RESUMEN
The rat mast cell granule chymotrypsinlike enzyme was purified to homogeneity from 1 M NaCl solubilized membrane and granule-rich fractions of concentrated rat peritoneal mast cells by a preparative technique utilizing chromatography on Dowex 1, filtration on Sephadex G-75, and affinity chromatography with D-tryptophan methyl ester. Acid disk gel electrophoresis of the purified chymase disclosed a single stained band with activity being eluted from a replicate sliced gel in the same region. SDS-polyacrylamide gel electrophoresis of purified protein gave a single stained band that did not change in position with reduction and alkylation. Mast cell chymase is thus a cationic protein of 25,000 mol wt composed of a single polypeptide chain. The apparent K(m) of the chymase for BTEE was 1.5 x 10(-3) M and the V(max) was 67.8 mumol/min per mg. The enzyme was inhibited by TPCK and not by TLCK. The chymase complexed with native macromolecular rat mast cell heparin in molar ratios of 12:1 and 16:1, and complete heparin uptake occurred at a 40:1 ratio of chymase to heparin. Chymase activity was partially masked by combination with heparin in the isolated granule or experimental chymase-heparin complex, and soluble purified chymase was inhibited by concentrations of 5-HT comparable to those present in mast cells. It is therefore possible that the active site of chymase in the mast cell granule is largely masked by the combined effects of macromolecular heparin and 5-HT.
Asunto(s)
Quimosina/aislamiento & purificación , Mastocitos/enzimología , Animales , Benzamidas/metabolismo , Cromatografía , Quimosina/antagonistas & inhibidores , Quimosina/metabolismo , Gránulos Citoplasmáticos/metabolismo , Femenino , Heparina/metabolismo , Histamina/farmacología , Masculino , Mastocitos/ultraestructura , Ratas , Serotonina/farmacología , Clorometilcetona Tosilisina/farmacología , Clorometilcetona de Tosilfenilalanila/farmacología , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
It has been demonstrated that C1 isolated in the unactivated form fails to inactivate C4 or C2 in the fluid phase, while the activated molecule, C1 rapidly converts C4 to hemolytically inactive C4i, but does not efficiently inactivate C2. The production and presence of C4i now confers on C1 the ability to rapidly inactivate C2. After heating at 56 degrees C, so as to destroy the hemolytic activity, heat inactivated C1 is still capable of inactivating C4 but the presence of C4i no longer confers an ability to inactivate C2. Studies with the subunits of C1-C1q, C1r, C1s, indicate that the action of C1s on C2 can be inhibited by C1r and that this inhibition is reversed by the presence of homologous C4. These studies indicate that the interaction of C4i with a heat labile receptor conformation in C1 uncovers a masked specificity for C2.
Asunto(s)
Proteínas del Sistema Complemento , Animales , Sitios de Unión , Fenómenos Químicos , Química Física , Proteínas Inactivadoras de Complemento , Cobayas , Hemólisis , Calor , Humanos , Inmunoelectroforesis , Cinética , Especificidad de la Especie , Ácidos Sulfónicos/farmacologíaRESUMEN
Five sequential steps interspaced between the antigen activation of human lung fragments sensitized with IgE and the release of the chemical mediators, histamine and slow-reacting substance of anaphylaxis (SRS-A), have been delineated. The experimental design that permits this analysis is based upon the capacity to maintain the serine esterase essential to mediator release in its diisopropylphosphofluoridate (DFP)-resistant precursor state despite antigen challenge and upon the ability to arrest reversibly the reaction sequence by various manipulations. When sensitized lung fragments are challenged with antigen in the presence of DFP, a serine esterase is converted to its active DFP-inhibitable state; this conversion is prevented if antigen challenge in the presence of DFP occurs in calcium-free buffer indicating that immunologic activation of the esterase requires extracellular calcium. The fact that calcium depletion alone does not impair antigen-induced histamine release implies that prevention of esterase activation