RESUMO
Saliva contains acidic proline-rich salivary proteins that are involved in the formation of the salivary pellicle coating supragingival tooth surfaces. However, human leukocyte elastase, arriving in gingival exudates from inflamed periodontal tissues, degrades the acidic proline-rich salivary proteins, preventing binding to hydroxylapatite surfaces. Here it is reported that high-molecular-weight non-immunoglobulin salivary agglutinin inhibited the proteolytic action of human leukocyte elastase on purified acidic proline-rich salivary proteins. Inhibition was eliminated with monoclonal antibody to a protein determinant on the salivary agglutinin. The addition of antibody against salivary agglutinin blocked the inhibitory effect of parotid saliva on exogenously applied human leukocyte elastase, allowing for the elastase-mediated digestion of the salivary acidic proline-rich salivary proteins. Salivary agglutinin, therefore, is a physiologically important inhibitor of human leukocyte elastase and is able to inhibit elastase-mediated digestion of salivary acidic proline-rich proteins.
Assuntos
Aglutininas/metabolismo , Elastase de Leucócito/antagonistas & inibidores , Peptídeos/metabolismo , Inibidores de Proteases/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Aglutininas/química , Aglutininas/farmacologia , Anticorpos Monoclonais , Reações Antígeno-Anticorpo , Película Dentária , Relação Dose-Resposta a Droga , Líquido do Sulco Gengival/enzimologia , Humanos , Imunoglobulina G , Peso Molecular , Periodontite/enzimologia , Domínios Proteicos Ricos em Prolina , Saliva/química , Proteínas Salivares Ricas em ProlinaRESUMO
Human leucocyte elastase from inflammatory gingival crevicular exudates (gingival crevicular fluid) contacts saliva and saliva-coated tooth surfaces coronal to the gingival margin. Major components of saliva are the salivary acidic proline-rich proteins (PRPs). These acidic PRPs, via the numerous negatively charged amino acid residues located predominantly within their amino-terminal region, bind to the hydroxyapatite mineral of the tooth surface and become part of the salivary pellicle. Thus the potential for human leucocyte elastase-mediated removal of the negatively charged amino-terminal region of acidic PRP variants (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f) was examined. It was determined that each of the acidic PRP variants was susceptible to fragmentation by human leucocyte elastase, in which the 16 amino-terminal segment was removed, leaving the respective residual fragment named as the transitional product (tr). The transitional products were termed PRP-1tr, PRP-2tr (PIF-str), PRP-3tr and PRP-4tr (PIF-ftr). Each of the residual transitional products of acidic PRP had an amino-terminal beginning with serine residue no. 17, determined by amino acid sequencing. When samples of human leucocyte elastase-treated acidic PRPs were placed on native polyacrylamide gels and electrophoresed, the respective transitional products moved more slowly than the parental acidic PRP molecules, reflecting the loss of a portion of the negatively charged section. In comparison to the acidic PRPs, the acidic PRP transitional products had markedly reduced binding to hydroxyapatite. The transitional products were resistant to further enzymatic digestion as a function of increased incubation time and appeared to exert an antihuman leucocyte elastase effect. However, when increased concentrations of human leucocyte elastase were incubated with the acidic PRP, a more extensive digestion occurred, leaving a residual peptide with an amino-terminal beginning with alanine residue no. 44. Interestingly, intact acidic PRPs if prebound to hydroxyapatite particles, resisted digestion by human leucocyte elastase. In summary, human leucocyte elastase was capable of digesting fluid-phase (unbound) acidic PRP in a manner that eliminated part of their negatively charged region, which subsequently reduced their binding to hydroxyapatite. High concentrations of human leucocyte elastase, arriving from inflammatory gingival crevicular exudates, may interrupt the normal binding of fluid-phase acidic PRPs to hydroxyapatite.
