RESUMO
The abnormal neurodevelopment secondary to in utero adversities, such as hypoxia, malnutrition and maternal infections, underlies schizophrenia (SZ) etiology. As the genes of MBL-associated serine proteases (MASP) of the complement lectin pathway, MASP1 and MASP2, are expressed in the developing cortex and are functionally important for neuronal migration, we hypothesize that the malfunction ofl-ficolin-MASP arm may also be involved in schizophrenia pathophysiology as it was shown for MBL-MASP complexes. We investigated serum l-ficolin and plasma MASP-2 levels, the activity of l-ficolin-bound MASP-2, as well as an array of the complement-related variables in chronic schizophrenic patients in the acute phase of the disease and controls without physical or mental diagnoses. The median concentration of l-ficolin in Armenian controls was 3.66 µg/ml and similar to those reported for other Caucasian populations. SZ-cases had â¼40 % increase in serum l-ficolin (median 5.08 µg/ml; P < 0.0024). In the pooled sample, l-ficolin level was higher in males than in females (P < 0.0031), but this gender dichotomy was not affecting the variable association with schizophrenia (P < 0.016). Remarkably, MASP-2 plasma concentration showed gender-dependent significant variability in the group of patients but not in controls. When adjusted for gender and gender*diagnosis interaction, a significantly high MASP-2 level in female patients versus female controls was observed (median: 362 ng/ml versus 260 ng/ml, respectively; P < 0.0020). A significant increase in l-ficolin-bound MASP-2 activity was also observed in schizophrenia (on the median, cases vs controls: 7.60 vs 6.50 RU; P < 0.021). Correlation analyses of the levels of l-ficolin and MASP-2, l-ficolin-(MASP-2) activity and the demographic data did not show any significant association with the age of individuals, family history, age at onset and duration of the illness, and smoking. Noteworthy, the levels of l-ficolin and MASP-2 in circulation were significantly associated with the type of schizophrenia (paranoid SZ-cases had much higher l-ficolin (P < 0.0035) and lower MASP-2 levels than the other types combined (P < 0.049)). Correlations were also found between: (i) the classical pathway functional activity and l-ficolin level (rs = 0.19, P < 0.010); (ii) the alternative pathway functional activity and MASP-2 level (rs = 0.26, P < 0.00035); (iii) the activity of l-ficolin-bound MASP2 and the downstream C2 component haemolytic activity (rs = -0.19, P < 0.017); and (iv) l-ficolin and the upstream C-reactive protein (CRP) serum concentrations (r = 0.28, P < 0.018). Overall, the results showed l-ficolin-related lectin pathway alterations in schizophrenia pathophysiology. It is likely that in addition to the MBL-MASP component over-activity reported previously, the alterations of the lectin pathway in schizophrenia also involve variations of l-ficolin-(MASP-2) on protein concentration and activity levels.
Assuntos
Lectina de Ligação a Manose , Esquizofrenia , Masculino , Humanos , Feminino , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Lectinas , Lectina de Ligação a Manose da Via do Complemento , Proteínas do Sistema Complemento , Lectina de Ligação a Manose/genética , FicolinasRESUMO
The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis.
Assuntos
Proteínas do Sistema Complemento/biossíntese , Glioblastoma/metabolismo , Biomarcadores , Ativação do Complemento , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Glioblastoma/imunologia , Hemólise/imunologia , Humanos , ProteóliseRESUMO
Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen-macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1ß, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-ß) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1ß, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host-pathogen interactions in tuberculosis.
Assuntos
Macrófagos/fisiologia , Mycobacterium bovis/fisiologia , Properdina/fisiologia , Trombospondinas/fisiologia , Citocinas/genética , Humanos , Células THP-1RESUMO
Development of nanoparticles as tissue-specific drug delivery platforms can be considerably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) via its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation.
