RESUMO
PURPOSE: To detect immunoglobulins in aqueous humour of AMD patients after repeated administration of intravitreal aflibercept. PATIENTS AND METHODS: Twenty-one patients (age: 77.85 ± 9.21 years) previously treated with intravitreal aflibercept due to wet type age-related macular degeneration (AMD group) and 18 age-matched control subjects (age: 69.75 ± 12.67 years) were included in this study. Patients in the AMD group received a mean of 5 intravitreal injections (min: 1 max: 17) prior to the cataract surgery. Samples of aqueous humour (50 µl) were obtained by anterior chamber paracentesis as the first step of routine cataract surgery. The IgG content of the samples was analysed by an in-house developed ELISA system. RESULTS: A significant increase in nonspecific IgG levels in the AMD group was detected compared to the control group (13.37 ± 6.65 vs. 9.44 ± 6.55 µg/ml; p = 0.03). In 11 patients, intraocular anti-aflibercept immunoglobulins could be detected (0.05 ± 0.01 µg/ml) which was significantly higher than the limit of detection for anti-aflibercept (0.04 µg/ml; p = 0.001). No correlation was found between the number of injections or the type of CNV and the aqueous level of anti-aflibercept (r = 0.02; p = 0.95). CONCLUSION: According to our results, penetration of non-specific systemic antibodies through the impaired blood-retinal barrier is higher in patients with neovascular AMD than in subjects with an intact structural barrier. Evaluation of neutralizing antibodies to anti-VEGF agents in the aqueous humour can lead us to understanding tachyphylaxis and changes in intraocular immune mechanisms due to AMD.
Assuntos
Catarata , Degeneração Macular Exsudativa , Humanos , Idoso , Idoso de 80 Anos ou mais , Pessoa de Meia-Idade , Inibidores da Angiogênese/uso terapêutico , Ranibizumab/uso terapêutico , Degeneração Macular Exsudativa/tratamento farmacológico , Degeneração Macular Exsudativa/diagnóstico , Fator A de Crescimento do Endotélio Vascular , Anticorpos Neutralizantes/uso terapêutico , Acuidade Visual , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Injeções Intravítreas , Catarata/tratamento farmacológico , Imunoglobulina G , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands.
Assuntos
Linfócitos B/metabolismo , Células Dendríticas/metabolismo , Granulócitos/metabolismo , Neutrófilos/metabolismo , Fagócitos/metabolismo , Ativação do Complemento , Fator H do Complemento/metabolismo , HumanosRESUMO
Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin- and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathway-related and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.
Assuntos
Lectina de Ligação a Manose da Via do Complemento/fisiologia , Proteínas do Sistema Complemento/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Periplásmicas/metabolismo , Serina Proteases/sangue , Ativação do Complemento/fisiologia , Escherichia coli/metabolismo , Humanos , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/metabolismo , Yersinia pestis/metabolismoRESUMO
The complement system is a major component of immune defense. Activation of the complement cascade by foreign substances and altered self-structures may lead to the elimination of the activating agent, and during the enzymatic cascade, several biologically active fragments are generated. Most immune regulatory effects of complement are mediated by the activation products of C3, the central component. The indispensable role of C3 in opsonic phagocytosis as well as in the regulation of humoral immune response is known for long, while the involvement of complement in T-cell biology have been revealed in the past few years. In this review, we discuss the immune modulatory functions of C3-derived fragments focusing on their role in processes which have not been summarized so far. The importance of locally synthesized complement will receive special emphasis, as several immunological processes take place in tissues, where hepatocyte-derived complement components might not be available at high concentrations. We also aim to call the attention to important differences between human and mouse systems regarding C3-mediated processes.