Assuntos
Elastase de Leucócito/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos/efeitos dos fármacos , Película Dentária , Relação Dose-Resposta a Droga , Durapatita/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Elastase de Leucócito/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Peptídeos/isolamento & purificação , Domínios Proteicos Ricos em Prolina , Saliva/química , Proteínas Salivares Ricas em Prolina , Análise de Sequência , Fatores de TempoRESUMO
A new function for C1 inhibitor (C1 INH) is reported. C1 inhibitor dislodged the entire activated C1 complex (C1qr2s2) from immobilized human IgG. C1 binding to doses of immobilized human IgG3, IgG1, or IgG2 was quantified as a function of time. When human serum, as a source of C1qr2s2, was added to relatively low doses of immobilized IgG, C1q binding peaked at 1.0 min then gradually decreased. However when purified C1q was applied to immobilized IgG, C1q binding did not diminish with time. The removal of C1q was duplicated by adding purified C1 INH to C1qr2s2 which had been bound to immobilized IgG. The dislodgement of C1q from immobilized IgG required the presence of intact C1qr2s2 and of C1 INH. This removal of C1q by purified C1 INH was prevented when activated C1s was used to neutralize C1 INH function or when relatively high levels of IgG were immobilized.
Assuntos
Proteínas Inativadoras do Complemento 1/fisiologia , Complemento C1q/metabolismo , Complemento C1r/metabolismo , Imunoglobulina G/metabolismo , Ligação Competitiva , Complemento C4/metabolismo , Humanos , Substâncias Macromoleculares , Ligação ProteicaRESUMO
Evidence is presented for a new C1 Inhibitor (C1 INH) function. C1 INH was capable of dislodging the entire C1qr2s2 complex from C1-activating substances that bound weakly to the globular heads of C1q. Two different mouse IgG1 monoclonal antibodies with different affinities for C1q globular heads were compared for their complement-activating properties in the presence of normal human serum. As expected the higher affinity monoclonal antibody (Qu) was more effective in binding C1q and causing C1-mediated C4b deposition. Unexpectedly, time responses of C1 (C1q) binding to immobilized 3C7 reached a peak then gradually decreased. However, C1q remained constantly bound to immobilized Qu. These results indicated that after C1 activation in human serum, the entire C1 complex (including C1q) was dislodged from 3C7, but not from immobilized Qu. The addition of purified C1 INH to purified C1, which had bound to immobilized 3C7, resulted in removal of C1 (C1q). Removal of the entire C1qr2s2 did not occur when C1 INH preparations were first neutralized by the addition of purified activated C1s. In summary, it is suggested that C1 INH plays a prominent role in dislodging the entire C1qr2s2 from immunoglobulin preparations which have a low binding affinity for the globular heads of C1q.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Ativação do Complemento , Proteínas Inativadoras do Complemento 1/farmacologia , Complemento C1q/imunologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , CamundongosRESUMO
A uniquely developed series of totally human monoclonal antibodies (mAbs) were examined for their complement fixing properties in comparison to human myeloma preparations and to commercially available human polyclonal immunoglobulins. C3b and C4b deposition was measured using a kinetic ELISA technique. When the IgG myeloma proteins were tested for classical pathway activation, our findings were similar to those previously described, where IgG1 and IgG3 were more potent activators of the classical pathway than IgG2 and IgG4. However, those same studies determined that IgG2 was the best activator of the alternative pathway followed by IgG1 and IgG3 while IgG4 does not activate complement via either pathway. In our studies of alternative pathway activation, the IgG2 myeloma exhibited strong activation of the alternative pathway, but, at levels lower than the other three IgG subtypes. Using this test system, we examined the complement activating potential of four totally human mAbs that were constructed from the peripheral blood lymphocytes of a colon carcinoma patient in long term remission. We found that our uniquely constructed totally human IgG2 mAbs (A3, E1, F6 and F8) were able to activate complement by both the classical and alternative pathways to varying degrees. In addition, we found that the complement activating ability of the human mAbs was greater than that of the human IgG2 myeloma immunoglobulins or normal human IgG2 preparations. This study represents the first report of complement activation by totally human mAbs and confirms more recent findings which indicate that levels of complement activation by human IgG immunoglobulins cannot be predicted based solely on their subclass identity.
