RESUMO
The evolutionary history of complement suggests that the alternative pathway arose prior to the arrival of the classical and lectin pathways. In these pathways, target specificity is provided by antibodies and sugar specific lectins. While these efficient initiation systems dominate activation on most targets, the alternative pathway produces most of the C3b and 80%-90% of the C5b-9. While the tickover process, originally proposed by Peter Lachmann, provided ancient hosts with a crude self/non-self-discriminatory system that initiated complement attack on everything foreign, tickover clearly plays a more minor role in complement activation in modern organisms possessing classical and lectin pathways. Spontaneous activation of the alternative pathway via tickover may play a major role in human pathologies where tissue damage is complement-mediated. The molecular mechanism of tickover is still not convincingly proven. Prevailing hypotheses include (a) spontaneous hydrolysis of the thioester in C3 forming the C3b-like C3(H2 O) in solution and (b) "enhanced tickover" in which surfaces cause specific or non-specific contact activated conformational changes in C3. Theoretical considerations, including computer simulations, suggest that the latter mechanism is more likely and that more research needs to be devoted to understanding interactions between biological surfaces and C3.
Assuntos
Complemento C3 , Complemento C3b , Humanos , Complemento C3/metabolismo , Complemento C3b/metabolismo , Ativação do Complemento , Anticorpos , Compostos de Enxofre , Via Alternativa do ComplementoRESUMO
C3 is the central effector molecule of the complement system, mediating its multiple functions through different binding sites and their corresponding receptors. We will introduce the C3 forms (native C3, C3 [H2 O], and intracellular C3), the C3 fragments C3a, C3b, iC3b, and C3dg/C3d, and the C3 expression sites. To highlight the important role that C3 plays in human biological processes, we will give an overview of the diseases linked to C3 deficiency and to uncontrolled C3 activation. Next, we will present a structural description of C3 activation and of the C3 fragments generated by complement regulation. We will proceed by describing the C3a interaction with the anaphylatoxin receptor, followed by the interactions of opsonins (C3b, iC3b, and C3dg/C3d) with complement receptors, divided into two groups: receptors bearing complement regulatory functions and the effector receptors without complement regulatory activity. We outline the molecular architecture of the receptors, their binding sites on the C3 activation fragments, the cells expressing them, the diversity of their functions, and recent advances. With this review, we aim to give an up-to-date analysis of the processes triggered by C3 activation fragments on different cell types in health and disease contexts.
Assuntos
Complemento C3 , Complemento C3b , Humanos , Complemento C3/análise , Complemento C3/metabolismo , Complemento C3b/metabolismo , Receptores de Complemento/análise , Sítios de Ligação , Ativação do ComplementoRESUMO
The alternative pathway (AP) is the phylogenetically oldest arm of the complement system and may have evolved to mark pathogens for elimination by phagocytes. Studies using purified AP proteins or AP-specific serum showed that C3b amplification on bacteria commenced following a lag phase of about 5 min and was highly dependent on the concentration of complement. Most pathogens have evolved several elegant mechanisms to evade complement, including expressing proteases that degrade AP proteins and secreting proteins that block function of C3 convertases. In an example of convergent evolution, many microbes recruit the AP inhibitor factor H (FH) using molecular mechanisms that mimic FH interactions with host cells. In most instances, the AP serves to amplify C3b deposited on microbes by the classical pathway (CP). The role of properdin on microbes appears to be restricted to stabilization of C3 convertases; scant evidence exists for its role as an initiator of the AP on pathogens in the context of serum. Therapeutic complement inhibition carries with it an increased risk of infection. Antibody (Ab)-dependent AP activation may be critical for complement activation by vaccine-elicited Ab when the CP is blocked, and its molecular mechanism is discussed.
Assuntos
Infecções Bacterianas , Ativação do Complemento , Via Alternativa do Complemento , Humanos , Ativação do Complemento/fisiologia , Properdina/metabolismo , Infecções Bacterianas/metabolismo , Complemento C3b/metabolismoRESUMO
As the most abundant component of the complement system, C3 and its proteolytic derivatives serve essential roles in the function of all three complement pathways. Central to this is a network of protein-protein interactions made possible by the sequential proteolysis and far-reaching structural changes that accompany C3 activation. Beginning with the crystal structures of C3, C3b, and C3c nearly twenty years ago, the physical transformations underlying C3 function that had long been suspected were finally revealed. In the years that followed, a compendium of crystallographic information on C3 derivatives bound to various enzymes, regulators, receptors, and inhibitors generated new levels of insight into the structure and function of the C3 molecule. This Review provides a concise classification, summary, and interpretation of the more than 50 unique crystal structure determinations for human C3. It also highlights other salient features of C3 structure that were made possible through solution-based methods, including Hydrogen/Deuterium Exchange and Small Angle X-ray Scattering. At this pivotal time when the first C3-targeted therapeutics begin to see use in the clinic, some perspectives are also offered on how this continually growing body of structural information might be leveraged for future development of next-generation C3 inhibitors.