Assuntos
Complemento C3/imunologia , Fígado/imunologia , Peptídeos/imunologia , Fagocitose/imunologia , Linfócitos T/imunologia , Animais , Humanos , Imunidade Celular , Imunidade Humoral , Imunomodulação , Camundongos , Modelos AnimaisRESUMO
Complement is an ancient and complex network of the immune system and, as such, it plays vital physiological roles, but it is also involved in numerous pathological processes. The proper regulation of the complement system is important to allow its sufficient and targeted activity without deleterious side-effects. Factor H is a major complement regulator, and together with its splice variant factor H-like protein 1 and the five human factor H-related (FHR) proteins, they have been linked to various diseases. The role of factor H in inhibiting complement activation is well studied, but the function of the FHRs is less characterized. Current evidence supports the main role of the FHRs as enhancers of complement activation and opsonization, i.e., counter-balancing the inhibitory effect of factor H. FHRs emerge as soluble pattern recognition molecules and positive regulators of the complement system. In addition, factor H and some of the FHR proteins were shown to modulate the activity of immune cells, a non-canonical function outside the complement cascade. Recent efforts have intensified to study factor H and the FHRs and develop new tools for the distinction, quantification and functional characterization of members of this protein family. Here, we provide an update and overview on the versatile roles of factor H family proteins, what we know about their biological functions in healthy conditions and in diseases.
Assuntos
Fator H do Complemento , Proteínas do Sistema Complemento , Humanos , Fator H do Complemento/metabolismo , Ativação do ComplementoRESUMO
Umbilical cord blood (UCB) is a promising alternative for the treatment of hematological malignancies. The lower immune reactivity of UCB lymphocytes is a well-known phenomenon; however, immune tolerance mechanisms are not fully elucidated. Galectin-1 has strong immunosuppressive properties and plays a key role in the regulation of immune reactivity. We aimed to determine the properties of intracellular galectin-1 (Gal-1)-producing cells within CD3, CD4, CD8, regulatory T (Treg), and natural killer (NK) cells in UCB compared to adult peripheral blood (APB). We took peripheral blood samples from 22 healthy adults and cord blood samples from 19 healthy, term neonates. Intracellular Gal-1 expression was determined by flow cytometry in the above subsets. Furthermore, we assessed the prevalence of naive and memory T cells that play a role in the regulation of immune reactivity. We also performed functional analyses to assess the effect of exogenous Gal-1 on the rate of proliferation of T lymphocytes isolated from APB and UCB. The prevalence of intracellular Gal-1-expressing CD3, CD4, CD8, Treg and NK lymphocytes was lower in UCB than in APB. However, their capability to produce Gal-1 reaches the level seen in adults. The prevalence of naive cells was higher, whereas that of central and effector memory T cells was lower in UCB compared with APB. Lower Gal-1-producing cell proportion might be due to the naivety of neonatal lymphocytes, as indicated by the positive correlation detected between the number of CD3 lymphocytes expressing intracellular Gal-1 and the prevalence of memory T cells. The intracellular expression of Gal-1 may be down-regulated in neonatal lymphocytes due to the already reduced immune reactivity of UCB. In contrast with previous findings, our results indicate that the administration of exogenous Gal-1 failed to decrease the rate of proliferation in T lymphocytes isolated from either APB or UCB. This suggests that Gal-1-expressing lymphocytes are unlikely to play a major role in mitigating the immune reactivity of UCB.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Sangue Fetal/metabolismo , Galectina 1/imunologia , Células Matadoras Naturais/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/imunologia , Citometria de Fluxo , Galectina 1/genética , Galectina 1/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Humanos , Memória Imunológica , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Contagem de Linfócitos , Masculino , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of the alternative pathway of complement activation, has been reported to bind to various immune cells and regulate their functions, beyond its role in modulating complement activation. In this study we investigated the effect of FH, its alternative splice product FH-like protein 1 (FHL-1), the FH-related (FHR) proteins FHR-1 and FHR-5, and the recently developed artificial complement inhibitor mini-FH, on two key innate immune cells, monocytes and neutrophilic granulocytes. We found that, similar to FH, the other factor H family proteins FHL-1, FHR-1 and FHR-5, as well as the recombinant mini-FH, are able to bind to both monocytes and neutrophils. As a functional outcome, immobilized FH and FHR-1 inhibited PMA-induced NET formation, but increased the adherence and IL-8 production of neutrophils. FHL-1 increased only the adherence of the cells, while FHR-5 was ineffective in altering these functions. The adherence of monocytes was increased