Assuntos
Anticorpos Monoclonais/metabolismo , Ativação do Complemento , Complemento C3/metabolismo , Complemento C4/metabolismo , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas , Imunoglobulinas/química , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Proteínas do Mieloma/química , Proteínas do Mieloma/imunologia , Proteínas do Mieloma/metabolismo , Sensibilidade e EspecificidadeRESUMO
Using normal human serum and EDTA-plasma as the two sources of S-protein (vitronectin) in an enzyme-linked immunosorbent assay, we determined that heparin pretreatment of immobilized rgp120 or of immobilized CD4 caused the serum form of S-protein to deposit in a dose-dependent manner. Interestingly, the EDTA-plasma form of S-protein (native form) had little or no interaction with either of the heparin-treated surfaces. Several other sulfated polysaccharides such as dextran sulfate, pentosan polysulfate, heparan sulfate, and fucoidan, likewise mediated the deposition of the serum form S-protein on immobilized rgp120 and CD4. These findings may explain why certain glycosaminoglycans are effective against HIV infectivity in cell culture where the serum form of S-protein is present, yet ineffective in vivo where the native form of S-protein is predominant. The elevated glycosaminoglycan levels in gingival crevicular exudates, coupled with the effects of the serum form of S-protein and salivary-mediated neutralization mechanisms may explain the reduced rates of salivary HIV transmission.
Assuntos
Antígenos CD4/metabolismo , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Heparina/farmacologia , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/química , Humanos , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , VitronectinaRESUMO
Non-Immunoglobulin Salivary Agglutinins (NIA) which directly bind to microbes [including HIV] were studied for their potential to activate the first complement component (C1). It was determined that NIA had the same specific activity as heat aggregated IgG in binding to C1q and in activating C1. In order to determine the region of C1q which bound to NIA, C1q globular heads and C1q stems (collagen-like regions) were prepared and separated via a Western blot procedure. NIA bound principally to the globular heads of C1q and weakly to the collagen-like stem region. NIA were also studied for their potential to activate native C1 in normal human serum. Heat-aggregated IgG and cardiolipin served as positive controls. It was observed that incubation of isolated NIA with fresh normal human serum resulted in the formation of sodium dodecyl sulfate (SDS)-irreversible complexes of activated C1r-C1 inhibitor and activated C1s-C1 inhibitor and in activated C1s mediated C4 conversion. This indicated that isolated NIA had the potential to directly and effectively mediate classical complement pathway activation. Preincubation of NIA with C1q, blocked NIA mediated C1r and C1s activation and C4 conversion. The concn of NIA required to activate C1r and C1s was similar to that of heat-aggregated human IgG. In kinetic ELISA, NIA or aggregated IgG (positive controls) were first immobilized on microtiter plates, blocked with gelatin then incubated with fresh human serum as a source of complement. Depositions of C4b, C3b and iC3b substantiated that the complement system was effectively activated by immobilized NIA. The optimal relative NaCl concn for C4b deposition was 0.11 M. While pre-incubation of NIA with C1q blocked the subsequent C1 fixing potential of NIA, pre-incubation of NIA with rgp160 [HIV-1] or fibronectin did not interfere with the potential of NIA to fix C1.