Assuntos
Complemento C3 , Complemento C3b , Humanos , Complemento C3b/química , Complemento C3b/metabolismo , Complemento C3/metabolismo , Ativação do ComplementoRESUMO
The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step in vitro reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.
Assuntos
Via Alternativa do Complemento , Humanos , Via Alternativa do Complemento/efeitos dos fármacos , Complemento C3/metabolismo , Complemento C3/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Complemento C3b/metabolismoRESUMO
Complement receptor 1 (CR1) is a membrane glycoprotein with a highly duplicated domain structure able to bind multiple ligands such as C3b and C4b, the activated fragments of complement components C3 and C4, respectively. We have previously used our knowledge of this domain structure to identify CSL040, a soluble extracellular fragment of CR1 containing the long homologous repeat (LHR) domains A, B, and C. CSL040 retains the ability to bind both C3b and C4b but is also a more potent complement inhibitor than other recombinant CR1-based therapeutics. To generate soluble CR1 variants with increased inhibitory potential across all three complement pathways, or variants with activity skewed to specific pathways, we exploited the domain structure of CR1 further by generating LHR domain duplications. We identified LHR-ABCC, a soluble CR1 variant containing a duplicated C3b-binding C-terminal LHR-C domain that exhibited significantly enhanced alternative pathway inhibitory activity in vitro compared to CSL040. Another variant, LHR-BBCC, containing duplications of both LHR-B and LHR-C with four C3b binding sites, was shown to have reduced classical/lectin pathway inhibitory activity compared to CSL040, but comparable alternative pathway activity. Interestingly, multiplication of the C4b-binding LHR-A domain resulted in only minor increases in classical/lectin pathway inhibitory activity. The CR1 duplication variants characterized in these in vitro potency assays, as well as in affinity in solution C3b and C4b binding assays, not only provides an opportunity to identify new therapeutic molecules but also additional mechanistic insights to the multiple interactions between CR1 and C3b/C4b.
Assuntos
Complemento C3b , Domínios Proteicos , Humanos , Complemento C3b/metabolismo , Complemento C3b/química , Complemento C3b/genética , Receptores de Complemento 3b/metabolismo , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/química , Complemento C4b/metabolismo , Complemento C4b/genética , Complemento C4b/química , Ligação ProteicaRESUMO
Rare variants (RVs) in the gene encoding the regulatory enzyme complement factor I (CFI; FI) that reduce protein function or levels increase age-related macular degeneration risk. A total of 3357 subjects underwent screening in the SCOPE natural history study for geographic atrophy secondary to age-related macular degeneration, including CFI sequencing and serum FI measurement. Eleven CFI RV genotypes that were challenging to categorize as type I (low serum level) or type II (normal serum level, reduced enzymatic function) were characterized in the context of pure FI protein in C3b and C4b fluid phase cleavage assays and a novel bead-based functional assay (BBFA) of C3b cleavage. Four variants predicted or previously characterized as benign were analyzed by BBFA for comparison. In all, three variants (W51S, C67R, and I370T) resulted in low expression. Furthermore, four variants (P64L, R339Q, G527V, and P528T) were identified as being highly deleterious with IC50s for C3b breakdown >1 log increased versus the WT protein, while two variants (K476E and R474Q) were â¼1 log reduced in function. Meanwhile, six variants (P50A, T203I, K441R, E548Q, P553S, and S570T) had IC50s similar to WT. Odds ratios and BBFA IC50s were positively correlated (r = 0.76, p < 0.01), while odds ratios versus combined annotation dependent depletion (CADD) scores were not (r = 0.43, p = 0.16). Overall, 15 CFI RVs were functionally characterized which may aid future patient stratification for complement-targeted therapies. Pure protein in vitro analysis remains the gold standard for determining the functional consequence of CFI RVs.