Assuntos
Proteínas ADAMTS/imunologia , Macrófagos/imunologia , Nanotubos de Carbono/química , Properdina/imunologia , Proteínas ADAMTS/genética , Carboximetilcelulose Sódica/química , Ativação do Complemento , Citocinas/genética , Células HEK293 , Humanos , Inflamação/imunologia , Properdina/genética , Ligação Proteica , Células THP-1RESUMO
Bovine conglutinin, the first animal collectin to be discovered, is structurally very similar to Surfactant Protein D (SP-D). SP-D is known to interact with Mycobacterium tuberculosis, and the closely-related M. bovis, the causative agent of bovine tuberculosis. We speculated that due to the overall similarities between conglutinin and SP-D, conglutinin is likely to have a protective influence in bovine tuberculosis. We set out to investigate the role of conglutinin in host-pathogen interaction during mycobacterial infection. We show here that a recombinant truncated form of conglutinin (rfBC), composed of the neck and C-type lectin domains, binds specifically and in a dose-dependent manner to the model organism Mycobacterium bovis BCG. rfBC showed a significant direct bacteriostatic effect on the growth of M. bovis BCG in culture. In addition, rfBC inhibited the uptake of M. bovis BCG by THP-1 macrophages (human monocyte lineage cell line) and suppressed the subsequent pro-inflammatory response. Conglutinin is well-known as a binder of the complement activation product, iC3b. rfBC was also able to inhibit the uptake of complement-coated M. bovis BCG by THP-1 macrophages, whilst modulating the pro-inflammatory response. It is likely that rfBC inhibits the phagocytosis of mycobacteria by two distinct mechanisms: firstly, rfBC interferes with mannose receptor-mediated uptake by masking lipoarabinomannan (LAM) on the mycobacterial surface. Secondly, since conglutinin binds iC3b, it can interfere with complement receptor-mediated uptake via CR3 and CR4, by masking interactions with iC3b deposited on the mycobacterial surface. rfBC was also able to modulate the downstream pro-inflammatory response in THP-1 cells, which is important for mobilizing the adaptive immune response, facilitating containment of mycobacterial infection. In conclusion, we show that conglutinin possesses complement-dependent and complement-independent anti-mycobacterial activities, interfering with both known mechanisms of mycobacterial uptake by macrophages. As mycobacteria are specialized intracellular pathogens, conglutinin may inhibit M. bovis and M. tuberculosis from establishing an intracellular niche within macrophages, and thus, negatively affect the long-term survival of the pathogen in the host.
Assuntos
Colectinas/imunologia , Proteínas do Sistema Complemento/imunologia , Mycobacterium bovis/imunologia , Soroglobulinas/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Animais , Biomarcadores , Bovinos , Colectinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/imunologia , Soroglobulinas/metabolismo , Células THP-1 , Tuberculose Bovina/metabolismoRESUMO
Carbon nanotubes (CNTs) are increasingly being developed for use in biomedical applications, including drug delivery. One of the most promising applications under evaluation is in treating pulmonary diseases such as tuberculosis. Once inhaled or administered, the nanoparticles are likely to be recognised by innate immune molecules in the lungs such as hydrophilic pulmonary surfactant proteins. Here, we set out to examine the interaction between surfactant protein D (SP-D), a key lung pattern recognition molecule and CNTs, and possible downstream effects on the immune response via macrophages. We show here that a recombinant form of human SP-D (rhSP-D) bound to oxidised and carboxymethyl cellulose (CMC) coated CNTs via its C-type lectin domain and enhanced phagocytosis by U937 and THP-1 macrophages/monocytic cell lines, together with an increased pro-inflammatory response, suggesting that sequestration of SP-D by CNTs in the lungs can trigger an unwanted and damaging immune response. We also observed that functionalised CNTs, opsonised with rhSP-D, continued to activate complement via the classical pathway, suggesting that C1q, which is the recognition sub-component of the classical pathway, and SP-D have distinct pattern recognition sites on the CNTs. Consistent with our earlier reports, complement deposition on the rhSP-D opsonised CNTs led to dampening of the pro-inflammatory immune response by THP-1 macrophages, as evident from qPCR, cytokine array and NF-κB nuclear translocation analyses. This study highlights the importance of understanding the interplay between innate immune humoral factors including complement in devising nanoparticle based drug delivery strategies.