Assuntos
Aglutininas/metabolismo , Ativação do Complemento , Complemento C1/metabolismo , Complemento C1q/metabolismo , Saliva/imunologia , Western Blotting , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Humanos , Peso MolecularRESUMO
Human saliva has been shown to reduce the infectivity of human immunodeficiency virus (HIV) particles in vitro. The factors in human saliva involved in this inhibition of HIV infectivity are unknown, although the salivary sediment of normal individuals has the major HIV neutralizing activity. Interestingly, the first complement component (C1) has been detected on the surface of the salivary sediment in the whole saliva of normal individuals. At the relatively low ionic strength of saliva, we determined that purified human C1q bound with high affinity to the envelope glycoprotein of HIV. Normally, the interaction of the C1q globular heads with immune complexes causes C1 activation. However, direct interactions between C1 and rgp120 (or rgp160) did not lead to C1 fixation, as determined by hemolytic studies with rate-limiting levels of C1, nor did rgp120 cause C1 activation as determined by activated C1s-mediated C4 conversion in normal human serum. Using ELISA, it was observed that intact C1, with the C1r2C1s2 tetramer associated with the collagen-like stem of C1q, did not bind to immobilized rgp120, whereas free C1q did bind. In addition, digestion of the C1q stem portion with collagenase completely eliminated its binding to rgp120. These findings suggest that the collagen-like stem region of C1q, rather than the globular heads, may participate in the binding to the envelope glycoprotein of HIV. Fibronectin, which is present in submandibular saliva, appeared to bind to rgp120 and to enhance the interaction of C1q with rgp120. It is conceivable that C1q and fibronectin, in binding and sequestering HIV particles (i.e. to the salivary sediment), may play an important role in the reduction of HIV transmission via saliva. Further studies will be needed to test the latter speculation.
Assuntos
Complemento C1q/metabolismo , Fibronectinas/metabolismo , Produtos do Gene env/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Precursores de Proteínas/metabolismo , Saliva/metabolismo , Western Blotting , Ativação do Complemento , Complemento C1/farmacologia , Complemento C4/biossíntese , Ensaio de Atividade Hemolítica de Complemento , Ensaio de Imunoadsorção Enzimática , Proteína gp160 do Envelope de HIV , Humanos , Imunoglobulina G/farmacologia , Técnicas In Vitro , Proteínas RecombinantesRESUMO
In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH.
Assuntos
Proteínas Inativadoras do Complemento 1/metabolismo , Neutrófilos/metabolismo , Ligação Competitiva/imunologia , Linhagem Celular , Ensaio de Atividade Hemolítica de Complemento , Endopeptidases/farmacologia , Humanos , Cinética , Microesferas , Neutrófilos/efeitos dos fármacos , Ácidos Siálicos/imunologiaRESUMO
When complement first contacts salivary secretions, as when gingival crevicular fluid first meets saliva at the gingival margin, complement function is enhanced. The immediate potentiation of the complement system at equal volume ratios of serum to saliva is due to several factors, including the lower ionic strength of saliva when compared with serum and the presence of certain salivary glyproteins such as the nonimmunoglobulin agglutinins that appear to simultaneously activate C1 and affect (sequester) certain complement control proteins, such as Factor H. This initial potentiation of the complement cascade by saliva may aid in defending the area immediately above the gingival crevice from oral microbiota that are being coated with a combination of serous exudate components and salivary components. As serum becomes much more diluted with saliva (i.e., crevicular fluid moves away from the supragingival area), the acidic proline-rich salivary proteins (APRP) begin to disrupt the unbound C1q-C1r2-C1s2 macromolecular complexes. Thus, the APRP along with other C1 fixing substances in saliva appear to restrict complement function, but only when the ratios of saliva to serum exceed 250:1. Since certain salivary glycoproteins bind to viruses, the potentiation of the complement system by saliva may also play a role in neutralizing certain viral infections on mucosal surfaces where tissue transudates containing complement begin to contact mucosal secretions such as saliva. Again, the ratio of serous fluid to mucosal secretion appears to be an important factor. This article also discusses some of our preliminary data and speculations concerning the binding of the self-associating high-molecular-weight nonimmunoglobulin salivary agglutinins (NIA) with the envelope of the human immunodeficiency virus (HIV) and the possible cooperative role of C1q and fibronectin in aiding neutralization of HIV infectivity.