Assuntos
Complemento C3b , Fator I do Complemento , Genótipo , Atrofia Geográfica , Humanos , Fator I do Complemento/genética , Fator I do Complemento/metabolismo , Atrofia Geográfica/genética , Atrofia Geográfica/sangue , Atrofia Geográfica/metabolismo , Feminino , Masculino , Complemento C3b/metabolismo , Complemento C3b/genética , Idoso , Estudos de Coortes , Degeneração Macular/genética , Degeneração Macular/metabolismo , Pessoa de Meia-IdadeRESUMO
Trypanosomes are known to activate the complement system on their surface, but they control the cascade in a manner such that the cascade does not progress into the terminal pathway. It was recently reported that the invariant surface glycoprotein ISG65 from Trypanosoma brucei interacts reversibly with complement C3 and its degradation products, but the molecular mechanism by which ISG65 interferes with complement activation remains unknown. In this study, we show that ISG65 does not interfere directly with the assembly or activity of the two C3 convertases. However, ISG65 acts as a potent inhibitor of C3 deposition through the alternative pathway in human and murine serum. Degradation assays demonstrate that ISG65 stimulates the C3b to iC3b converting activity of complement factor I in the presence of the cofactors factor H or complement receptor 1. A structure-based model suggests that ISG65 promotes a C3b conformation susceptible to degradation or directly bridges factor I and C3b without contact with the cofactor. In addition, ISG65 is observed to form a stable ternary complex with the ligand binding domain of complement receptor 3 and iC3b. Our data suggest that ISG65 supports trypanosome complement evasion by accelerating the conversion of C3b to iC3b through a unique mechanism.
Assuntos
Trypanosoma brucei brucei , Camundongos , Animais , Humanos , Trypanosoma brucei brucei/metabolismo , Complemento C3b/metabolismo , Receptores de Complemento 3b , Ativação do Complemento , Fator H do Complemento/metabolismo , Fibrinogênio , Via Alternativa do Complemento , Convertases de Complemento C3-C5/metabolismoRESUMO
Hyperactivation of the complement system, a major component of innate immunity, has been recognized as one of the core clinical features in severe covid-19 patients. However, how the virus escapes the targeted elimination by the network of activated complement pathways still remains an enigma. Here, we identified SARS-CoV-2-encoded ORF8 protein as one of the major binding partners of human complement C3/C3b components and their metabolites. Our results demonstrated that preincubation of ORF8 with C3/C3b in the fluid phase has two immediate functional consequences in the alternative pathway; this preincubation inhibits factor I-mediated proteolysis and blocks factor B zymogen activation into active Bb. ORF8 binding results in the occlusion of both factor H and factor B from C3b, rendering the complexes resistant to factor I-mediated proteolysis and inhibition of pro-C3-convertase (C3bB) formation, respectively. We also confirmed the complement inhibitory activity of ORF8 in our hemolysis-based assay, where ORF8 prevented human serum-induced lysis of rabbit erythrocytes with an IC50 value of about 2.3 µM. This inhibitory characteristic of ORF8 was also supported by in-silico protein-protein docking analysis, as it appeared to establish primary interactions with the ß-chain of C3b, orienting itself near the C3b CUB (C1r/C1s, Uegf, Bmp1) domain like a peptidomimetic compound, sterically hindering the binding of essential cofactors required for complement amplification. Thus, ORF8 has characteristics to act as an inhibitor of critical regulatory steps in the alternative pathway, converging to hasten the decay of C3-convertase and thereby, attenuating the complement amplification loop.
Assuntos
COVID-19 , Fator B do Complemento , Animais , Humanos , Coelhos , Ativação do Complemento , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Fator B do Complemento/metabolismo , Fator H do Complemento/metabolismo , Via Alternativa do Complemento/fisiologia , SARS-CoV-2/metabolismo , Ligação Proteica , Simulação por ComputadorRESUMO
Psoriasis is a chronic inflammatory skin disease with a characteristic isomorphic reaction, i.e. the Köbner reaction, induced by slight epidermal trauma. In this study, the tape-stripping technique was used to induce the development of Köbner reaction in 18 subjects with psoriasis. Eight subjects developed a positive reaction. To study the early cellular changes, skin biopsies were taken at the baseline and subsequent time points of 2 h, 1 d, 3 d, and 7 d for the immunostaining of complement C3c, iC3b, and cells expressing complement receptor 3 (CD11b/CD18; a receptor of iC3b) or CD14. The results show that the positive Köbner reaction is associated with rapid (2 h-1 d) and sustained (3-7 d) increase in the expression of epidermal C3c and iC3b and dermal C3c. In addition, there was a positive correlation between CD11b+ and CD14+ cells in baseline and 2 h-1 d biopsies with a subsequent increase in CD11b+ and CD14+ cells in 3-7 d biopsies in the Köbner-positive group. In the Köbner-negative group, only a transient increase in epidermal iC3b at 2 h-1 d, as well as rapid (2 h-1 d) and sustained increase (3-7 d) in dermal iC3b and CD14+ cells, was observed. In experiments with cultured monolayer keratinocytes, a slight cell damage already at 30 mJ/cm2 ultraviolet B irradiation led to increased expression of C3c, but not iC3b. Therefore, there are marked differences between Köbner groups in respect to the expression of C3c, iC3b, and cells expressing CD11b or CD14. Of note is the rapid and sustained increase in epidermal C3c and iC3b in the positive Köbner reaction.