on FH, recombinant mini-FH and FHL-1 covered surfaces and, except for FHL-1, the same molecules also enhanced secretion of the inflammatory cytokines IL-1ß and TNFα. When monocytes were stimulated with LPS in the presence of immobilized FH family proteins, FH, FHL-1 and mini-FH enhanced whereas FHR-1 and FHR-5 decreased the secretion of TNFα; FHL-1 and mini-FH also enhanced IL-10 release compared to the effect of LPS alone. Our results reveal heterogeneous effects of FH and FH family members on monocytes and neutrophils, altering key features involved in pathogen killing, and also demonstrate that FH-based complement inhibitors, such as mini-FH, may have effects beyond their function of inhibiting complement activation. Thus, our data provide new insight into the non-canonical functions of FH, FHL-1, FHR-1 and FHR-5 that might be exploited during protection against infections and in vaccine development.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Monócitos/metabolismo , Proteínas Musculares/metabolismo , Neutrófilos/metabolismo , Animais , Proteínas Sanguíneas/genética , Forma Celular , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Inativadores do Complemento/farmacologia , Proteínas do Sistema Complemento/genética , Citocinas/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteínas Musculares/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fenótipo , Células Sf9 , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Células U937RESUMO
Maturation of dendritic cells (DCs) is known to be induced by several stimuli, including microbial products, inflammatory cytokines and immobilized IgG, as demonstrated recently. Since immune complexes formed in vivo also contain C1q, moreover apoptotic cells and several pathogens fix C1q in the absence of antibodies, we undertook to investigate whether this complement protein has an impact on various functions of human DCs. Maturation of monocyte-derived immature DCs (imMDCs) cultured on immobilized C1q was followed by monitoring expression of CD80, CD83, CD86, MHCII and CCR7. The functional activity of the cells was assessed by measuring cytokine secretion and their ability to activate allogeneic T lymphocytes. Cytokine production by T cells co-cultured with C1q-matured DCs was also investigated. C1q, but not the structurally related mannose-binding lectin was found to bind to imMDC in a dose-dependent manner and induced NF-kappaB translocation to the nucleus. Immobilized C1q induced maturation of MDCs and enhanced secretion of IL-12 and TNF-alpha, moreover, elevated their T-cell stimulating capacity. As IFN-gamma levels were increased in supernatants of MDC-T cell co-cultures, our data suggest that C1q-induced DC maturation generates a Th1-type response. Interestingly, IL-10 levels were elevated by C1q-treated MDCs but not in the supernatant of their co-cultures with allogeneic T cells. Taken together, these results indicate that C1q-opsonized antigens may play a role in the induction and regulation of immune response. Moreover our data are relevant in view of the role of C1q in removal of apoptotic cells and the association between C1q-deficiency and autoimmunity.
Assuntos
Diferenciação Celular/imunologia , Complemento C1q/fisiologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Animais , Células Cultivadas , Técnicas de Cocultura , Humanos , CoelhosRESUMO
CR3 and CR4 belong to the family of ß2-integrins and play an important role in phagocytosis, cellular adherence and migration. CR3 and CR4 are generally expected to mediate similar functions due to their structural homology, overlapping ligand specificity and parallel expression on human phagocytes. Although the different signalling pathways of these receptors suggest distinct functions, possible differences are just being revealed. Previously we proved that CR3 plays a key role in the uptake of iC3b-opsonized particles by human dendritic cells. Now, besides measuring the overall phagocytic capacity of cells including the assessment of surface bound as well as internalized particles, we extended our investigations and studied the digestion of the iC3b opsonized antigen by various human phagocytes. The participation of CR3 and CR4 was compared in the process of binding, internalization and digestion of iC3b opsonized Staphylococcus aureus by monocytes, monocyte derived macrophages (MDMs), monocyte derived dendritic cells (MDDCs), and neutrophils. Comparing the activity of the two ß2-integrin type complement receptors we found that CR3 plays a dominant role in the phagocytosis of iC3b opsonized S. aureus by all of these cell types. Studying another important integrin-mediated function we demonstrated earlier that CR4 is dominant in the adhesion of monocytes, MDMs and MDDCs to fibrinogen. Here we studied the participation of CR3 and CR4 in podosome formation by human phagocytes, since these structures are known to play an essential role in cell migration. Our confocal microscopy analysis revealed that both CD11b and CD11c concentrate in the podosome adhesion ring. In summary our data highlight differences in the function of human CR3 and CR4 in the process of uptake and digestion of complement opsonized antigen, while in the process of podosome formation, connected to cellular motility, both receptors equally take part.