Assuntos
Proteínas do Sistema Complemento/fisiologia , Saliva/fisiologia , Ativação do Complemento/fisiologia , Humanos , Modelos Biológicos , Saliva/imunologia , Estomatite/imunologiaAssuntos
Proteínas do Sistema Complemento , Angioedema/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Adesão Celular , Membrana Celular/metabolismo , Ativação do Complemento , Proteínas do Sistema Complemento/deficiência , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Hemoglobinúria Paroxística/imunologia , Humanos , Fagocitose , Conformação Proteica , Receptores de Complemento/imunologia , Diálise Renal/efeitos adversosRESUMO
The relative complement-activating properties of several dental cements were investigated. After the cements were incubated with fresh human serum as a source of complement, the percent of the electrophoretic conversion was assessed by means of the C3 crossed-immunoelectrophoresis technique. It was determined that the ZnO-containing cements--which include zinc phosphate, zinc polycarboxylate, zinc oxide eugenol, and zinc hexyl vanillate--each caused C3 conversion, indicative of complement activation. Glass-ionomer cement, which does not contain ZnO, did not activate the complement system. In dose-response studies, ZnO at relatively low concentrations was effective in causing C3 conversion, while at higher concentrations ZnO appeared to block C3 conversion. Supernatants from ZnO suspensions also caused C3 conversion. Cement particle size, as well as soluble degradative products containing ZnO or Zn++, were suggested as possible factors contributing to the differential effects of the dental cements on complement activation and/or function.
Assuntos
Ativação do Complemento , Cimentos Dentários , Via Alternativa do Complemento , Cimentos de Ionômeros de Vidro , Óxido de ZincoRESUMO
Samples of four brands of gutta-percha and the nine ingredients that make up one brand were studied in vitro to observe their interaction with the serum complement system, thus allowing for assessment of their possible inflammatory potential. Crossed immunoelectrophoresis of the third complement component was used as an indicator of complement activation. The four different brands of gutta-percha showed comparable complement activation as determined by C3 conversion. When the ingredients of one brand of gutta-percha were examined for complement-activating properties, major activities were associated with gutta-percha compound, agerite stalite-antioxidant, and titanium oxide food grade. The significance of the possible inflammatory potential of gutta-percha and its ingredients, as it relates to endodontic therapy, is discussed.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Complemento C3/imunologia , Guta-Percha/farmacologia , Materiais Restauradores do Canal Radicular/farmacologia , Humanos , Imunoeletroforese , InflamaçãoRESUMO
Non-immunoglobulin salivary agglutinins (SBA) for bacteria which bind to Streptococcus milleri TJ7 were isolated from parotid saliva and their interactions with human IgG studied. Purified SBA showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular weight of approx. 500,000. Heat aggregated human IgG (63 degrees C, 30 min), but not native IgG, interacted with SBA and thereby interfered with the ability of the SBA to agglutinate Strep. milleri. Immune complexes prepared from tetanus toxoid and isolated human IgG anti-tetanus toxoid antibody also inhibited salivary bacterial agglutination by SBA; antigen (tetanus toxoid) alone or antibody (anti-tetanus toxoid antibody) alone did not have this effect. Direct-binding studies with immobilized SBA on nitrocellulose paper showed that aggregated IgG bound to immobilized SBA and that this binding was inhibited by EDTA. Thus it appears that heat or specific antigen is able to induce an aggregation of IgG which results in the binding of the aggregated form of IgG to SBA.
Assuntos
Aglutininas/imunologia , Complexo Antígeno-Anticorpo/imunologia , Imunoglobulina G/imunologia , Saliva/imunologia , Streptococcus/imunologia , Aglutinação , Temperatura Alta , Humanos , Toxoide Tetânico/imunologiaRESUMO
In order to clarify the mechanism of interaction of serum amyloid P component (SAP) with complement, the interaction of SAP with C1q and with IgG was studied. It is known that SAP binds Sepharose in the presence of calcium. When purified 125I-C1q was incubated with SAP prior to Sepharose affinity chromatography, 125I-C1q was retained. However, in the absence of SAP, the 125I-C1q was not retained. To further examine the interaction of SAP with C1q, isolated SAP was incubated at varying ratios with C1q in the presence of 1.5 mM Ca2+. These mixtures were subsequently examined via crossed immunoelectrophoresis against goat anti-SAP. A change in the electrophoretic behavior of SAP was observed in the presence of C1q. In other studies, it was observed that SAP might interact with the collagen-like stem of C1q. In these latter studies, 125I-SAP was incubated with pepsin digests of C1q in a microtitre solid-phase binding assay. In addition, a microtitre solid-phase binding assay was utilized in order to investigate the possible binding of isolated 125I-SAP with IgG. Interestingly in the presence of Ca2+, human IgG and Fab gamma, but not Fc gamma, were found to bind 125I-SAP.
