RESUMEN
Complement C1q, the initiation molecule of the classical pathway, exerts various immunomodulatory functions independent of complement activation. Non-classical functions of C1q include the clearance of apoptotic cells and cholesterol crystals (CC), as well as the modulation of cytokine secretion by immune cells such as macrophages. Moreover, C1q has been shown to act as a binding partner for von Willebrand factor (vWF), initiation molecule of primary hemostasis. However, the consequences of this C1q-vWF interaction on the phagocytosis of CC by macrophages has remained elusive until now. Here, we used CC-C1q-vWF complexes to study immunological effects on human monocyte-derived macrophages (HMDMs). HMDMs were investigated by analyzing surface receptor expression, phagocytosis of CC complexes, cytokine secretion, and caspase-1 activity. We found that vWF only bound to CC in a C1q-dependent manner. Exposure of macrophages to CC-C1q-vWF complexes resulted in an upregulated expression of phagocytosis-mediating receptors MerTK, LRP-1, and SR-A1 as well as CD14, LAIR1, and PD-L1 when compared to CC-C1q without vWF, whereas phagocytosis of CC-C1q complexes was hampered in the presence of vWF. In addition, we observed a diminished caspase-1 activation and subsequent reduction in pro-inflammatory IL-1ß cytokine secretion, IL-1ß/IL-1RA ratio and IL-1α/IL-1RA ratio. In conclusion, our results demonstrate that vWF binding to C1q substantially modulates the effects of C1q on HMDMs. In this way, the C1q-vWF interaction might be beneficial in dampening inflammation, e.g., in the context of atherosclerosis.
Asunto(s)
Colesterol/metabolismo , Complemento C1q/inmunología , Complemento C1q/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Fagocitosis/inmunología , Factor de von Willebrand/metabolismo , Biomarcadores , Caspasa 1/metabolismo , Quimiocinas CC/metabolismo , Colesterol/química , Citocinas/metabolismo , Humanos , Inmunidad InnataRESUMEN
In several types of antigen-presenting cells (APCs), Cathepsin S (CatS) plays a crucial role in the regulation of MHC class II surface expression and consequently influences antigen (Ag) presentation of APCs to CD4+ T cells. During the assembly of MHC class II-Ag peptide complexes, CatS cleaves the invariant chain p10 (Lip10) - a fragment of the MHC class II-associated invariant chain peptide. In this report, we used a selective, high-affinity CatS inhibitor to suppress the proteolytic activity of CatS in lymphoid and myeloid cells. CatS inhibition resulted in a concentration-dependent Lip10 accumulation in B cells from both healthy donors and patients with systemic lupus erythematosus (SLE). Furthermore, CatS inhibition led to a decreased MHC class II expression on B cells, monocytes, and proinflammatory macrophages. In SLE patient-derived peripheral blood mononuclear cells, CatS inhibition led to a suppressed secretion of IL-6, TNFα, and IL-10. In a second step, we tested the effect of CatS inhibition on macrophages being exposed to patient-derived autoantibodies against C1q (anti-C1q) that are known to be associated with severe lupus nephritis. As shown previously, those SLE patient-derived high-affinity anti-C1q bound to immobilized C1q induce a proinflammatory phenotype in macrophages. Using this human in vitro model of autoimmunity, we found that CatS inhibition reduces the inflammatory responses of macrophages as demonstrated by a decreased secretion of proinflammatory cytokines, the downregulation of MHC class II and CD80. In summary, we can show that the used CatS inhibitor is able to block Lip10 degradation in healthy donor- and SLE patient-derived B cells and inhibits the induction of proinflammatory macrophages. Thus, CatS inhibition seems to be a promising future treatment of SLE.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Inflamación/metabolismo , Lupus Eritematoso Sistémico/inmunología , Macrófagos/metabolismo , Adulto , Anciano , Antígenos CD20/genética , Antígenos CD20/metabolismo , Antígenos de Superficie , Linfocitos B , Células Cultivadas , Citocinas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Antibodies against C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE). The anti-C1q antibodies strongly correlate with the occurrence of lupus nephritis and low-circulating C1q levels. Previous studies have demonstrated that myeloid cells, i.e., dendritic cells and macrophages, are a major source of C1q. However, a direct effect of anti-C1q on C1q secretion by macrophages has not yet been established. In the present study, we investigated the C1q secretion profile of in vitro human monocyte-derived macrophages (HMDMs) obtained from healthy donors and from patients with SLE. The effect of SLE patient-derived anti-C1q bound to immobilized C1q (imC1q) and imC1q alone on HMDMs was investigated by C1q secretion levels, the expression of membrane-bound and intracellular C1q using flow cytometry and ImageStreamX technology, and testing the ability of secreted C1q to activate the classical pathway (CP) of the complement. Bound anti-C1q induced significantly greater C1q secretion levels as compared with imC1q alone or healthy donor IgG. The extent of C1q secretion by HMDMs correlated with IgG anti-C1q levels of patients with SLE but not of healthy controls. Furthermore, bound autoantibodies and imC1q induced continuous and de novo C1q synthesis as evident by the intracellular C1q content, which correlated with C1q secretion levels. Finally, secreted C1q was able to activate the CP, as reflected by C4b deposition. Interestingly, anti-C1q-dependent C1q secretion could also be observed in SLE patient-derived cells. In conclusion, our data indicate that imC1q-bound anti-C1q strongly stimulate the C1q production by HMDMs. Anti-C1q-induced C1q secretion might be an important immune-modulatory factor in SLE.