Assuntos
Antígeno CD11b , Complemento C3b , Receptores de Lipopolissacarídeos , Psoríase , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Psoríase/imunologia , Psoríase/metabolismo , Feminino , Antígeno CD11b/metabolismo , Adulto , Pessoa de Meia-Idade , Complemento C3b/metabolismo , Complemento C3b/imunologia , Pele/patologia , Pele/imunologia , Pele/metabolismo , Pele/efeitos da radiação , Biópsia , Epiderme/metabolismo , Epiderme/imunologia , Epiderme/patologiaRESUMO
The ß protein from group B Streptococcus (GBS) is a â¼132-kDa, cell-surface exposed molecule that binds to multiple host-derived ligands, including complement factor H (FH). Many details regarding this interaction and its significance to immune evasion by GBS remain unclear. In this study, we identified a three-helix bundle domain within the C-terminal half of the B75KN region of ß as the major FH-binding determinant and determined its crystal structure at 2.5 Å resolution. Analysis of this structure suggested a role in FH binding for a loop region connecting helices α1 and α2, which we confirmed by mutagenesis and direct binding studies. Using a combination of protein cross-linking and mass spectrometry, we observed that B75KN bound to complement control protein (CCP)3 and CCP4 domains of FH. Although this binding site lies within a complement regulatory region of FH, we determined that FH bound by ß retained its decay acceleration and cofactor activities. Heterologous expression of ß by Lactococcus lactis resulted in recruitment of FH to the bacterial surface and a significant reduction of C3b deposition following exposure to human serum. Surprisingly, we found that FH binding by ß was not required for bacterial resistance to phagocytosis by neutrophils or killing of bacteria by whole human blood. However, loss of the B75KN region significantly diminished bacterial survival in both assays. Although our results show that FH recruited to the bacterial surface through a high-affinity interaction maintains key complement-regulatory functions, they raise questions about the importance of FH binding to immune evasion by GBS as a whole.
Assuntos
Proteínas de Bactérias/metabolismo , Evasão da Resposta Imune/imunologia , Proteínas de Membrana/metabolismo , Streptococcus agalactiae/imunologia , Sítios de Ligação/fisiologia , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Humanos , Neutrófilos/imunologia , Opsonização/imunologia , Ligação Proteica/imunologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/patologiaRESUMO
The integrin receptor αMß2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of αMß2 signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human αMß2 headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H αM variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside-inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the αM subunit in an Mg2+-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the αM subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound αMß2 with iC3b, suggesting that it may represent a novel high-affinity proteinaceous αMß2-specific agonist. Taken together, our data suggest that the iC3b-αMß2 complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to αMß2.
Assuntos
Complemento C3b , Antígeno de Macrófago 1 , Antígenos CD18/metabolismo , Complemento C3b/metabolismo , Humanos , Integrinas , Leucócitos/metabolismo , Ligantes , Antígeno de Macrófago 1/metabolismoRESUMO
Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis, the causative agent of plague. Here, the contribution of Ail for Y. pestis survival in the flea vector was examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δail mutant at 28°C in vitro. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescent microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δail mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6+ wild type, a Δail mutant, and the Δail/ail+ control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (AilF100V E108_S109insS) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6+ Δail mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain. IMPORTANCE This work contributes to our understanding of how highly virulent Y. pestis evolved from its innocuous enteric predecessor. Among identified virulence factors is the attachment invasion locus protein, Ail, that is required to protect Y. pestis from serum complement in all mammals tested except mice. Murine sera is not bactericidal. In this study, we asked, is bactericidal sera from humans active in Y. pestis colonized fleas? We found it was not. The importance of this observation is that it identifies a protective niche for the growth of serum sensitive and nonsensitive Y. pestis strains.