Assuntos
Enzimas Ativadoras do Complemento/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias gama de Imunoglobulina/imunologia , Componente Amiloide P Sérico/imunologia , Reações Antígeno-Anticorpo , Complemento C1q , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina G/imunologiaRESUMO
When human serum is diluted and pre-incubated at 37 degrees C in low ionic strength buffer (LIS, u = 0.07; made iso-osmotic with dextrose), a spontaneous activation of complement (C) is observed as determined by C4 and C3 electrophoretic conversion. In this paper it is postulated that most species of glycosaminoglycans (GAG) restricted non-specific fluid phase complement consumption induced by LIS, an effect which conserved complement and thereby enhanced the subsequent residual serum C mediated hemolytic activity. The capacity of glycosaminoglycans, to have modulated the hemolytic activity at low ionic strength, depended on the charge of the GAG species tested. In general, the GAG regulatory effects may have been due to GAG mediated restriction of spontaneous non-specific fluid phase C1 autoactivation, and/or restriction of activated C1 activity. Such effects would result in the subsequent reduction of the spontaneous fluid phase C4 and C3 consumption. Although the precise mechanisms responsible for the effects were not identified, it is speculated that the potentiation of C1 inhibitor function and direct effects on C1 might be involved. Overall, the relative specific activities of the glycosaminoglycans, on a weight basis, in mediating the fluid phase C regulatory effect were heparin greater than dermatan sulfate greater than chondroitin-6-sulfate greater than chondroitin-4-sulfate greater than hyaluronic acid and keratan sulfate. When much higher concns of heparin (greater than or equal 0.2 micrograms/ml) were used, complement mediated lysis of EA was inhibited, probably due to the direct inhibition of C1, even C1 which may have bound to the sensitized erythrocytes (EA). Results similar to that of heparin were obtained using greater than 1 mg/ml of dermatan sulfate or dextran sulfate. In contrast, pre-incubation of human serum in LIS with high concns (up to 10 mg/ml) of hyaluronic acid or chondroitin-4-sulfate, which are much less charged, continued to result only in the restriction of hemolytically non-specific (fluid phase) C consumption, resulting in a higher residual complement hemolytic activity. A theory is developed that the binding of polyionic GAG to C1 and to C1 INH may provide a charged local environment which simulates a relatively higher ionic strength. Chemical degradation of hyaluronic acid or chondroitin-4 or -6 sulfate resulted in lowering of this C modulating effect, indicative of the importance of the structural integrity of these charged glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Proteínas do Sistema Complemento/imunologia , Glicosaminoglicanos/farmacologia , Hemólise/efeitos dos fármacos , Saliva/imunologia , Ativação do Complemento/efeitos dos fármacos , Complemento C3/metabolismo , Relação Dose-Resposta Imunológica , Temperatura Alta , Humanos , Ácido Hialurônico/farmacologia , Concentração OsmolarRESUMO
The dilution of human serum in relatively low ionic strength buffer (mu = 0.070) caused the spontaneous activation of C1 and a limited activation of C4 and C3 in the fluid phase. The unusual degree of activation of the complement system in the fluid phase suggested that the optimal functions of complement-regulatory systems such as C1 inhibitor might be reduced. As a function of the time of preincubation (PI) of diluted serum at 37 degrees C, under low ionic strength conditions, an unusual complement-mediated hemolytic kinetic pattern was observed upon adding sensitized erythrocytes (EA). For a 1:36 dilution of human serum, there was an initial progressive decrease in complement hemolytic activity (from 3 to 20 min PI, phase I), followed by an apparent functional reversal (increase) in hemolytic activity (20-50 min PI, phase II) and finally a gradual irreversible depletion of the hemolytic activity (after 50 min PI, phase III). This hemolytic pattern could only be adequately demonstrated using a kinetic assay which followed the course of lysis of EA in the presence of low dilutions of human serum as a complement source. Others might have missed this observation due to the use of end-point