Assuntos
Macrófagos/citologia , Nanotubos de Carbono/química , Fagocitose , Proteína D Associada a Surfactante Pulmonar/química , Complemento C1q/química , Via Clássica do Complemento , Citocinas/metabolismo , Humanos , Proteínas Recombinantes/química , Células THP-1 , Fatores de Transcrição/metabolismo , Transcriptoma , Células U937RESUMO
Nanoparticles are attractive drug delivery vehicles for targeted organ-specific as well as systemic therapy. However, their interaction with the immune system offers an intriguing challenge to the success of nanotherapeutics in vivo. Recently, we showed that pristine and derivatised carbon nanotubes (CNT) can activate complement mainly via the classical pathway leading to enhanced uptake by phagocytic cells, and transcriptional down-regulation of pro-inflammatory cytokines. Here, we report the interaction of complement-activating CC-CNT and RNA-CNT, and non-complement-activating gold-nickel (Au-Ni) nanowires with cell lines representing macrophage, B and T cells. Complement deposition considerably enhanced uptake of CNTs by immune cells known to overexpress complement receptors. Real-Time qPCR and multiplex array analyses showed complement-dependent down-regulation of TNF-α and IL-1ß and up-regulation of IL-12 by CMC- and RNA-CNTs, in addition to revealing IL-10 as a crucial regulator during nanoparticle-immune cell interaction. It appears that complement system can recognize molecular patterns differentially displayed by nanoparticles and thus, modulate subsequent processing of nanoparticles by antigen capturing and antigen presenting cells, which can shape innate and adaptive immune axes.
Assuntos
Linfócitos B/imunologia , Citocinas/imunologia , Imunidade Celular/imunologia , Macrófagos/imunologia , Nanotubos de Carbono/toxicidade , Linfócitos T/imunologia , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/toxicidade , Humanos , Imunidade Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Teste de Materiais , Nanotubos de Carbono/química , Linfócitos T/efeitos dos fármacosRESUMO
Mycobacterium tuberculosis is an accomplished intracellular pathogen, particularly within the macrophage and this is of the utmost importance in the host-pathogen stand-off observed in the granuloma during latent tuberculosis. Contact with innate immune molecules is one of the primary interactions that can occur with the pathogen M. tuberculosis once inhaled. Complement proteins may play a role in facilitating M. tuberculosis interactions with macrophages. Here, we demonstrate that factor H, a complement regulatory protein that down-regulates complement alternative pathway activation, binds directly to the model organism M. bovis BCG. Binding of factor H reaches saturation at 5-10µg of factor H/ml, well below the plasma level. C4 binding protein (C4BP) competed with factor H for binding to mycobacteria. Factor H was also found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Real-time qPCR analysis showed stark differential responses of pro- and anti-inflammatory cytokines during the early stages of phagocytosis, as evident from elevated levels of TNF-α, IL-1ß and IL-6, and a concomitant decrease in IL-10, TGF-ß and IL-12 levels, when THP-1:BCG interaction took place in the presence of factor H. Our results suggest that factor H can interfere with mycobacterial entry into macrophages and modulate inflammatory cytokine responses, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. Our results offer novel insights into complement activation-independent functions of factor H during the host-pathogen interaction in tuberculosis.
Assuntos
Citocinas/imunologia , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Mycobacterium bovis/fisiologia , Linhagem Celular Tumoral , Proteína de Ligação ao Complemento C4b/imunologia , Fator H do Complemento/imunologia , Citocinas/genética , Humanos , Macrófagos/imunologia , FagocitoseRESUMO
Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. FROM THE CLINICAL EDITOR: Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting.
Assuntos
Complemento C1q/imunologia , Fator H do Complemento/imunologia , Imunidade Inata , Macrófagos/imunologia , Nanotubos de Carbono/efeitos adversos , Linhagem Celular , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Ativação do Complemento , Proteínas do Sistema Complemento , Humanos , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestrutura , FagocitoseRESUMO
Carbon nanotubes (CNTs) have promised a range of applications in biomedicine. Although influenced by the dispersants used, CNTs are recognized by the innate immune system, predominantly by the classical pathway of the complement system. Here, we confirm that complement activation by the CNT used continues up to C3 and C5, indicating that the entire complement system is activated including the formation of membrane-attack complexes. Using recombinant forms of the globular regions of human C1q (gC1q) as inhibitors of CNT-mediated classical pathway activation, we show that C1q, the first recognition subcomponent of the classical pathway, binds CNTs via the gC1q domain. Complement opsonisation of CNTs significantly enhances their uptake by U937 cells, with concomitant downregulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines in both U937 cells and human monocytes. We propose that CNT-mediated complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response. FROM THE CLINICAL EDITOR: This study highlights the importance of the complement system in response to carbon nanontube administration, suggesting that the ensuing complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Fagocitose/efeitos dos fármacos , Linhagem Celular , Complemento C1q/imunologia , Citocinas/imunologia , Humanos , Macrófagos/imunologia , Nanotubos de Carbono/ultraestruturaRESUMO
Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin αMß2, or CD11b/CD18) is expressed on all macrophages and recognizes iC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of iC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for iC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site-bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d:CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes.
