RESUMO
BACKGROUND: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. METHODS: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), ßIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. RESULTS: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVß3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. CONCLUSIONS: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells.
Assuntos
Helicobacter pylori , Camundongos , Animais , Helicobacter pylori/metabolismo , Astrócitos , Urease/metabolismo , Urease/farmacologia , NF-kappa B/metabolismo , Fator B do Complemento/metabolismo , Fator B do Complemento/farmacologia , Modelos Animais de Doenças , Espectrometria de Massas em Tandem , NeurôniosRESUMO
Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease characterized by involuntary bizarre movements, psychiatric symptoms, dementia, and early death. Several studies suggested neuroprotective activities of inosine; however its role in HD is yet to be elucidated. The current study aimed to demonstrate the neuroprotective effect of inosine in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats while investigating possible underlying mechanisms. Rats were randomly divided into five groups; group 1 received i.p. injections of 1% DMSO, whereas groups 2, 3, 4, and 5 received 3-NP (10 mg/kg, i.p.) for 14 days, concomitantly with inosine (200 mg/kg., i.p.) in groups 3, 4, and 5, SCH58261, a selective adenosine 2A receptor (A2AR) antagonist, (0.05 mg/kg, i.p.) in group 4, and PD98059, an extracellular signal-regulated kinase (ERK) inhibitor, (0.3 mg/kg, i.p.) in group 5. Treatment with inosine mitigated 3-NP-induced motor abnormalities and body weight loss. Moreover, inosine boosted the striatal brain-derived neurotrophic factor (BDNF) level, p-tropomyosin receptor kinase B (TrKB), p-ERK, and p-cAMP response element-binding protein (CREB) expression, which subsequently suppressed oxidative stress biomarkers (malondialdehyde and nitric oxide) and pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin-1ß) and replenished the glutathione content. Similarly, histopathological analyses revealed decreased striatal injury score, the expression of the glial fibrillary acidic protein, and neuronal loss after inosine treatment. These effects were attenuated by the pre-administration of SCH58261 or PD98059. In conclusion, inosine attenuated 3-NP-induced HD-like symptoms in rats, at least in part, via the activation of the A2AR/BDNF/TrKB/ERK/CREB signaling pathway.
Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator B do Complemento/metabolismo , Fator B do Complemento/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Doença de Huntington/induzido quimicamente , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Inosina/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Nitrocompostos , Propionatos/farmacologia , Ratos , Transdução de SinaisRESUMO
Tamoxifen is an anti-estrogenic agent for the treatment of breast cancer, while exhibiting estrogenic activity in such tissues as the uterus. This study aimed to test whether these opposite properties of tamoxifen in the uterus can be evaluated separately in vivo. We employed two transgenic murine models named, respectively, the ERE-EGFP Ar+/+ mouse and ERE-EGFP Ar-/- mouse. Both types of mice possess an enhanced green fluorescent protein (EGFP) gene regulated by four copies of estrogen response elements (EREs), while the latter lacks a functional aromatase gene, which encodes an enzyme catalyzing conversion of androgens to estrogens. Tamoxifen clearly exhibited estrogenic activity in the uteri of ERE-EGFP Ar-/- mice, as it caused uterine wet weight gain and E2-target gene induction, as 17beta-estradiol (E2) did. However, tamoxifen did not enhance the EGFP expression in ERE-EGFP Ar-/- mice, although E2 induced it significantly. In ERE-EGFP Ar+/+ mice, tamoxifen suppressed the EGFP expression in a time- and dose-dependent manner. Thus, the present study demonstrated that estrogenic and anti-estrogenic activities of tamoxifen can be evaluated by using ERE-EGFP Ar-/- and ERE-EGFP Ar+/+ mice, respectively. Furthermore, these animal models are useful to select and evaluate estrogenic and anti-estrogenic activities of chemical compounds.