titration methods which required the use of relatively elevated serum dilutions at the time of EA addition. Mechanisms which governed the variations in hemolytic activity at low ionic strength were not clear. Speculatively, partial consumption of early complement components, generation of free C1q and generation of complement fragments might have accounted for the initial decrease in the hemolytic activity observed in phase I. The apparent functional reversal of hemolytic activity observed in phase II might have involved a critical depletion of C1 inhibitor which occurred secondary to C1 inhibitor binding to C1 (activated by low ionic strength effects) and to the C1 activated at the time of EA addition. Without sufficient regulation, a rapid unrestricted C1-mediated complement activation could have occurred, which resulted in a rapid deposition of complement on the EA. Finally, prolonged exposure of serum to low ionic strength effects appeared to induce a significant complement consumption, which caused a time-dependent irreversible depletion of complement hemolytic activity (phase III). Excess exogenous C1 inhibitor, when co-incubated with diluted serum at low ionic strength, reversed the time-dependent effects of low ionic strength and enhanced the subsequent specific complement-mediated hemolytic activity as compared to controls.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Proteínas do Sistema Complemento/metabolismo , Hemólise , Ativação do Complemento , Complemento C1/metabolismo , Humanos , Íons , Cinética , Concentração Osmolar , Fatores de TempoRESUMO
Native hyaluronic acid (HA) is reported to be a weak anticomplementary agent. However, the normal buffer systems used for complement tests incorporate gelatin, Ca2+ and Mg2+, which may bind to HA, influence its conformation and interfere with its anticomplementary reactions with complement components such as Cl. In this study, metal ions (Ca2+ and Mg2+), gelatin and fibronectin appeared to react with native HA preparations and block their anticomplementary effects on Cl. In previous studies, we obtained evidence for a relationship between reversible heat-induced HA conformational changes and a subsequent reversible increase in anticomplementary activity. The anticomplementary activity of heat-treated HA preparations was also reduced by gelatin.
Assuntos
Cátions Bivalentes/farmacologia , Proteínas Inativadoras do Complemento/antagonistas & inibidores , Gelatina/farmacologia , Ácido Hialurônico/antagonistas & inibidores , Sítios de Ligação , Soluções Tampão , Cálcio/farmacologia , Complemento C1/metabolismo , Fibronectinas/farmacologia , Hemólise/efeitos dos fármacos , Temperatura Alta , Humanos , Ácido Hialurônico/farmacologia , Magnésio/farmacologia , Conformação ProteicaRESUMO
The in vitro interaction of hyaluronic acid (HA) with complement (C) classical-pathway activity has been investigated. It was found that native HA, even at a high concn (greater than 3 mg/ml), has a relatively weak anticomplementary activity. However, we report here that native HA can be reversibly altered by heat treatment such that C-inhibitory properties are manifested. We have determined in this study that a potent C-inhibitory activity can be obtained if HA solutions are thermally treated (100 degrees C), and stabilized by prompt freezing with prompt thawing just prior to the interaction with human serum complement. Several investigators have proposed that the intermolecular-associated strands of HA undergo a reversible decoupling upon thermal treatment and this decoupled state of HA can be semi-stabilized by quickly cooling the sample. This heat-treated HA strongly inhibits C1 as well as classical-pathway-mediated C3 conversion. However, if heat-treated HA samples are not stabilized but, rather, slowly cooled after heating or if heated HA samples are snapfrozen and then slowly thawed, the anticomplementary activity is gradually lost. Interestingly, the activity for this same sample can be regenerated by retreatment of the same sample with heat followed by low-temp stabilization, indicating the reversibility of the physical state of HA responsible for the anticomplementary effect. Since no detectable molecular degradation of thermally-treated HA was found, it was assumed that a heat-induced physical transition of HA (decoupled state) was responsible for the C-inhibitory effect.