depends upon both the absence of extracellular calcium and the inactivation of any active esterase by DFP to prevent an autocatalytic feedback activation. Arresting the antigen-induced activation of the serine esterase by the combination of DFP in calcium-free buffer precludes the sequence from reaching the labile, 2-deoxyglucose (2-DG)-inhibitable, energy-requiring step, indicating that proesterase activation precedes this energy-requiring stage. The 2-DG-inhibitable step precedes a second calcium-requiring, EDTA-inhibitable stage, as EDTA prevents glucose reversal of 2-DG inhibition of antigen-challenged tissue, while the presence of 2-DG does not prevent calcium reversal of EDTA inhibition. The finding that isoproterenol prevents calcium reversal of EDTA inhibition of mediator release suggests that the inhibitory site of action of increased concentrations of cyclic AMP is coincident with or subsequent to the second calcium-requiring, EDTA-inhibitable step. Therefore, the sequence of biochemical events initiated by the interaction of antigen with tissue-fixed IgE antibodies appears to proceed from the calcium-requiring activation of a DFP-sensitive serine esterase; the further autocatalytic activation of the esterase; a 2-DG-inhibitable energy requirement; a second calcium-requiring, EDTA-inhibitable stage; and a cyclic AMP-inhibitable step to the release of histamine and SRS-A.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Liberación de Histamina , Pulmón/inmunología , SRS-A/metabolismo , Calcio/metabolismo , AMP Cíclico/farmacología , Ácido Edético/farmacología , Esterasas/antagonistas & inhibidores , Esterasas/metabolismo , Humanos , Inmunoquímica , Inmunoglobulina E , Isoflurofato/farmacología , Isoproterenol/farmacología , Pulmón/enzimología , Pulmón/metabolismo , Factores de TiempoRESUMEN
The fluid phase inactivation of C2(hu) by C1(hu) is markedly enhanced by the presence of C4(hu). The enhancement is afforded by C1 inactivated C4(hu), namely C4i(hu), and requires the simultaneous presence of enzymatically active C1. Heterologous C4 of guinea pig origin protects C2(hu) from the inactivation by C1(hu). Thus, in both the fluid phase and on the cellular intermediate, C4(hu) is essential to the specific action of C1(hu) on C2(hu). It is possible that C4i alters C2 so as to present a more suitable substrate to the C1 enzyme or that C4i acts on the C1 to uncover a specificity for native C2.
Asunto(s)
Proteínas del Sistema Complemento , Hemólisis , Animales , Sitios de Unión , Proteínas del Sistema Complemento/aislamiento & purificación , Cobayas , Humanos , CinéticaRESUMEN
Activation of a plasma fraction containing unactivated Hageman factor and prekallikrein followed by chromatography of this fraction on DEAE-cellulose revealed four peaks having bradykinin-generating activity. Peak 1 contained kallikrein; peaks 2-3, 4, and 5 each contained prekallikrein-activating activity. Elution of peaks 2-3, 4, and 5 from disc gels after electrophoresis at pH 9.3 revealed peaks of prekallikrein-activating activity located at 5-8, 11-12, 15-16, and 20-26 mm, each of which was associated with a peak of clot-promoting activity which specifically corrected Hageman factor deficiency. Conversion of peak 2 to peaks 3, 4, and 5 was associated with a progressive decrease in size, increase in net negative charge, increased prekallikrein-activating activity, and decreased ability to correct Hageman factor deficiency. Plasminogen and plasmin were found on a DEAE-cellulose chromatogram of serum overlapping peaks 2 and 3. Incubation of active Hageman factor with streptokinase-activated plasminogen resulted in enhanced ability of the mixture to activate prekallikrein. Assessment of the products of this reaction by disc gel electrophoresis demonstrated the formation of the prealbumin prekallikrein activator corresponding to the major prekallikrein activator generated by contact activation of human plasma. The conversion of plasminogen to plasmin and the subsequent cleavage of Hageman factor by plasmin to form activators of prekallikrein represents one pathway in which coagulation, fibrinolysis, and inflammation are linked.