Assuntos
Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Podossomos/ultraestrutura , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Adesão Celular , Diferenciação Celular , Movimento Celular , Células Cultivadas , Complemento C3b/metabolismo , Humanos , Interleucina-4/metabolismo , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Monócitos/microbiologia , Monócitos/ultraestrutura , Neutrófilos/microbiologia , Neutrófilos/ultraestrutura , Fagocitose , Agregação Patológica de ProteínasRESUMO
Dendritic cells (DCs) are professional antigen presenting cells, which take up pathogens/foreign structures in peripheral tissues, then migrate to secondary lymphoid organs where they initiate adaptive immune responses by activating naive T-cells. In the early phase of antigen uptake pattern recognition receptors (including mannose-, scavenger- and toll-like receptors) that recognize pathogen-associated molecular patterns play an important role. Later receptors binding opsonized antigen are also involved in phagocytosis. These cell membrane molecules include various Fc-receptors, recognizing different isotypes of antibodies and various complement-receptors, such as CR3, CR4 and the C1q-binding complex of calreticulin and CD91. Here we aim to summarize how these immunecomplex binding receptors are involved in the initiation of DC maturation, and how they influence antigen presentation as well as some additional functions of these cells.
Assuntos
Células Dendríticas/imunologia , Receptores de Complemento/metabolismo , Receptores Fc/metabolismo , Humanos , Imunoglobulinas/imunologiaRESUMO
Factor H (FH) is a major inhibitor of the alternative pathway of complement activation in plasma and on certain host surfaces. In addition to being a complement regulator, FH can bind to various cells via specific receptors, including binding to neutrophil granulocytes through complement receptor type 3 (CR3; CD11b/CD18), and modulate their function. The cellular roles of FH are, however, poorly understood. Because neutrophils are important innate immune cells in inflammatory processes and the host defense against pathogens, we aimed at studying the effects of FH on various neutrophil functions, including the generation of extracellular traps. FH co-localized with CD11b on the surface of neutrophils isolated from peripheral blood of healthy individuals, and cell-bound FH retained its cofactor activity and enhanced C3b degradation. Soluble FH supported neutrophil migration and immobilized FH induced cell spreading. In addition, immobilized but not soluble FH enhanced IL-8 release from neutrophils. FH alone did not trigger the cells to produce neutrophil extracellular traps (NETs), but NET formation induced by PMA and by fibronectin plus fungal ß-glucan were inhibited by immobilized, but not by soluble, FH. Moreover, in parallel with NET formation, immobilized FH also inhibited the production of reactive oxygen species induced by PMA and by fibronectin plus ß-glucan. Altogether, these data indicate that FH has multiple regulatory roles on neutrophil functions. While it can support the recruitment of neutrophils, FH may also exert anti-inflammatory effects and influence local inflammatory and antimicrobial reactions, and reduce tissue damage by modulating NET formation.
Assuntos
Fator H do Complemento/imunologia , Armadilhas Extracelulares , Neutrófilos/imunologia , Movimento Celular , Complemento C3b/imunologia , Humanos , Interleucina-8/metabolismo , Ativação de Neutrófilo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 µm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 µm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.
Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Processamento de Imagem Assistida por Computador , Robótica/métodos , Feminino , Humanos , MasculinoRESUMO
Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at â¼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell-extracellular matrix interactions.
Assuntos
Técnicas Biossensoriais/métodos , Adesão Celular , Células Dendríticas/fisiologia , Macrófagos/fisiologia , Monócitos/fisiologia , Materiais Revestidos Biocompatíveis , Determinação de Ponto Final , Humanos , Fatores de TempoRESUMO
Complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) belong to the family of beta2 integrins and are expressed mainly by myeloid cell types in humans. Previously, we proved that CR3 rather than CR4 plays a key role in phagocytosis. Here we analysed how CD11b and CD11c participate in cell adhesion to fibrinogen, a common ligand of CR3 and CR4, employing human monocytes, monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) highly expressing CD11b as well as CD11c. We determined the exact numbers of CD11b and CD11c on these cell types by a bead-based technique, and found that the ratio of CD11b/CD11c is 1.2 for MDDCs, 1.7 for MDMs and 7.1 for monocytes, suggesting that the function of CD11c is preponderant in MDDCs and less pronounced in monocytes. Applying state-of-the-art biophysical techniques, we proved that cellular adherence to fibrinogen is dominated by CD11c. Furthermore, we found that blocking CD11b significantly enhances the attachment of MDDCs and MDMs to fibrinogen, demonstrating a competition between CD11b and CD11c for this ligand. On the basis of the cell surface receptor numbers and the measured adhesion strength we set up a model, which explains the different behavior of the three cell types.