Assuntos
Antagonistas de Estrogênios/farmacologia , Proteínas de Fluorescência Verde/biossíntese , Elementos de Resposta , Tamoxifeno/farmacologia , Animais , Complemento C3/efeitos dos fármacos , Fator B do Complemento/farmacologia , Dieta , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Lactoferrina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Testículo/efeitos dos fármacos , Ativação Transcricional , Útero/efeitos dos fármacosRESUMO
Deficiency of complement components within the classical pathway is associated with increased risk for immune complex disease. However, C2-deficient individuals often have a mild disease and about 50% are healthy. To study the importance of the different components for immune complex clearance, bovine serum albumin (BSA)/anti-BSA complexes were opsonized in human serum and the binding to erythrocyte complement receptor type 1 (CR1, CD35) was measured in vitro. In C2-depleted serum the complexes were opsonized and bound to CR1 but the reaction needed a longer opsonization time than in normal human serum (NHS). In contrast, serum reagent lacking C1q, C4 or C3 did not promote binding in this assay system. We also demonstrated that elevated levels of factor B could restore binding of complexes to erythrocytes in C2-depleted serum via alternative pathway activation. These results indicate that in spite of lack of a complete classical pathway, C2-deficient individuals could retain some immune complex opsonizing activity via the alternative pathway. This finding could contribute to the understanding of differences in association between complement deficiency and immune-complex disease.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Complemento C2/fisiologia , Via Alternativa do Complemento , Via Clássica do Complemento , Eritrócitos/metabolismo , Receptores de Complemento 3b/metabolismo , Fator B do Complemento/farmacologia , HumanosRESUMO
C3 nephritic factor (C3NeF) is an autoantibody produced by patients with membranoproliferative glomerulonephritis (MPGN); it is thought to be responsible for the continued breakdown of C3 in these patients. We have studied three patients with MPGN for periods of 7 to 17 years who were noted to develop a normal C3 and factor B level in the face of persistent circulating C3NeF. In addition, C3NeF obtained from other sera were inactive when added to these patients' sera. When C3NeF was isolated from these patients' sera, however, it was completely active when added to normal human serum as a source of C3 and factor B. Removal of the C3 and factor B from these patients' sera by passage over anti-C3 and anti-factor B columns followed by reconstitution with C3 and factor B isolated from normal serum allowed C3NeF to be active in these sera. Replacement of C3 alone did not restore activity to these sera but replacement with factor B alone restored full C3NeF activity. These data suggest some alteration in autoantigen (factor B) as a mechanism for reduction in autoantibody (C3NeF) activity.
Assuntos
Autoanticorpos/farmacologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Fator Nefrítico do Complemento 3/farmacologia , Glomerulonefrite Membranoproliferativa/imunologia , Adulto , Reações Antígeno-Anticorpo/efeitos dos fármacos , Autoanticorpos/sangue , Autoanticorpos/metabolismo , Autoantígenos/sangue , Complemento C3/metabolismo , Fator Nefrítico do Complemento 3/metabolismo , Fator B do Complemento/metabolismo , Fator B do Complemento/farmacologia , Feminino , HumanosRESUMO
Vitronectin occupies the metastable binding site of C5b-7, which is unable to insert membranes as part of the complement lytic attack. Some evidence has been presented that vitronectin inhibits also membrane-associated pore formation by inhibiting C9 polymerization in the terminal complement complex (TCC). The authors wished to add to this background by studying the effect of vitronectin on formation of TCC on a carbohydrate surface like agarose beads, an alternative complement pathway activator. Bound TCC was detected by monoclonal and polyclonal antibodies to C9-neoepitopes. Soluble SC5b-7 and TCC (SC5b-9) did not bind to the agarose beads. Using serum or isolated complement factors for the alternative and terminal pathways, the authors found that vitronectin reduced the density of C9-neoepitopes on the beads. As there was no convincing evidence for association of vitronectin with the factors C5b-8 of the agarose-bound TCC, it was concluded that vitronectin bound directly to C9 in TCC and inhibited C9 polymerization within the complex. The authors have shown that TCC can bind to a carbohydrate surface like agarose (an alternating polymer of galactose moieties) in the absence of lipid. These results suggest that vitronectin can limit the lytic effect of membrane-bound TCC by inhibiting C9 polymerization.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Microesferas , Sefarose , Animais , Complemento C9/metabolismo , Fator B do Complemento/farmacologia , Complexo de Ataque à Membrana do Sistema Complemento/efeitos dos fármacos , Glicoproteínas/sangue , Glicoproteínas/farmacologia , Humanos , Polímeros , Solubilidade , VitronectinaRESUMO
Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.