Asunto(s)
Precursores Enzimáticos , Factor XII , Fibrinolisina , Calicreínas , Bradiquinina/biosíntesis , Fenómenos Químicos , Química , Cromatografía DEAE-Celulosa , Electroforesis Discontinua , Activación Enzimática , Humanos , Metilcelulosa , Plasminógeno , EstreptoquinasaRESUMEN
An activity designated Kf can be separated from human serum and shown to give a 100-300% enhancement in the hemolytic activity of fully activated, fractionated C1. The enhancement of C1 activity is not because of activation of precursor C1 and it is not attributable to an effect on C1 binding. EAC42 or EAC4 intermediates interacted with C1Kf exhibit a greater T(max) and shorter Z(max) than when such intermediates are reacted with the same number of hemolytic units of C1. C3 consumption by the EAC1Kf42 intermediate greatly exceeds that of the EAC142 intermediate produced from the same EAC4 cells by comparable inputs of the other two complement components. Taken together, these findings suggest that Kf-treated C1 achieves more efficient utilization of C4 and C2 to create a larger number of 42 sites as appreciated on the intermediates by shorter T(max) and a greater Z(max), and an increased capacity to utilize C3. The capacity of Kf to enhance C1 upon introduction into whole serum of a patient with hereditary angioedema (HAE) in a manner comparable to its effect on fractionated C1 suggests that the effect of Kf may be pertinent to certain pathophysiologic conditions of man.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Hemólisis , Calicreínas , Angioedema/inmunología , Animales , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cobayas , Humanos , Inmunidad Celular , Inmunoquímica , Cininas/sangre , Ovinos , Glycine max , Espectrofotometría , Inhibidores de TripsinaRESUMEN
The conversion of the plasminogen proactivator to plasminogen activator by activated Hageman factor or its fragments has been recognized as an essential step in the conversion of plasminogen to plasmin. The plasminogen proactivator has been completely separated from prekallikrein and pre-PTA, two other proenzyme substrates of activated Hageman factor or its fragments. Plasminogen proactivator, free of any contaminating proteins as assessed by disc gel electrophoresis or isoelectric focusing, revealed a single band with an isoelectric point of 8.9 corresponding in position to the Hageman factor activatable material eluted from replicate unstained gels. After conversion of plasminogen proactivator by Hageman factor fragments to the plasminogen activator, the active site of the plasminogen activator is not inhibited by C1INH and is thus readily distinguished from that of kallikrein or PTA. The plasminogen activator is susceptible to inactivation by DFP while the plasminogen proactivator is not, as has been the case for esterases having a serine in the active site. Its interaction with plasminogen is inhibited by epsilon-aminocaproic acid.
Asunto(s)
Factores de Coagulación Sanguínea , Factor XII , Fibrinolisina , Fibrinólisis , Aminocaproatos/farmacología , Factores de Coagulación Sanguínea/aislamiento & purificación , Cromatografía en Gel , Proteínas del Sistema Complemento , Electroforesis Discontinua , Factor XII/aislamiento & purificación , Fibrinolisina/antagonistas & inhibidores , Histidina/antagonistas & inhibidores , Humanos , Focalización Isoeléctrica , Calicreínas , Cininas , Peso Molecular , Plasminógeno/antagonistas & inhibidores , Serina/antagonistas & inhibidores , Estreptoquinasa , TromboplastinaRESUMEN
The immunologic release of histamine and slow reacting substance of anaphylaxis (SRS-A) from human lung tissue can be enhanced by stimulation with either alpha adrenergic agents (phenylephrine or norepinephrine in the presence of propranolol) or cholinergic agents (acetylcholine or Carbachol). The finding that atropine prevents cholinergic but not comparable alpha adrenergic enhancement is consistent with the view that cholinergic and alpha adrenergic agonists interact with separate receptor sites on the target cells involved in the immunologic release of chemical mediators. The consistent qualitative relationship between the antigen-induced release of mediators and the level of cyclic adenosine monophosphate (cyclic AMP) as measured by the isolation of (14)C-labeled cyclic AMP after incorporation of adenine-(14)C into the tissues or by the cyclic AMP binding protein assay suggests that changes in the level of this cyclic nucleotide mediate adrenergic modulation of the release of histamine and SRS-A. The addition of 8-bromo-cyclic guanosine monophosphate (cyclic GMP) produces an enhancement of the immunologic release of mediators while dibutyryl cyclic AMP is inhibitory. As cholinergic-induced enhancement was not associated with a measurable change in the levels of cyclic AMP, the possibility is suggested that cyclic GMP may be the intracellular mediator of cholinergic-induced enhancement of the immunologic release of histamine and SRS-A.