Assuntos
Peste , Sifonápteros , Yersinia pestis , Animais , Humanos , Camundongos , Ratos , Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Mamíferos , Peste/microbiologia , Sifonápteros/metabolismo , Sifonápteros/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Yersinia pestis/genética , Yersinia pestis/metabolismo , Complemento C3b/metabolismo , Complemento C3b/farmacologiaRESUMO
Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The generation of FXIa in plasma induced the cleavage of CFH. Moreover, FXIa reduced the cleavage of C3b by factor I in serum. Conversely, we observed that CFH inhibited FXI activation by either thrombin or FXIIa. Our study provides, to our knowledge, a novel molecular link between the contact pathway of coagulation and the complement system. These results suggest that FXIa generation enhances the activity of the complement system and thus may potentiate the immune response.
Assuntos
Plaquetas/metabolismo , Fator H do Complemento/metabolismo , Células Endoteliais/metabolismo , Fator XIa/metabolismo , Inflamação/metabolismo , Animais , Coagulação Sanguínea , Complemento C3b/metabolismo , Via Alternativa do Complemento , Fibrinogênio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Papio , Ligação Proteica , Receptor Cross-TalkRESUMO
AIMS: To use experimental periodontitis models in rats to investigate the correlation between local expression of the complement components C3b and C4b in periodontal tissues and disease severity, and to assess the therapeutic effects of targeting C3b/C4b on inflammatory bone loss. MATERIALS AND METHODS: The gingival expression of C3, C3b, and C4b in animal experimental periodontitis models were analysed immunohistochemically. The therapeutic effects of the C3b/C4b inhibitor (SB002) on ligation-induced experimental periodontitis was examined using biochemical, histological, and immunohistochemical analyses. RESULTS: The gingival expression levels of C3, C3b, and C4b were positively correlated with the severity of periodontitis. Moreover, both single and multiple injections of the C3b/C4b inhibitor had preventive and therapeutic effects on alveolar bone loss in ligation-induced experimental periodontitis with no associated adverse consequences. CONCLUSIONS: The association between C3b/C4b and periodontitis may provide a basis for the development of novel therapeutic strategies for periodontitis and other inflammatory diseases.
Assuntos
Complemento C4b , Periodontite , Ratos , Animais , Complemento C4b/metabolismo , Complemento C3b/metabolismo , Convertases de Complemento C3-C5/metabolismo , Inflamação , Periodontite/complicações , Periodontite/tratamento farmacológicoRESUMO
Human Complement Receptor 1 (HuCR1) is a potent membrane-bound regulator of complement both in vitro and in vivo, acting via interaction with its ligands C3b and C4b. Soluble versions of HuCR1 have been described such as TP10, the recombinant full-length extracellular domain, and more recently CSL040, a truncated version lacking the C-terminal long homologous repeat domain D (LHR-D). However, the role of N-linked glycosylation in determining its pharmacokinetic (PK) and pharmacodynamic (PD) properties is only partly understood. We demonstrated a relationship between the asialo-N-glycan levels of CSL040 and its PK/PD properties in rats and non-human primates (NHPs), using recombinant CSL040 preparations with varying asialo-N-glycan levels. The clearance mechanism likely involves the asialoglycoprotein receptor (ASGR), as clearance of CSL040 with a high proportion of asialo-N-glycans was attenuated in vivo by co-administration of rats with asialofetuin, which saturates the ASGR. Biodistribution studies also showed CSL040 localization to the liver following systemic administration. Our studies uncovered differential PD effects by CSL040 on complement pathways, with extended inhibition in both rats and NHPs of the alternative pathway compared with the classical and lectin pathways that were not correlated with its PK profile. Further studies showed that this effect was dose dependent and observed with both CSL040 and the full-length extracellular domain of HuCR1. Taken together, our data suggests that sialylation optimization is an important consideration for developing HuCR1-based therapeutic candidates such as CSL040 with improved PK properties and shows that CSL040 has superior PK/PD responses compared with full-length soluble HuCR1.