Assuntos
Proteínas Sanguíneas/farmacologia , Via Alternativa do Complemento/efeitos dos fármacos , Eosinófilos/química , Ribonucleases , Fator B do Complemento/farmacologia , Fator D do Complemento/farmacologia , Relação Dose-Resposta a Droga , Proteínas Granulares de Eosinófilos , Humanos , Técnicas In Vitro , Peroxidases/farmacologiaRESUMO
High molecular weight B cell growth factor (HMW-BCGF) and the complement component, Factor B, are antigenically related. HMW-BCGF and the physiologic Factor B activation fragment Bb, are both mitogenic for B lymphocytes and compete for binding to the B cell plasma membrane (Peters, M., Ambrus, J. L., Jr., Fauci, A., and Brown, E. (1988) J. Exp. Med. 168, 1225-1235). To understand which second messengers that occur after ligand-receptor interaction are associated with mitogenesis, we have examined the early signaling events after stimulation of activated B cells with these related growth factors. HMW-BCGF but not Bb increased [cAMP]i with a maximum between 45 and 60 min after stimulation. The increase in [cAMP]i was inhibited by indomethacin, suggesting that prostaglandin synthesis is involved in this response. Increase in [cAMP]i induced by HMW-BCGF, cholera toxin, or dibutyryl cAMP was associated with increased expression of the HMW-BCGF receptor, but there was no increase in proliferation of activated B cells when they were stimulated with cAMP agonists other than HMW-BCGF. These data suggest that cAMP is associated with regulation of receptor expression but is neither necessary nor sufficient for induction of proliferation. Both HMW-BCGF and Bb increased cellular levels of diacylglycerol and a water-soluble molecule which could be labeled with both [3H]myoinositol and [14C] glucosamine. However, only HMW-BCGF induced increases in intracellular calcium. Thus, two antigenically related B cell growth factors, HMW-BCGF and Bb, produce overlapping but distinct sets of second messengers after incubation with Sac-activated B cells. Since both induced increases in diacylglycerol and water-soluble inositol, one or both of these molecules may be involved in the proliferative signal generated by the related growth factors. In contrast, the increase in [cAMP]i caused by HMW-BCGF but not Bb is involved in the signal to increase HMW-BCGF receptor expression, but is unrelated to proliferation.
Assuntos
Linfócitos B/efeitos dos fármacos , Fator B do Complemento/farmacologia , Interleucina-4/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos B/citologia , Linfócitos B/metabolismo , Bucladesina/farmacologia , Divisão Celular , Toxina da Cólera/farmacologia , AMP Cíclico/biossíntese , Diglicerídeos/biossíntese , Ácidos Graxos/metabolismo , Glucosamina/metabolismo , Humanos , Inositol/metabolismo , Peso Molecular , Sistemas do Segundo MensageiroRESUMO
In a previous study, we reported that fragment Bb of bovine complement factor B activated bovine monocytes, as demonstrated by the uptake of 3H-deoxyglucose. In the present study, the effects of Bb on the production of superoxide anion and hydrogen peroxide by bovine monocytes was investigated. The production of superoxide was measured by the superoxide dismutase inhibitable reduction of cytochrome c. The change in absorbance was determined by a spectrophotometer at a wavelength of 550 nm. Hydrogen peroxide production was measured by the horse-radish peroxidase-dependent oxidation of phenol red. The resulting color change was measured by a spectrophotometer at a wavelength of 620 nm. Fragment Bb (20 micrograms/mL) induced the generation of 0.96 +/- 0.2 (mean +/- SEM) nanomoles of superoxide/2.5 x 10(5) monocytes at 5 min. The production of superoxide peaked at 15 min (1.24 +/- 0.3 nanomoles). The production of hydrogen peroxide was also rapid: 0.195 +/- 0.05 nanomoles/2.5 x 10(5) monocytes at 5 min with a peak at 15 min (0.250 +/- 0.04 nanomoles). These observations show that fragment Bb, which has serine protease activity, induces bovine monocytes to generate reactive oxygen intermediates such as superoxide and hydrogen peroxide.