RESUMO
The opportunistic pathogenic yeast Candida albicans employs several mechanisms to interfere with the human complement system. This includes the acquisition of host complement regulators, the release of molecules that scavenge complement proteins or block cellular receptors, and the secretion of proteases that inactivate complement components. Secreted aspartic protease 2 (Sap2) was previously shown to cleave C3b, C4b and C5. C. albicans also recruits the complement inhibitor factor H (FH), but yeast-bound FH can enhance the antifungal activity of human neutrophils via binding to complement receptor type 3 (CR3). In this study, we characterized FH binding to human monocyte-derived macrophages. Inhibition studies with antibodies and siRNA targeting CR3 (CD11b/CD18) and CR4 (CD11c/CD18), as well as analysis of colocalization of FH with these integrins indicated that both function as FH receptors on macrophages. Preincubation of C. albicans yeast cells with FH induced increased production of IL-1ß and IL-6 in macrophages. Furthermore, FH enhanced zymosan-induced production of these cytokines. C. albicans Sap2 cleaved FH, diminishing its complement regulatory activity, and Sap2-treatment resulted in less detectable CR3 and CR4 on macrophages. These data show that FH enhances the activation of human macrophages when bound on C. albicans. However, the fungus can inactivate both FH and its receptors on macrophages by secreting Sap2, which may represent an additional means for C. albicans to evade the host innate immune system.
Assuntos
Ácido Aspártico Endopeptidases/imunologia , Candida albicans/imunologia , Fator H do Complemento/imunologia , Proteínas Fúngicas/imunologia , Integrina alfaXbeta2/imunologia , Antígeno de Macrófago 1/imunologia , Ácido Aspártico Endopeptidases/metabolismo , Western Blotting , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Antígenos CD18/genética , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Candida albicans/enzimologia , Candida albicans/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Fator H do Complemento/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/metabolismo , Interferência de RNARESUMO
Adhesion and spreading of primary monocytes isolated from human blood were monitored utilizing optical waveguide lightmode spectroscopy (OWLS); a highly sensitive label-free biosensor technique using evanescent optical waves generated at a biocompatible surface. Appropriate development on a custom built setup enabled the OWLS cuvette to be operated as a 1.5 ml mini-incubator, controlling both temperature and CO2 levels. The incubator-equipped OWLS is readily applicable for delicate and long-term studies on sensitive primary cells, demonstrated here through monitoring the serum dependence of the adhesion and spreading of human monocytes. Moreover, the custom-built setup enables the simultaneous monitoring of the position and overall width of the OWLS resonant peaks. This unique feature makes it possible to distinguish the refractive index variations induced by the adsorption of secreted material from refractive index changes provoked by cellular spreading. A definite attachment and spreading activity was observed on the substratum (glassy silica-titania), when the serum level of the culturing medium was 0.0-0.01%. Increasing serum concentration resulted in a steep fall in monocyte surface adhesion and spreading. 1.0% serum level practically abolished all spreading activity measured by OWLS, and the number of attached cells was significantly decreased, too. Serum addition to fully spread cells provoked a reduction in the cell-substratum contact area, clearly detectable by the biosensor. Cell spreading was inhibited by pre-coating the sensor surface with considerable amounts of serum proteins. These findings suggest that monocyte spreading is inhibited by the adsorption of serum biomolecules to the substratum, rather than by soluble factors present in the serum. All of these results were obtained completely noninvasively with real time monitoring; demonstrating the capabilities of OWLS to sensitively monitor the adhesion properties of immune cells isolated from human blood. The current study is, therefore, a significant step towards the application of label-free optical biosensors in medical diagnostics.
Assuntos
Técnicas Biossensoriais/instrumentação , Monócitos/citologia , Adesão Celular , Células Cultivadas , Desenho de Equipamento , Humanos , Microscopia de Contraste de Fase/instrumentação , Soro/químicaRESUMO
Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (â¼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes.