Asunto(s)
Acetilcolina/farmacología , Carbacol/farmacología , Liberación de Histamina , Metabolismo de los Lípidos , Pulmón/inmunología , Norepinefrina/farmacología , Fenilefrina/farmacología , Propranolol/farmacología , Receptores de Droga , Acetilcolina/antagonistas & inhibidores , Adenina/metabolismo , Anafilaxia , Atropina/farmacología , Autacoides/metabolismo , Carbacol/antagonistas & inhibidores , Isótopos de Carbono , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Antagonismo de Drogas , Humanos , Técnicas In Vitro , Pulmón/efectos de los fármacos , Receptores Adrenérgicos/efectos de los fármacos , Receptores Colinérgicos/efectos de los fármacos , Estimulación QuímicaRESUMEN
A factor has been derived from human leukocytes which irreversibly inhibits the response of human neutrophils to diverse chemotactic stimuli without impairing their viability. It is released by both polymorphonuclear and mononuclear leukocytes during incubation in acidic medium, after endotoxin exposure and subsequent incubation in low potassium medium, and during phagocytosis of particles. It is extractable from both leukocyte types and therefore must be preformed. This chemotactic inhibitor is completely separable from contaminating chemotactic activity in the crude supernatants, has a mol wt of 5000, and is inactivated by digestion with trypsin or chymotrypsin. It has been termed a neutrophil-immobilizing factor because it inhibits neutrophils directly and independently of the chemotactic stimulus, and has relatively little effect on human monocyte chemotaxis.
Asunto(s)
Quimiotaxis , Proteínas del Sistema Complemento , Inflamación , Leucocitos/inmunología , Neutrófilos/inmunología , Pruebas Serológicas , Plaquetas , Inhibición de Migración Celular , Relación Dosis-Respuesta a Droga , Endotoxinas , Humanos , Calicreínas/farmacología , Fagocitosis/efectos de los fármacos , Estimulación QuímicaRESUMEN
Cleavage of C3 by the alternative complement pathway occurs in at least two distinct phases: continuous low grade generation of C3b by the interaction of native C3, B, D, and P, and subsequent amplified cleavage of C3 by the interaction of C3b, B, D, and P which forms the amplification convertase, P,C3b,Bb. Transition to C3b-dependent amplification is necessary to achieve substantial C3 cleavage and is normally limited by the combined action of C3b inactivator (C3bINA) and betalH. An activator of the alternative pathway, such as rabbit erythrocytes (E(r)), provides sites that protect bound C3b and P,C3b,Bb from the action of these regulatory proteins and permits C3b deposited by the low grade fluid phase reaction to assemble a membrane-associated amplification convertase which can deposit additional protected C3b. Under conditions in which the control proteins, C3bINA and beta1H, almost completely inactivated C3b bound to sheep erythrocytes (E(s)), which does not activate the alternative pathway, the function of C3b bound to E(r) was diminished by less than one-fifth. Further, the P- stabilized amplification convertase on E(r) was 10-fold less sensitive to beta1H-mediated decay-dissociation than the convertase on E(s). The addition of E(r) to a regulated mixture of purified C3, B, D, P, C3bINA, and beta1H resulted in amplified inactivation of C3 and B by formation of the amplification convertase on E(r) as indicated by its lysis with subsequent exposure to C3-C9. In contrast, E(s) did not advance the low grade fluid phase inactivation of C3 and B to amplified inactivation and the cell was not converted to an intermediate susceptible to lysis