Assuntos
Fator B do Complemento/farmacologia , Peróxido de Hidrogênio/metabolismo , Monócitos/metabolismo , Fragmentos de Peptídeos/farmacologia , Superóxidos/metabolismo , Animais , Bovinos , Células Cultivadas , Fator B do Complemento/fisiologia , Grupo dos Citocromos c/metabolismo , Feminino , Monócitos/imunologia , Oxirredução , Fragmentos de Peptídeos/fisiologia , Superóxido Dismutase/metabolismo , Acetato de TetradecanoilforbolRESUMO
We have previously shown that the Bb fragment of bovine complement factor B activates bovine monocytes and neutrophils. The activation was demonstrated by the enhanced uptake of 3H-deoxyglucose. To investigate the potential effect of fragment Bb on the microbicidal activity of bovine monocytes, a direct method was used. This method involves an initial ingestion period at 37 degrees C followed by repeated washing. The decrease in the total number of viable intracellular Staphylococcus aureus during the reincubation of the bacteria with bovine monocytes determines the intracellular killing. Maximal intracellular killing was seen when the monocytes containing the ingested S. aureus was incubated with fresh bovine serum (mean +/- SEM = 73.4 +/- 1.4%). On incubation of the monocytes, containing the ingested bacteria with heat-inactivated bovine serum, 32.5 +/- 0.7% of the intracellular bacteria were killed. When affinity-purified bovine factor Bb was added to the heat-inactivated serum, the intracellular killing capacity was almost restored (65.8 +/- 1.5%). When monocytes were incubated with medium alone, they killed 22.4% of the intracellular microorganisms. When fragment Bb (25 micrograms/mL) was added to the medium, the intracellular killing of S. aureus doubled (46 +/- 1.29%). We conclude that the Bb fragment of bovine complement factor B stimulates bovine monocytes in their microbicidal activity.
Assuntos
Atividade Bactericida do Sangue/imunologia , Fator B do Complemento/farmacologia , Monócitos/imunologia , Fragmentos de Peptídeos/farmacologia , Staphylococcus aureus/imunologia , Animais , Bovinos , Células Cultivadas , Fator B do Complemento/imunologia , Relação Dose-Resposta Imunológica , Feminino , Fragmentos de Peptídeos/imunologia , FagocitoseRESUMO
Normal human B lymphocyte function is finely regulated by both positive and negative signals at each stage of activation, proliferation, and differentiation. Activation signals include antigen and surface Ig cross-linking agents such as anti-mu or anti-delta. Signals inducing proliferation include IL-2, high m.w.-B cell growth factor (BCGF), and low m.w.-BCGF. IL-2 as well as IL-6 and other partially characterized B cell differentiation factors can induce terminal differentiation of proliferating B cells into Ig-secreting plasma cells. Various C components have been described to regulate B cell function including Bb that enhances proliferation, C5a that enhances Ig production, and C3a that inhibits Ig production. In our study, we examined the ability of the factor B cleavage fragment Ba to influence human B cell function. Ba did not affect the activation of resting B cells but inhibited the proliferation of activated B cells stimulated with either high m.w.-BCGF or low m.w.-BCGF. The inhibition occurred with doses of Ba as low as 1 microgram/ml (29 nM). Ba was found to bind to activated human B lymphocytes in a saturable manner with an apparent K of approximately 25 nM and an apparent Bmax of 56,000 sites/cell. A peptide made of the carboxy terminal 10 amino acids of Ba (GHGPGEQQKR), was also found to inhibit growth factor induced proliferation of activated B cells but at an ID50 of approximately 5 microM. Finally, Ba was found to inhibit the terminal differentiation of Staphylococcus aweus Cowan-activated B cells stimulated with B cell differentiation factors but not Ig secretion by the partially differentiated EBV-transformed cell line SKW.6. Thus, concentrations of Ba achievable in vivo at sites of active inflammation were found to act on human B lymphocytes by inhibiting their proliferation. This may act to limit the immune response to a specific antigenic challenge.