Assuntos
Complemento C3b/metabolismo , Imunidade Inata/imunologia , Antígeno de Macrófago 1/química , Macrófagos/metabolismo , Modelos Moleculares , Proteínas Opsonizantes/química , Fagocitose/imunologia , Biologia Computacional , Escherichia coli , Humanos , Antígeno de Macrófago 1/metabolismo , Proteínas Opsonizantes/metabolismo , Conformação ProteicaRESUMO
Properdin and factor H are two key regulatory proteins having opposite functions in the alternative complement pathway. Properdin up-regulates the alternative pathway by stabilizing the C3bBb complex, whereas factor H downregulates the pathway by promoting proteolytic degradation of C3b. While factor H is mainly produced in the liver, there are several extrahepatic sources. In addition to the liver, factor H is also synthesized in fetal tubuli, keratinocytes, skin fibroblasts, ocular tissue, adipose tissue, brain, lungs, heart, spleen, pancreas, kidney, muscle, and placenta. Neutrophils are the major source of properdin, and it is also produced by monocytes, T cells and bone marrow progenitor cell line. Properdin is released by neutrophils from intracellular stores following stimulation by N-formyl-methionine-leucine-phenylalanine (fMLP) and tumor necrosis factor alpha (TNF-α). The HEP G2 cells derived from human liver has been found to produce functional properdin. Endothelial cells also produce properdin when induced by shear stress, thus is a physiological source for plasma properdin. The diverse range of extrahepatic sites for synthesis of these two complement regulators suggests the importance and need for local availability of the proteins. Here, we discuss the significance of the local synthesis of properdin and factor H. This assumes greater importance in view of recently identified unexpected and novel roles of properdin and factor H that are potentially independent of their involvement in complement regulation.
RESUMO
The laminated layer (LL) is the massive carbohydrate-rich structure that protects Echinococcus larvae, which cause cystic echinococcosis (hydatid disease) and alveolar echinococcosis. Increased understanding of the biochemistry of the LL is allowing a more informed analysis of its immunology. The LL not only protects the parasite against host attack but also shapes the overall immune response against it. Because of its dense glycosylation, it probably contains few T-cell epitopes, being important instead in T-cell independent antibody responses. Crucially, it is decoded in non-inflammatory fashion by innate immunity, surely contributing to the strong immune-regulation observed in Echinococcus infections. Defining the active LL molecular motifs and corresponding host innate receptors is a feasible and promising goal in the field of helminth-derived immune-regulatory molecules.
Assuntos
Equinococose/imunologia , Echinococcus/imunologia , Mucinas/imunologia , Imunidade Adaptativa , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Equinococose/parasitologia , Epitopos de Linfócito T/imunologia , Glicosilação , Interações Hospedeiro-Parasita , Humanos , Larva/química , Larva/imunologia , Mucinas/químicaRESUMO
Carbon nanotubes possess interesting physicochemical properties which make them potentially usable in medicine. Single-walled carbon nanotubes and multi-walled carbon nanotubes, for example, may carry and deliver anticancer drugs, such as cisplatin. Magnetic nanoparticles, like iron filled MWCNT, can be used in hyperthermia therapy. However, their hydrophobic character is a major difficulty, as preparation of stable dispersions of carbon nanotubes in biological buffers is an essential step towards biomedical applications. Recently, a novel treatment using the glycolipid, Galactosyl-beta1-sphingosine (psychosine), was employed to make stable suspensions of psychosine-functionalized carbon nanotubes in biological buffers. In this paper, the interactions of psychosine-functionalized carbon nanotubes with a part of the human immune system, complement, is presented. To investigate if human serum complement proteins can interact with psychosine-functionalized carbon nanotubes, complement consumption (depletion) assays were conducted. Moreover, direct protein binding studies, to analyze the interaction of plasma proteins with the psychosine-functionalized carbon nanotubes, using affinity chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis techniques, were applied. The psychosine-functionalized carbon nanotubes activate human complement via the classical pathway. Interestingly, as the hydrophilic part of the glycolipid may bind to ficolins, the lectin pathway could also be involved. Binding of human plasma proteins is very selective as only very few proteins adsorb to the psychosine-functionalized carbon nanotube surface, when placed in contact with human plasma. Bovine serum albumin-coated carbon nanotubes were used as a standard to find the differences in complement activation and protein adsorption patterns, caused by various non-covalent coatings of carbon nanotubes.