by C3- C9. Since E(r) and E(s) did not differ in their inefficient fixation of C3b generated during an unregulated fluid phase reaction, the activating capacity of E(r) must reside in its protection of bound C3b and P, C3b,Bb from the regulatory proteins rather than in enhanced capacity to bind C3b from the fluid phase. When the reaction is limited to low grade fluid phase turnover, introduction of E(r) but not E(s) results in a 100-fold increase in the deposition of C3b, indicating that surface-dependent activation of the alternative pathway is characterized by efficient deposition of C3b on the initiating surface. Thus, the activating surfaces advance the interaction of the alternative pathway proteins to the amplification phase because of the selective inability of the regulatory proteins to deal with their substrates when deposited on these surfaces and results in a specificity that is not necessarily dependent on adaptive immunity.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Eritrocitos/metabolismo , Properdina/fisiología , Animales , Complemento C3/metabolismo , Esterasas/metabolismo , Genes Reguladores , Unión Proteica , Conejos , OvinosRESUMEN
A function of P in the alternative complement pathway is to prolong the first order decay of the hemolytic sites on EAC43B in a dose-dependent manner. As the number of initial convertase sites is not changed, even when activated properdin (P) increases the t1/2 10-fold or more, P acts to stabilize rather than to uncover additional sites. P binds to EAC43 to generate EAC43P in a reaction that proceeds slightly more rapidly at 15 degrees C than at 0 degrees C, but reaches the same plateau and does not require divalent cations. The presence of P on EAC43P not only stabilizes the convertase subsequently formed on that cell, but, alternatively, permits transfer to convertase sites on other cells with the stability of the recipient intermediate being dependent on the P available for transfer. The capacity of P to bind to C3b and stabilize C3B contrasts with the inhibitory effect of the C3b inactivator on formation of this amplification convertase.
Asunto(s)
Complemento C3/metabolismo , Proteínas del Sistema Complemento/metabolismo , Esterasas/metabolismo , Properdina/metabolismo , Animales , Sitios de Unión de Anticuerpos , Eritrocitos/enzimología , Eritrocitos/inmunología , Cobayas , HemólisisRESUMEN
The activity of properdin factor D was measured by the generation of the hemolytically active cellular intermediate, EAC43B(D), bearing the C3b-dependent alternate pathway C3 convertase. Treatment of factor D with DFP prevented formation of EAC43B(D); thus, a serine esterase is essential for the generation of the alternate pathway C3 convertase, a situation analogous to the role of C1 in the formation of the classical C3 convertase, C42. The definition of factor D as a serine esterase prompted a search for its proenzyme form, and resulted in the chromatographic isolation from plasma of a single peak of trypsin-inducible factor D activity, distinct from activated factor D. Analytical gel filtration indicated an apparent mol wt of 25,000. This protein from which trypsin elaborated factor D activity, as assessed by the formation of EAC43B(D), the generation of the CoVF-dependent C3 convertase, and the cleavage of factor B in the presence of C3b, was designated "precursor factor D." The DFP resistance of precursor factor D, and the susceptibility of its trypsin-activated form to inactivation by DFP is analogous to the behavior of other plasma serine esterases, including C1.