Assuntos
Linfócitos B/imunologia , Fator B do Complemento/farmacologia , Precursores Enzimáticos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Sequência de Aminoácidos , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Interleucina-4/farmacologia , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de PeptídeosRESUMO
The reaction of radiolabeled C3b-binding proteins with C3b-coated particles has been investigated. CR1 binding was inhibited by factor H and factor B (in the presence of properdin), but not by properdin alone. CR2 and MCP binding were also inhibited by factor H. Therefore factor H, factor B, CR1, CR2 and MCP probably comprise a group of mutually competitive proteins with similar or overlapping binding sites on C3b. These results correlate with their structural homology and suggest that they all evolved from a single C3b-binding molecule. Factor H, CR1 and MCP are also cofactors for the factor-I-mediated cleavage of C3b. A species incompatibility between rat factor I and human CR1 for the cleavage of human C3b suggests the possibility that cofactors may also function by interacting directly with factor I.
Assuntos
Antígenos CD , Proteínas Inativadoras do Complemento C3b/metabolismo , Complemento C3b/metabolismo , Fator B do Complemento/metabolismo , Precursores Enzimáticos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Proteínas Inativadoras do Complemento C3b/farmacologia , Fator B do Complemento/farmacologia , Fator H do Complemento , Humanos , Proteína Cofatora de Membrana , Properdina/farmacologia , Ratos , OvinosAssuntos
Enzimas Ativadoras do Complemento/antagonistas & inibidores , Convertases de Complemento C3-C5/antagonistas & inibidores , Properdina/farmacologia , Ligação Competitiva , Convertases de Complemento C3-C5/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Fator B do Complemento/farmacologia , Fator H do Complemento , Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Humanos , Técnicas In Vitro , Properdina/metabolismoRESUMO
Two separate complement pathway activities and their interaction in the opsonization of mutant strains of Salmonella typhimurium by human neutrophils were investigated in 1% and 10% sera. In the absence of antibody, all mutant strains of S. typhimurium could activate complement by either the classical or the alternative pathway. The presence of antibody did not alter alternative pathway activity but enhanced the classical pathway activity, especially for those strains possessing intact core structure and in the presence of 10% serum. This effect of antibody on classical pathway activation decreased as the lipopolysaccharide chain length decreased. The finding that the presence of antibody reduced the classical pathway activity in the opsonization of SL 1032 (Rd1) strain in 10% serum was unexpected. This suggests that if complement activity in the absence of antibody is already marked, the presence of antibody cannot increase it further. Inhibition was also found in the interaction between classical and alternative pathways. For SL 1032 (Rd1) strain, in the absence of antibody, either classical or alternative pathway activity alone could be maximally activated in 10% serum and when present together were in competition with each other. The interaction, either enhancement or inhibition, was not only dependent on the individual strain but also on the concentration of serum. In general, enhancement occurred in the presence of 1% serum while inhibition was observed in the presence of 10% serum. Such antagonistic interaction has not previously been reported.