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/metabolismo , Nanotubos de Carbono/química , Psicosina/farmacologia , Adsorção/efeitos dos fármacos , Animais , Proteínas Sanguíneas/metabolismo , Bovinos , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Microscopia de Força Atômica , Ligação Proteica/efeitos dos fármacos , Psicosina/química , Soroalbumina Bovina/metabolismoRESUMO
Dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN; CD209) has an important role in mediating adherence of Mycobacteria species, including M. tuberculosis and M. bovis BCG to human dendritic cells and macrophages, in which these bacteria can survive intracellularly. DC-SIGN is a C-type lectin, and interactions with mycobacterial cells are believed to occur via mannosylated structures on the mycobacterial surface. Recent studies suggest more varied modes of binding to multiple mycobacterial ligands. Here we identify, by affinity chromatography and mass-spectrometry, four novel ligands of M. bovis BCG that bind to DC-SIGN. The novel ligands are chaperone protein DnaK, 60 kDa chaperonin-1 (Cpn60.1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and lipoprotein lprG. Other published work strongly suggests that these are on the cell surface. Of these ligands, lprG appears to bind DC-SIGN via typical proteinglycan interactions, but DnaK and Cpn60.1 binding do not show evidence of carbohydrate-dependent interactions. LprG was also identified as a ligand for DC-SIGNR (L-SIGN; CD299) and the M. tuberculosis orthologue of lprG has been found previously to interact with human toll-like receptor 2. Collectively, these findings offer new targets for combating mycobacterial adhesion and within-host survival, and reinforce the role of DCSIGN as an important host ligand in mycobacterial infection.
Assuntos
Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/metabolismo , Mycobacterium bovis/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Moléculas de Adesão Celular/genética , Cromatografia de Afinidade , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Técnicas In Vitro , Lectinas Tipo C/genética , Ligantes , Macrófagos/metabolismo , Macrófagos/microbiologia , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Proteína A Associada a Surfactante Pulmonar/metabolismo , Receptores de Superfície Celular/genéticaRESUMO
Mannan-binding lectin (MBL) is a soluble innate immune protein that binds to glycosylated targets. MBL acts as an opsonin and activates complement, contributing to the destruction and clearance of infecting microorganisms. Hepatitis C virus (HCV) encodes two envelope glycoproteins E1 and E2, expressed as non-covalent E1/E2 heterodimers in the viral envelope. E1 and E2 are potential ligands for MBL. Here we describe an analysis of the interaction between HCV and MBL using recombinant soluble E2 ectodomain fragment, the full-length E1/E2 heterodimer, expressed in vitro, and assess the effect of this interaction on virus entry. A binding assay using antibody capture of full length E1/E2 heterodimers was used to demonstrate calcium dependent, saturating binding of MBL to HCV glycoproteins. Competition with various saccharides further confirmed that the interaction was via the lectin domain of MBL. MBL binds to E1/E2 representing a broad range of virus genotypes. MBL was shown to neutralize the entry into Huh-7 cells of HCV pseudoparticles (HCVpp) bearing E1/E2 from a wide range of genotypes. HCVpp were neutralized to varying degrees. MBL was also shown to neutralize an authentic cell culture infectious virus, strain JFH-1 (HCVcc). Furthermore, binding of MBL to E1/E2 was able to activate the complement system via MBL-associated serine protease 2. In conclusion, MBL interacts directly with HCV glycoproteins, which are present on the surface of the virion, resulting in neutralization of HCV particles.