Asunto(s)
Proteínas del Sistema Complemento , Precursores Enzimáticos/aislamiento & purificación , Esterasas/metabolismo , Hemólisis , Animales , Sitios de Unión , Cloro/farmacología , Activación Enzimática , Esterasas/antagonistas & inhibidores , Globulinas/antagonistas & inhibidores , Globulinas/aislamiento & purificación , Isoflurofato/farmacología , Lisina/farmacología , Serpientes , Compuestos de Tosilo/farmacología , Tripsina/farmacología , PonzoñasRESUMEN
The protein in the properdin pathway responsible for conversion of precursor factor D to D has been isolated and found to be identical with properdin. Sequential ion exchange and gel filtration chromatography demonstrated identity between properdin protein, measured by radial immunodiffusion, and the capacity to activate D to D, assessed by formation of the intermediate, EAC43B(D). Properdin, purified in this manner, was homogeneous on acid polyacrylamide disc gel electrophoretic analysis, with the band of protein corresponding to the position of eluates in the replicate gel capable of activating highly purified D. Demonstration of the homogeneity of purified D by alkaline disc gel electrophoresis, coupled with the linear stoichiometric hemolytic titrations of each factor, indicates that direct interaction between properdin and D generates D. Thus, activation of D by properdin represents a mechanism in the properdin pathway by which D becomes available for formation of the C3b-dependent C3 convertase.
Asunto(s)
Properdina/farmacología , Seroglobulinas/farmacología , Animales , Fenómenos Químicos , Química , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Proteínas del Sistema Complemento/análisis , Electroforesis Discontinua , Precursores Enzimáticos , Cobayas , Hemólisis , Humanos , Inmunodifusión , Matemática , Properdina/análisis , Conejos/inmunología , Solubilidad , Estimulación QuímicaRESUMEN
The acidic tetrapeptides of ECF-A, Ala/Val-Gly-Ser-Glu, exhibit peak in vitro chemotactic activity for human eosinophils at concentrations of 3 X 10(-8) M to 10(-6) M, and rapidly deactivate eosinophils to homologous and other stimuli at concentrations as low as 10(-10) M. The analogue Leu-Gly-Ser-Glu reaches peak activity at 10(-8)M-10(-7)M, while Phe-Gly-Ser-Glu requires 10(-4)M to elicit a peak response. Although inversion of the order of glycine and serine does not alter the eosinophil chemotactic activity of the tetrapeptides, deletion of glycine increases by 10-fold the concentration required for peak chemotactic activity, indicating the critical nature of the spacing between NH2- and COOH-terminal residues. The substituent COOH-terminal tripeptide, which is only marginally chemotactic, irreversibly suppresses eosinophil chemotactic responsiveness at a concentration 10,000-fold higher than concentrations necessary for deactivation by the intact tetrapeptide. The high concentration of tripeptide required for this cell directed effect, which is assumed to be analogous to deactivation, is attributed to the absence of the NH2-terminal residue which would facilitate effective interaction with the eosinophil. A substituent NH2-terminal tripeptide and amides of the NH2-terminal amino acids, which are devoid of chemotactic and deactivating activities, reversibly inhibit the tetrapeptide stimulus in a dose-response fashion. The additional finding that the NH2-terminal tripeptide protects the eosinophil from deactivation by the intact tetrapeptide confirms that the competitive interaction is stimulus specific.
Asunto(s)
Quimiotaxis de Leucocito , Eosinófilos/fisiología , Oligopéptidos/farmacología , Alanina , Secuencia de Aminoácidos , Anafilaxia/inmunología , Sitios de Unión , Quimiotaxis de Leucocito/efectos de los fármacos , Eosinófilos/ultraestructura , Humanos , Modelos Biológicos , Neutrófilos/fisiología , Solubilidad , Relación Estructura-Actividad , ValinaRESUMEN
The present studies demonstrate that the factor B-dependent C3 convertase can be affixed to an erythrocyte by use of an intermediate bearing C3b and that this convertase brings the hemolytic reaction to completion with an efficiency comparable to that of classical convertase. The evidence that the EAC43 intermediate was lysed by a new pathway includes requirements for factors B and D and cell-bound C3b for subsequent lysis by the terminal components, C3-C9. The linear stoichiometry of the effective molecule titrations of C3b and factor B, and the first-order kinetics displayed by the generation and decay of the factor B-dependent hemolytic site are characteristics consistent with the one-hit theory as initially developed for the classical complement system. The use of hemolytically active cellular intermediates to examine the reactions occurring with C3b and factors B and D has allowed extension of the one-hit theory to this molecular sequence, development of effective molecule titrations, recognition of the analogies to the functional characteristics of the classical C3 convertase, and discrimination of the probable mechanism of terminal complement activation from reactive lysis.
