbumina Bovina/imunologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Quimiocina CCL11 , Quimiocinas CC/metabolismo , Dinitrofenóis/administração & dosagem , Esquema de Medicação , Eosinófilos/imunologia , Histamina/farmacologia , Imunoglobulina E/administração & dosagem , Mediadores da Inflamação/metabolismo , Mastócitos/imunologia , Ratos , Ratos Wistar , Soroalbumina Bovina/administração & dosagemRESUMO
We have previously demonstrated that there is a factor present in some human serum which inhibits the passive cutaneous anaphylaxis reaction mediated by IgE. The present study analyzes the effect of this factor on mast cell IgE-dependent tumor necrosis factor (TNF)-alpha release. Rat peritoneal mast cells and RBL-2H3 cells treated with monoclonal mouse IgE anti-dinitrophenol (anti-DNP) followed by DNP-bovine serum albumin (DNP-BSA) were used and TNF-alpha release was measured at different time points. Similarly the percentage of rat peritoneal mast cell degranulation was determined. Results show a period of 30 min as optimal incubation time for TNF-alpha release in both mast cell populations. Human serum anaphylaxis inhibitory factor enriched fraction inhibited TNF-alpha release when it was in contact with IgE before the antigen treatment. Under these conditions the percentage of mast cell degranulation decreased. Mast cells incubated before IgE treatment with the factor alone do not release TNF-alpha and the percentage of degranulation increases due to a non-IgE-dependent process. A possible role of the inhibitory factor in the later phase reaction in addition to immediate hypersensitivity described previously is suggested.
Assuntos
Imunoglobulina E/imunologia , Mastócitos/imunologia , Anafilaxia Cutânea Passiva/imunologia , Adulto , Animais , Células Cultivadas , Dinitrofenóis/imunologia , Humanos , Camundongos , Ratos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/análiseRESUMO
In the present report we established antigen dosages that induce oral tolerance of Th1 and Th2 lymphocytes or instead prime B- and Th2-dependent immune response and induce the tolerance of Th1 lymphocytes. Using different hapten-carrier systems, we found that low doses of OVA-DNP administered orally primed B and Th2 cells. On the other hand, no priming of B or Th2 cells was found in high-dose-OVA-DNP-fed rats. Low-dose-OVA-DNP-fed rats showed a strong mucosal immune response, with a high number of IgA anti-DNP antibody-forming cells in the lamina propria, while no mucosal immune response was observed in high-dose-OVA-DNP-fed rats. Thirty days after the immunization, tolerization of Th1 lymphocytes was confirmed in low- and high-dose-OVA-DNP-fed rats by diminished antigen-specific proliferation in vitro, reduced titers of anti-DNP IgG2a in serum, reduced expression of CD25 and CD134 molecules in cultured cells exposed to the antigen, reduced DTH reaction, and reduced IL-2 synthesis in culture. On the other hand, a high dose of OVA-DNP led to Th1 and Th2 tolerance, with an inhibition of specific IgG1 and IgG2a anti-DNP antibodies in serum after a parenteral challenge with OVA in CFA. This functional evidence was supported by the direct examination of IL-2 and IL-4 production. Furthermore, whereas in vitro assays seem to indicate that active suppression could be the responsible for Th1 tolerization in low-dose-OVA-DNP-fed rats, the results obtained after the transference of spleen or MLN cells to naive recipients support the idea that a subtractive mechanism is behind the tolerization of Th1 lymphocytes.
Assuntos
Antígenos/administração & dosagem , Linfócitos B/imunologia , Dinitrofenóis/administração & dosagem , Dinitrofenóis/imunologia , Tolerância Imunológica , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Células Th2/imunologia , Administração Oral , Transferência Adotiva , Animais , Formação de Anticorpos , Citocinas/biossíntese , Relação Dose-Resposta Imunológica , Feminino , Imunidade nas Mucosas , Técnicas In Vitro , Linfonodos/citologia , Linfonodos/imunologia , Ativação Linfocitária , Ratos , Ratos Wistar , Baço/citologia , Baço/imunologia , Células Th1/imunologiaRESUMO
PROBLEM: The in vitro immunomodulating effect of placental culture supernatants (PSs) obtained from two H-2k x H-2d allogeneic crossbreedings, the CBA/J x DBA/2 abortion-prone mating combination, and the reproductively normal pregnancy CBA/J x BALB/c crossbreeding were compared, and the influence of previous deliveries was evaluated. The behavior of placentae obtained from CBA/J females with two previous pregnancies by BALB/c males was also investigated. METHOD OF STUDY: Supernatants of cultures of murine placentae were added to a mouse immunoglobulin (Ig) G1 hybridoma culture which produced anti-dinitrophenol (anti-DNP) antibodies. The quantity of monoclonal antibody produced, the nature of these antibodies, and the proliferation of the hybridoma cells were studied. RESULTS: CBA/J x DBA/2 placental factors obtained from multiparous females induced a diminished asymmetric IgG antibody production without varying the quantity of antibody produced. In contrast, PSs obtained from the nonresorption-prone CBA/J x BALB/c mating combination with the same number of previous deliveries enhanced the production of both symmetric and asymmetric anti-DNP molecules and also increased the proportion of asymmetric blocking monoclonal antibodies (mAbs) synthesized by the hybridoma. Both of the PSs analyzed had induced similar inhibition of 3H-thymidine uptake. PSs obtained from the abortion-prone mating combination whose CBA/J females had two previous pregnancies by BALB/c males showed similar immunomodulating effects to those observed using multiparous CBA/J x BALB/c placentae. CONCLUSIONS: We propose that the placenta produces soluble factors that participate in the regulation of antibody synthesis by the mother during gestation. Such a placental immunomodulating effect appears to be altered in the CBA/J x DBA/2 abortion-prone mating combination and could be corrected by previous pregnancies by BALB/c males. These observations suggest that placental factors would be relevant to the protection of the fetus and might play an important role in the immune equilibrium between mother and fetus. Asymmetric antibody production as a Th2 responsiveness was also discussed.