Assuntos
Hepacivirus/fisiologia , Lectina de Ligação a Manose/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Proteínas do Envelope Viral/metabolismo , Ligação Competitiva , Glicosilação , Hepacivirus/genética , Hepacivirus/patogenicidade , Humanos , Monossacarídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Células Tumorais Cultivadas , Vírion/patogenicidade , Vírion/fisiologia , Internalização do VírusRESUMO
The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP-1 has thrombin-like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP-2 has factor Xa-like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L-FCN-MASPs complexes, bound from serum to N-acetylcysteine-Sepharose, or MBL-MASPs complexes, bound to mannan-agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti-D-dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L-FCN-MASPs complexes captured on N-acetylcysteine-Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP-2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP-1 and MASP-2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP-mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP-catalysed deposition and polymerization of fibrin on the surface of micro-organisms may be protective by limiting the dissemination of infection.
Assuntos
Coagulação Sanguínea/fisiologia , Fibrina/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Acetilcisteína/imunologia , Coagulação Sanguínea/imunologia , Fibrinopeptídeo A/antagonistas & inibidores , Fibrinopeptídeo A/imunologia , Fibrinopeptídeo B/antagonistas & inibidores , Fibrinopeptídeo B/imunologia , Hirudinas/farmacologia , Humanos , Protrombina/imunologia , Sefarose/imunologiaRESUMO
Macrophage scavenger receptor A (SR-A) is a multifunctional, multiligand pattern recognition receptor with roles in innate immunity, apoptotic cell clearance, and age-related degenerative pathologies, such as atherosclerosis and Alzheimer's disease. Known endogenous SR-A ligands are polyanionic and include modified lipoproteins, advanced glycation end products, and extracellular matrix proteins. No native plasma ligands have been identified, but it is known that SR-A recognition of unidentified serum components mediates integrin-independent macrophage adhesion, which may drive chronic local inflammation. In this study, we used a high-throughput fractionation and screening method to identify novel endogenous SR-A ligands that may mediate macrophage adhesion. SR-A was found to recognize the exchangeable apolipoproteins A-I and E (apo A-I and apo E, respectively) in both lipid-free and lipid-associated form, suggesting the shared amphipathic alpha-helix as a potential recognition motif. Adhesion of RAW 264.7 macrophages to surfaces coated with apo A-I and apo E4 proved to be integrin-independent and could be blocked by anti-SR-A antibodies. The presence of apo A-I and apo E in pathological deposits, such as atherosclerotic lesions and neurotoxic Alzheimer's plaques, suggests a possible contribution of SR-A-dependent adhesion of macrophages to an inflammatory microenvironment.
Assuntos
Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Macrófagos/metabolismo , Receptores Depuradores Classe A/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/química , Apolipoproteínas E/química , Linhagem Celular , Humanos , Ligantes , Macrófagos/química , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Depuradores Classe A/química , Receptores Depuradores Classe A/deficiência , Receptores Depuradores Classe A/genética , Receptores Depuradores Classe A/metabolismoRESUMO
Mycobacterium tuberculosis is the leading cause of infectious disease in humans in the world. It evades the host immune system by being phagocytosed by macrophages and residing intracellularly. Complement-dependent opsonisation of extracellular mycobacteria may assist them to enter macrophages. This work examines in detail the mechanisms of complement activation by whole mycobacteria using Mycobacterium bovis BCG as a model organism. M. bovis BCG directly activates the classical, lectin and alternative pathways, resulting in fixation of C3b onto macromolecules of the mycobacterial surface. Investigation into the classical pathway has shown direct binding of human C1q to whole mycobacteria in the absence of antibodies. Most human sera contain IgG and IgM-anti-(M. bovis BCG), and pre-incubation with human immunoglobulin enhances C1q binding to the bacteria. Therefore classical pathway activation is both antibody-independent and dependent. The bacteria also activate the alternative pathway in an antibody-independent manner, but Factor H also binds, suggesting some regulation of amplification by this pathway. For the lectin pathway we have demonstrated direct binding of both MBL and L-ficolin from human serum to whole mycobacteria and subsequent MASP2 activation. H-ficolin binding was not observed. No M. bovis BCG cell surface or secreted protease appears likely to influence complement activation. Together, these data provide a more detailed analysis of the mechanisms by which M. bovis BCG interacts with the complement system.
Assuntos
Anticorpos Antibacterianos/imunologia , Ativação do Complemento/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Complemento C1q/imunologia , Complemento C3b/imunologia , Fator H do Complemento/imunologia , Humanos , Lectinas/imunologia , Lectina de Ligação a Manose/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , FicolinasRESUMO
Less than 6 feet under: Serum proteins C3, C4, and alpha(2)M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.