Asunto(s)
Proteínas del Sistema Complemento , Eritrocitos/inmunología , Hemólisis , Properdina , Humanos , Inmunoquímica , Inmunodifusión , Inmunoelectroforesis , Cinética , Properdina/aislamiento & purificaciónRESUMEN
c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of > 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5-lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Factores de Crecimiento de Célula Hematopoyética/farmacología , Leucotrieno C4/biosíntesis , Mastocitos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Fosfolipasas A/metabolismo , Transducción de Señal , Animales , Ácido Araquidónico/metabolismo , Transporte Biológico , Células de la Médula Ósea , Inhibidores de la Ciclooxigenasa/metabolismo , Citocinas/farmacología , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Inmunoglobulina E/inmunología , Interleucina-1/farmacología , Interleucina-10/farmacología , Masculino , Mastocitos/metabolismo , Lípidos de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Fosfolipasas A2 , Fosforilación/efectos de los fármacos , Prostaglandina D2/biosíntesis , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes/farmacología , Factor de Células Madre , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The ability of a number of p-nitrophenylethyl, alkyl phenylalkyl, chloroalkyl, and aminoalkyl phosphonates to inhibit the homocytotropic antibody-mediated release of histamine from rat peritoneal mast cells has been tested. The effectiveness of these same phosphonates against the activated first component of rat complement (C'1a) has also been investigated. The rat mast cell esterase activated by the reaction of antigen and homocytotropic antibody resembles chymotrypsin in its reactivity with the phenylalkyl and chloroalkyl phosphonate, but is unlike this protease in its greater responsiveness to the 5-aminopentyl phosphonate relative to the pentyl phosphonate. The antigen-homocytotropic antibody-activated mast cell esterase and chymotrypsin, thus, appear to be similar, but different enzymes; i.e., they are parazymes (see reference 4, p. 501). There are distinct differences in the pattern of inhibition given by the phenylalkyl and aminoalkyl and alkyl phosphonates of the homocytotropic antibody-mediated histamine release from rat peritoneal mast cells and from guinea pig lung slices. On the basis of these differences it is concluded that the esterases activated by the combination of antigen and homocytotropic antibody on the mast cells of the two species are not the same. The arithmetic dose response curve found for the action of the phosphonates on the antigen-induced histamine release from rat peritoneal mast cells contrasted sharply with the logarithmic relationship found when these same inhibitors acted on the guinea pig lung system. This suggests that in addition to the antigen-antibody-activated esterases being unlike, the detailed mode of histamine release from the mast cells of the guinea pig lung differs from that of the mast cells of the rat peritoneum. Distinct and large differences were found in the pattern of inhibition of histamine release from rat peritoneal mast cells and of rat C'1a given by the phenylalkyl, and chloroalkyl and alkyl phosphonates implying that esterase activated by the combination of antigen with the sensitized rat peritoneal mast cells is not C'1a. Thus, the results with the peritoneal mast cells lead to the same conclusion as the previous work with guinea pig lung slices; i.e., the antigen-antibody-activated esterase involved in the homocytotropic antibody-mediated release of histamine is not part of the complement system.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Proteínas del Sistema Complemento , Liberación de Histamina/fisiopatología , Mastocitos/efectos de los fármacos , Mastocitos/fisiopatología , Organofosfonatos/farmacología , Ancylostomatoidea/inmunología , Animales , Esterasas/metabolismo , Cobayas , Técnicas In Vitro , Pulmón/citología , Cavidad Peritoneal , RatasRESUMEN