Asunto(s)
Autoanticuerpos/inmunología , Complemento C1q/biosíntesis , Complemento C1q/inmunología , Lupus Eritematoso Sistémico/inmunología , Macrófagos/metabolismo , Adulto , Anciano , Femenino , Citometría de Flujo , Humanos , Proteínas Inmovilizadas/metabolismo , Inmunoglobulina G/metabolismo , Espacio Intracelular/metabolismo , Cinética , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/patología , Masculino , Persona de Mediana Edad , Monocitos/patologíaRESUMEN
Anti-C1q autoantibodies (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE) and correlate with the occurrence of proliferative lupus nephritis. A previous study of anti-C1q in experimental lupus nephritis demonstrated an important role for FcγRs in the pathogenesis of lupus nephritis, suggesting a direct effect on phagocytes. Therefore, we developed an in vitro model to study the effect of SLE patient-derived anti-C1q bound to immobilized C1q (imC1q) on human monocyte-derived macrophages (HMDMs) obtained from healthy donors and SLE patients. HMDMs were investigated by analyzing the cell morphology, LPS-induced cytokine profile, surface marker expression, and phagocytosis rate of apoptotic Jurkat cells. Morphologically, bound anti-C1q induced cell aggregations of HMDMs compared with imC1q or IgG alone. In addition, anti-C1q reversed the effect of imC1q alone, shifting the LPS-induced cytokine release toward a proinflammatory response. FcγR-blocking experiments revealed that the secretion of proinflammatory cytokines was mediated via FcγRII. The anti-C1q-induced inflammatory cytokine profile was accompanied by a downregulation of CD163 and an upregulation of LPS-induced CD80, CD274, and MHC class II. Finally, HMDMs primed on bound anti-C1q versus imC1q alone displayed a significantly lower phagocytosis rate of early and late apoptotic cells accompanied by a reduced Mer tyrosine kinase expression. Interestingly, anti-C1q-dependent secretion of proinflammatory cytokines was similar in SLE patient-derived cells, with the exception that IL-10 was slightly increased. In conclusion, anti-C1q induced a proinflammatory phenotype in HMDMs reversing the effects of imC1q alone. This effect might exacerbate underlying pathogenic mechanisms in lupus nephritis.
Asunto(s)
Autoanticuerpos/inmunología , Complemento C1q/inmunología , Lupus Eritematoso Sistémico/inmunología , Macrófagos/inmunología , Adulto , Autoantígenos/inmunología , Femenino , Humanos , Inflamación/inmunología , Nefritis Lúpica/inmunología , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Autoantibodies against complement C1q (anti-C1q) strongly correlate with the occurrence of lupus nephritis and hypocomplementemia in systemic lupus erythematosus (SLE). Although a direct pathogenic role of anti-C1q has been suggested, the assumed complement-activating capacity remains to be elucidated. Using an ELISA-based assay, we found that anti-C1q activate the classical (CP) and lectin pathways (LP) depending on the anti-C1q immunoglobulin-class repertoire present in the patient's serum. IgG anti-C1q resulted in the activation of the CP as reflected by C4b deposition in the presence of purified C1 and C4 in a dose-dependent manner. The extent of C4b deposition correlated with anti-C1q levels in SLE patients but not in healthy controls. Our data indicate that SLE patient-derived anti-C1q can activate the CP and the LP but not the alternative pathway of complement. These findings are of importance for the understanding of the role of anti-C1q in SLE suggesting a direct link to hypocomplementemia.
Asunto(s)
Autoanticuerpos/inmunología , Complemento C1q/inmunología , Vía Clásica del Complemento/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Lupus Eritematoso Sistémico/inmunología , Adulto , Anciano , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Nefritis Lúpica/inmunología , Masculino , Persona de Mediana EdadRESUMEN
Valerenic acid (VA), a sesquiterpenoid, is one of the major secondary bioactive metabolites of VALERIANA OFFICINALIS L. Until now IN VIVO studies on the absorption, bioavailability, disposition, and metabolism of VA are limited. We established and validated an LC-MS/MS assay for the determination of VA in rat plasma and successfully used this method for pharmacokinetic studies in rats after intravenous (i. v.) and oral administrations. The plasma concentration-time data was analyzed by both non-compartmental and compartmental approaches using WinNonlin software. Following i. v. administration, the disposition of VA in rat plasma was biphasic, subdivided into a fast distribution and a slow elimination phase. The half-life of the distribution phase was 6-12 min, and that of the terminal elimination phase 6-46 h, indicating a possible large tissue binding. Disposition PK of valerenic acid after oral treatment was also described by a two-compartment model with a clearance (CL/F) of 2-5 L · h (-1) · kg (-1) and volume of distribution of (V (d)) 17-20 L · kg (-1). The extent of absorption (F) after oral administration was estimated to be 33.70 % with a half-life of 2.7-5 h. Dose proportionality was observed in terms of dose and AUCs, suggesting linear pharmacokinetics at the dose levels studied in rats.