Assuntos
Computadores , Monócitos/citologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Automação , Adesão Celular/efeitos dos fármacos , Células Dendríticas/citologia , Fibrinogênio/farmacologia , Humanos , Hidrodinâmica , Macrófagos/citologia , Microfluídica , Monócitos/efeitos dos fármacos , Análise Numérica Assistida por Computador , Polietilenoglicóis/farmacologia , Polilisina/análogos & derivados , Polilisina/farmacologia , Resistência ao CisalhamentoRESUMO
Microarrayed antigens are used for identifying serum antibodies with given specificities and for generating binding profiles. Antibodies bind to these arrayed antigens forming immune complexes and are conventionally identified by secondary labelled antibodies.In the body immune complexes are identified by bone marrow derived phagocytic cells, such as monocytes. In our work we were looking into the possibility of replacing secondary antibodies with monocytoid cells for the generation of antibody profiles. Using the human monocytoid cell line U937, which expresses cell surface receptors for immune complex components, we show that cell adhesion is completely dependent on the interaction of IgG heavy chains and Fcγ receptors, and this recognition is susceptible to differences between heavy chain structures and their glycosylation. We also report data on a possible application of this system in autoimmune diagnostics.Compared to secondary antibodies, fluorescent monocytesas biosensors are superior in reflecting biological functions of microarray-bound antibodies and represent an easy and robust alternative for profiling interactions between serum proteins and antigens.
Assuntos
Autoantígenos/metabolismo , Imunoglobulina G/sangue , Monócitos/imunologia , Análise Serial de Proteínas/métodos , Anticorpos Imobilizados/química , Autoantígenos/química , Autoantígenos/imunologia , Adesão Celular , Linhagem Celular Tumoral , Proteínas do Sistema Complemento/metabolismo , Corantes Fluorescentes/química , Humanos , Imunoglobulina M/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Ligação Proteica , Receptores de IgG/metabolismoRESUMO
Dendritic cells (DCs) play a decisive role in immunity; they interact with various pathogens via several pattern recognition and different opsonophagocytotic receptors, including Fc- and complement-receptors. ß2-integrins, including complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) participate in many immunological processes, especially those involving cell migration, adherence, and phagocytosis. Human monocyte derived dendritic cells (MDCs) are known to express CR3 as well as CR4, however possible differences regarding the role of these receptors has not been addressed so far. Our aim was to explore whether there is a difference between the binding and uptake of various complement-opsonized microorganisms, mediated by CR3 and CR4. Studying the expression of receptors during differentiation of MDCs we found that the appearance of CD11b decreased, whereas that of CD11c increased. Interestingly, both receptors were present in the cell membrane in an active conformation. Here we demonstrate that ligation of CD11b directs MDCs to enhanced phagocytosis, while the maturation of the cells and their inflammatory cytokine production are not affected. Blocking CD11c alone did not change the uptake of opsonized yeast or bacteria by MDCs. We confirmed these results using siRNA; namely downregulation of CD11b blocked the phagocytosis of microbes while silencing CD11c had no effect on their uptake. Our data clearly demonstrate that complement C3-dependent phagocytosis of MDCs is mediated mainly by CR3.
Assuntos
Movimento Celular/fisiologia , Células Dendríticas/imunologia , Antígeno de Macrófago 1/imunologia , Monócitos/imunologia , Fagocitose/fisiologia , Adesão Celular/fisiologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Inativação Gênica , Humanos , Integrina alfaXbeta2/biossíntese , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/imunologia , Antígeno de Macrófago 1/biossíntese , Antígeno de Macrófago 1/genética , Masculino , Monócitos/citologia , Monócitos/metabolismoRESUMO
Although the complement system is thought to be mainly involved in innate immunity and in the humoral arm of adaptive responses, evidence implicating that complement impacts T cell responses are accumulating recently. The role of the various activation products of the major complement component C3 were mainly studied so far in animal systems, and investigations regarding the effect of different C3-fragments on human T cells are sparse. Here we show that anti-CD3 activated human T lymphocytes derived from the blood and tonsil of healthy individuals produce C3, and the major cleavage fragment that appears on the T cell surface is iC3b. Based on studies carried out in allogenic system we demonstrate that the T cell membrane bound iC3b binds to the CR3 and probably to CR4 receptors expressed on monocyte-derived dendritic cells, and this interaction leads to significantly enhanced T-cell proliferation. Since neither C3aR and nor C3a binding could be detected on the membrane of anti-CD3 activated T cells, our findings indicate that in humans in contrast to mice the C3a peptide is most probably not involved directly in the T cell activation process.