There is an absolute requirement for C'1, C'2, C'4, C'3, and C'5 in releasing histamine from rat peritoneal mast cells sensitized with rabbit anti-rat gamma globulin. This conclusion is based upon the restoration of histamine-releasing capacity by adding highly purified complement components to sera deficient in one or more of these components. Of special advantage was the availability of sera from humans with inborn or acquired deficiencies in a single component. The p-nitrophenyl ethyl phosphonates block this reaction by inhibiting an antigen-antibody-activated esterase which exists in a phosphonate resistant precursor state until activated by the interaction of the sensitized mast cell and serum complement. There is almost complete disparity between the ability of the phosphonates to inhibit complement-dependent histamine release by rabbit anti-RGG and to inactivate C'1a. Even though C'1a is required for complement-dependent histamine release by rabbit anti-RGG, this is not the esterase being blocked by the phosphonates under the experimental conditions used. The pattern of inhibition by the phosphonates of the antigen-antibody-activated esterase required for complement-dependent, noncytotoxic histamine release is remarkably similar to that of the esterase required for homocytotropic antibody-mediated histamine release. One possible implication is that these two quite different modes of carrying out antigen-antibody-induced histamine release from rat peritoneal mast cells lead to activation of the same esterase and share a common pathway.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Proteínas del Sistema Complemento , Liberación de Histamina , Mastocitos/efectos de los fármacos , Mastocitos/fisiopatología , Organofosfonatos/farmacología , Animales , Reacciones Antígeno-Anticuerpo , Esterasas/metabolismo , Técnicas In Vitro , Cavidad Peritoneal , Conejos , RatasRESUMEN
The capacity of actively or passively sensitized guinea pig lung to react with antigen to release a factor specifically chemotactic for eosinophil leukocytes (ECF-A) has been demonstrated. The release of ECF-A was also accompanied by the elaboration of both histamine and SRS-A and the appearance of all these mediators exhibited a similar response in terms of the time course of passve sensitization, the effect of antigen dose, the time course of release, divalent cation dependence and enhancement by the presence of succinate or maleate. Decomplementation by the administration of purified cobra venom factor had no effect on the antigen-induced release of ECF-A from actively or passively sensitized lung fragments. When fragments of guinea pig lung were passively sensitized with fractions of guinea pig 7S IgG, only the IgG(1)-containing fractions prepared tissue for the antigen-induced release of ECF-A. Histamine, SRS-A, bradykinin, serotonin, and the prostaglandins PGE(1), PGE(2), and PGF(2alpha) were not eosinophilotactic per se; neither was ECF-A detected following the incubation of these agents with sensitized lung in the absence of antigen. Both eosinophilotactic activity and SRS-A survived extraction in 80% ethanol and evaporation to dryness. SRS-A, however, withstood boiling in alkaline solution for 20 min, whereas ECF-A activity was abolished by this procedure. SRS-A and ECF-A could also be separated by gel filtration. ECF-A activity was completely recovered following its passage through a column of Sephadex G-25 and had an estimated molecular weight of between 500 and 1000. On the basis of size and a formation mechanism independent of the complement system, ECF-A is distinguishable from a previously described complement-dependent eosinophilotactic factor (ECF-C). Thus, ECF-A represents a hitherto undescribed agent which selectively attracts eosinophil leukocytes.
Asunto(s)
Anafilaxia/inmunología , Quimiotaxis , Eosinófilos/inmunología , Pulmón/inmunología , Músculo Liso/fisiología , Animales , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Proteínas del Sistema Complemento , Ácido Edético/farmacología , Filtración , Cobayas , Liberación de Histamina , Inmunoglobulina G , Técnicas In Vitro , Maleatos/farmacología , SRS-A , Serpientes , Succinatos/farmacología , PonzoñasRESUMEN
The optimum conditions for antigen-induced release of histamine in the rat IgE and IgGa antibody-mediated systems were studied in vitro. The IgE antibody-mediated reaction could be separated into two steps: preparation of target cells with antibody and challenge with antigen. The optimal conditions for these two steps were distinctly different. Release of histamine by IgGa antibody and antigen could not be separated into two steps, and the optimal conditions for the total reaction were identical to those of the antigen challenge step of the IgE antibody-mediated system.