Assuntos
Ativação do Complemento , Fagocitose , Salmonella typhimurium/imunologia , Anticorpos/imunologia , Ativação do Complemento/efeitos dos fármacos , Fator B do Complemento/farmacologia , Via Alternativa do Complemento , Via Clássica do Complemento , Ácido Egtázico/farmacologia , Temperatura Alta , Humanos , MutaçãoRESUMO
Human peripheral blood mononuclear phagocytes are induced by activated Factor B (Bb) of the complement alternative pathway to undergo morphological shape changes in vitro which have been described as "spreading." The spreading reaction induced by Bb has previously been shown to depend upon the enzymatic activity of Bb and to be inhibited by Fab' antibody fragments directed to C5 (but not anti-C3 Fab'). The possibility that Bb may exert its effect on monocytes by initiating assembly of terminal complement complexes comprised of C5b, 6, 7, C5b-8, or C5b-9 was addressed in the present study. The effects were tested of Fab' and F(ab')2 antibody fragments directed to C5, C6, C7, and C8 and to neoantigens expressed in the assembling terminal complement complexes on the monocyte spreading reaction induced by Bb. Differential effects of monovalent Fab' and divalent F(ab')2 antibody fragments were observed. Anti-C5, C6, and C7 Fab' were found to inhibit the spreading reaction induced by Bb in an immunologically specific manner. Divalent F(ab')2 fragments directed to these same proteins (but not to C3, C4, C8, or C9) induced monocyte spreading in the complete absence of Bb or other recognized inducing agents. Monocyte spreading induced by hybridoma immunoglobulin (Ig) directed to C5 and C7 was found to be correlated with the binding of 10(6) molecules Ig per cell. These findings support the notion that C5, C6, and C7 (or an analogous system of cellular proteins) are associated with the surface of human peripheral blood monocytes and that these proteins may play a role in certain reactions by which mononuclear phagocytes are induced to altered states of cellular physiology.
Assuntos
Complemento C5/imunologia , Complemento C6/imunologia , Fator B do Complemento/farmacologia , Precursores Enzimáticos/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Monócitos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Anti-Idiotípicos/farmacologia , Complemento C7/imunologia , Humanos , Monócitos/efeitos dos fármacos , Ligação ProteicaAssuntos
Anticorpos Monoclonais/imunologia , Complemento C3/imunologia , Epitopos/análise , Animais , Sítios de Ligação de Anticorpos , Ligação Competitiva , Complemento C3/metabolismo , Complemento C3a , Complemento C3b/imunologia , Complemento C3b/metabolismo , Complemento C3c , Complemento C5/metabolismo , Fator B do Complemento/farmacologia , Venenos Elapídicos/farmacologia , Ensaio de Imunoadsorção Enzimática , Cobaias , Testes de Hemaglutinação , Hemólise , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Activated Factor B (Bb), the central serine esterase of the alternative pathway of complement activation, exhibits restricted substrate specificity in the complement system for C3 and C5. The results presented here indicate that Bb can cleave and activate plasminogen in an experimental system containing purified plasminogen and Bb; complement cellular intermediate bearing the Bb-enzyme; or cobra venom factor-stabilized Bb-enzyme (CVF,Bb). Cleavage of plasminogen by Factor Bb generated 2 disulfide-linked polypeptides with apparent m.w. of 64,000 and 25,000 to 32,000 (SDS-PAGE). Complement cellular intermediates containing the C3b, Bb-enzyme cleave 40 to 80% of 4.5 micrograms of 125I-labeled plasminogen during 30 min of incubation at 37 degrees C; native Factor B was inactive; and anti-Factor Blg inhibited by 100% the plasminogen cleavage mediated by complement cellular intermediates bearing the Bb-enzyme. Fibrinolytic activity was detected in plasminogen activator (PA) assays when purified plasminogen and 125I-labeled fibrin tubes were incubated with Bb, CVF, Bb, or complement cellular intermediates bearing the C3b,Bb-enzyme: 10 micrograms Bb released 40 to 65% of the 125I-fibrin released by 5 micrograms urokinase in 4 hr at 37 degrees C. Plasminogen activator activity of the C3b,Bb-enzyme was found to be regulated in serum. At dilutions of NHS 1:50, the PA-activity of 1.6 micrograms Bb was 100% inhibited, and at a 1:250 dilution, 50% inhibition was observed. This report describes a novel activity for the Bb-enzyme, which constitutes the C3/C5-convertase of the alternative pathway of complement activation.