Assuntos
Lectinas , Polissacarídeos , Animais , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Glicosilação , Lectinas/metabolismo , Ratos , Receptores de Complemento/metabolismo , Receptores de Complemento 3b/metabolismo , Distribuição TecidualRESUMO
While angiotensin-converting enzyme (ACE) regulates blood pressure by producing angiotensin II as part of the renin-angiotensin system, we recently reported that elevated ACE in neutrophils promotes an effective immune response and increases resistance to infection. Here, we investigate if such neutrophils protect against renal injury in immune complex (IC)-mediated crescentic glomerulonephritis (GN) through complement. Nephrotoxic serum nephritis (NTN) was induced in wild-type and NeuACE mice that overexpress ACE in neutrophils. Glomerular injury of NTN in NeuACE mice was attenuated with much less proteinuria, milder histological injury, and reduced IC deposits, but presented with more glomerular neutrophils in the early stage of the disease. There were no significant defects in T and B cell functions in NeuACE mice. NeuACE neutrophils exhibited enhanced IC uptake with elevated surface expression of FcγRII/III and complement receptor CR1/2. IC uptake in neutrophils was enhanced by NeuACE serum containing elevated complement C3b. Given no significant complement activation by ACE, this suggests that neutrophil ACE indirectly preactivates C3 and that the C3b-CR1/2 axis and elevated FcγRII/III play a central role in IC elimination by neutrophils, resulting in reduced glomerular injury. The present study identified a novel renoprotective role of ACE in glomerulonephritis; elevated neutrophilic ACE promotes elimination of locally formed ICs in glomeruli via C3b-CR1/2 and FcγRII/III, ameliorating glomerular injury.NEW & NOTEWORTHY We studied immune complex (IC)-mediated crescentic glomerulonephritis in NeuACE mice that overexpress ACE only in neutrophils. Such mice show no significant defects in humoral immunity but strongly resist nephrotoxic serum nephritis (less proteinuria, milder histological damage, reduced IC deposits, and more glomerular neutrophils). NeuACE neutrophils enhanced IC uptake via increased surface expression of CR1/2 and FcgRII/III, as well as elevated serum complement C3b. These results suggest neutrophil ACE as a novel approach to reducing glomerulonephritis.
Assuntos
Glomerulonefrite , Nefrite , Angiotensina II/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Complemento C3b/metabolismo , Glomerulonefrite/metabolismo , Camundongos , Nefrite/metabolismo , Neutrófilos/metabolismo , Proteinúria/metabolismoRESUMO
Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.
Assuntos
Subpopulações de Linfócitos B/citologia , Linfócitos T CD4-Positivos/imunologia , Catepsina L/metabolismo , Diferenciação Celular , Ativação do Complemento/fisiologia , Complemento C3/metabolismo , Homeostase/fisiologia , Adulto , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Sobrevivência Celular/imunologia , Criança , Complemento C3/imunologia , Complemento C3a/metabolismo , Complemento C3b/metabolismo , Regulação da Expressão Gênica/imunologia , HumanosRESUMO
The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.
Assuntos
Anticorpos/metabolismo , Síndrome Hemolítico-Urêmica Atípica/genética , Complemento C3b/metabolismo , Fator H do Complemento/imunologia , Genótipo , Animais , Linhagem Celular , Ativação do Complemento , Fator H do Complemento/agonistas , Fator H do Complemento/genética , Predisposição Genética para Doença , Humanos , Camundongos , Mutação/genética , Polimorfismo Genético , Ligação ProteicaRESUMO
Although CRIg was originally identified as a macrophage receptor for binding complement C3b/iC3b in vitro, recent studies reveal that CRIg functions as a pattern recognition receptor in vivo for Kupffer cells (KCs) to directly bind bacterial pathogens in a complement-independent manner. This raises the critical question of whether CRIg captures circulating pathogens through interactions with complement in vivo under flow conditions. Furthermore, the role of CRIg during parasitic infection is unknown. Taking advantage of intravital microscopy and using African trypanosomes as a model, we studied the role of CRIg in intravascular clearance of bloodborne parasites. Complement C3 is required for intravascular clearance of African trypanosomes by KCs, preventing the early mortality of infected mice. Moreover, antibodies are essential for complement-mediated capture of circulating parasites by KCs. Interestingly, reduced antibody production was observed in the absence of complement C3 during infection. We further demonstrate that CRIg but not CR3 is critically involved in KC-mediated capture of circulating parasites, accounting for parasitemia control and host survival. Of note, CRIg cannot directly catch circulating parasites and antibody-induced complement activation is indispensable for CRIg-mediated parasite capture. Thus, we provide evidence that CRIg, by interacting with complement in vivo, plays an essential role in intravascular clearance of bloodborne parasites. Targeting CRIg may be considered as a therapeutic strategy.