Assuntos
Ativação do Complemento , Fator B do Complemento/farmacologia , Via Alternativa do Complemento , Precursores Enzimáticos/farmacologia , Ativadores de Plasminogênio/farmacologia , Fator B do Complemento/metabolismo , Eletroforese em Gel de Poliacrilamida , Fibrinólise , Humanos , Ativadores de Plasminogênio/metabolismoRESUMO
The central serine esterase of the alternative pathway of complement (APC) activation, activated factor B (Bb), has been shown recently to induce murine macrophages and human monocytes to become spread on a glass substrata. It has also been established that to induce the spreading reaction, the catalytic site of the Bb enzyme must be structurally intact since treatment of Bb with heat (56 degrees C for 30 min) or diisopropylfluorophosphate (10(-3) M) destroyed both enzymatic and spreading activities. In the C3b,Bb complex, Bb exhibits restricted substrate specificity for C3 and C5. With this in mind, the role of C3 and C5 in the monocyte spreading reaction was explored in the present study. Expression of C3 and C5 on the surface of human peripheral blood monocytes was investigated by the direct fluorescent antibody technique employing fluorescein isothiocyanate-conjugated anti-C3 or C5 F(ab')2 antibody fragments. It was found that C3 and C5 were present on 6 +/- 7% of freshly prepared monocytes and that expression of C5, but not C3, increased to 70 +/- 6% when monocytes were incubated for 3 d in serum-free medium. Biosynthesis of C5 was indicated when it was found that under serum-free conditions, monocytes incorporated [3H]leucine into immunoprecipitable C5 with an apparent mol wt of 180,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The role of C3 and C5 in the monocyte spreading reaction induced by factor Bb was explored by testing for the ability of anti-C3 and anti-C5 Fab' antibody fragments to block monocyte spreading. It was found that anti-C5 Fab' inhibited by up to 100% the 3-h human monocyte spreading reaction induced by Bb; in contrast, anti-C3 Fab' or anti-C4 Fab' inhibited by less than 10%. That the inhibitory effect of anti-C5 Fab' was exerted directly on the monocyte was established when it was found that the 3-h monocyte spreading reaction was significantly inhibited by pretreating monocytes with anti-C5 Fab' for 20 min and then washing before the addition of Bb. The specificity of the inhibitory effect of anti-C5 Fab' was established by quantitatively absorbing the antibody fragments with polyacrylamide gel-purified C5 antigen: greater than 4 microgram of C5 absorbed by 100% the inhibitory activity of 10-20 microgram of anti-C5 Fab'. That factor Bb exerted its effect on monocytes by interacting directly with cell surface C5 was indicated when it was found that purified C5 inhibited the monocyte spreading reaction induced by Bb; greater than 25 microgram of C5 inhibited by 100% the spreading reaction induced by 3 microgram factor Bb.
Assuntos
Ativação do Complemento , Complemento C3b/farmacologia , Complemento C5/farmacologia , Fator B do Complemento/farmacologia , Via Alternativa do Complemento , Precursores Enzimáticos/farmacologia , Monócitos/citologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Complemento C3/análise , Humanos , Monócitos/imunologia , Fragmentos de Peptídeos/farmacologiaRESUMO
Two cobra-venom factors, one from Naja n. naja (CVFn), the other from Naja h. haje venom (CVFh), have been purified and compared, functionally and structurally. Both factors interacted with human factors B and DS to form a potent C3 convertase, CVFBb. However, while the convertase formed with CVFn did also efficiently cleave C5, CVFhBb had very little C5-cleaving potency only, in particular when human C5 was used as substrate. Studies with agarose-linked CVF preparations indicated that CVFh has only low binding affinity for C5gp and C5hu whereas CVFn binds to both C5 species with much higher affinity. Since C5-binding (to CVF or to C3b) is a prerequisite for its cleavage by C3/C5 convertases, the difference in binding potency explains the different C5-cleaving activity of the two CVF preparations. When a ligand for C5, surface-fixed C3b, is present, CVFhBb is also capable of cleaving C5. The difference in activities of CVFn and CVFh is reflected in their different potency to interfere with immune haemolysis and in causing indirect lysis by their complexes with activated factor B. By gel chromatography of the CVF preparations in C5-containing medium, a stoichiometric complex CVFn-C5 (1 + 1) could be demonstrated. An analogous complex of C5 was neither found with CVFh, nor with C3hu or soluble C3bhu. Structural differences between CVFn and CVFh were revealed by immunodiffusion analysis and by polyacrylamide-gel electrophoresis in presence of SDS. The data available so far provide, however, no clear information about the structure of the C5 binding site.