RESUMO
The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Esquelético/irrigação sanguínea , Vasculite/imunologia , Doença Aguda , Animais , Adesão Celular , Forma Celular , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Leucotrieno B4/farmacologia , Ligantes , Antígeno de Macrófago 1/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/deficiência , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Neutrófilos/patologia , Quimera por Radiação , Proteínas Recombinantes de Fusão/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/etiologia , Vênulas/patologiaRESUMO
As leukotriene D4 receptor CysLT1R upregulation is an early event in inflammatory processes, specific detection of CysLT1R via molecular imaging might be a promising diagnostic tool for inflammatory diseases. We coupled a specific anti-CysLT1R IgG antibody to near-infrared (NIR) hemicyanine fluorophore DY-734. The fluorophore was also coupled to unspecific rabbit-IgG antibody or corresponding Fab fragments. Expression of CysLT1R in HL-60 human promyelocytic leukemia cells in vitro could be proven by reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, and flow cytometry. Detection of the probes by flow cytometry showed that CysLT1R*DY-734 probe binds distinctly stronger to HL-60 cells than IgG*DY-734. Induction of ear edema in mice was conducted to test signaling of the synthesized probes in vivo. A markedly higher fluorescence intensity was observed in the edematous region than in the healthy region by a whole-body imaging system. Semiquantitative analysis showed that CysLT1R*DY-734 and Fab-CysLT1R*DY-734 probes bind 1.9- and 1.2-fold stronger, respectively, than the unspecific probes. Biodistribution studies revealed an enrichment of full-length IgG probes in liver and spleen, whereas Fab-containing probes are mostly found in liver and kidneys. Taken together, we present an approach that might improve early diagnosis of inflammatory diseases in the long term.
Assuntos
Meios de Contraste , Edema/metabolismo , Inflamação/metabolismo , Raios Infravermelhos , Imagem Molecular/métodos , Receptores de Leucotrienos/metabolismo , Animais , Edema/patologia , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HL-60 , Humanos , Imunoglobulina G/imunologia , Inflamação/patologia , Masculino , Camundongos , Coelhos , Distribuição TecidualRESUMO
BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody-based antigen-capture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies. STUDY DESIGN AND METHODS: This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time. RESULTS: Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (approximately 490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of low-avidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA. CONCLUSIONS: Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies.
Assuntos
Antígenos de Plaquetas Humanas/imunologia , Isoanticorpos/sangue , Ressonância de Plasmônio de Superfície/métodos , Trombocitopenia Neonatal Aloimune/diagnóstico , Trombocitopenia Neonatal Aloimune/imunologia , Reações Falso-Negativas , Humanos , Imunoensaio/métodos , Recém-Nascido , Integrina beta3RESUMO
BACKGROUND AND OBJECTIVES: Human anti-mouse antibodies (HAMAs) are relatively common in human serum and may interfere with therapeutic and diagnostic mouse monoclonal antibodies (MoAbs). We developed a simple particle agglutination test (PaGIA) for the detection of HAMAs. DESIGN AND METHODS: Red-dyed high density particles were coated with monoclonal mouse IgG. These particles were incubated in the reaction chamber of a gel-card together with serum samples obtained from healthy blood donors (n=32), and patients with clinically proven autoimmune thrombocytopenia (AITP; n=26). Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed throughout the gel matrix. Furthermore, MoAb-coated particles were subjected to flow cytometry and the results were compared with the new HAMA PaGIA. RESULTS: HAMAs were detectable in 33% of serum samples tested (n=58). Results from flow cytometric analysis revealed a high parallel to those obtained by the PaGIA. Interestingly, we observed an increased incidence of HAMAs in AITP patients (42%) compared to healthy blood donors (26%). INTERPRETATION AND CONCLUSION: The new HAMA PaGIA allows a specific and easy, rapid detection of HAMAs and is suitable for large scale testing.
Assuntos
Testes de Aglutinação/métodos , Anticorpos Heterófilos/sangue , Anticorpos Monoclonais/sangue , Imunoglobulina G/sangue , Camundongos/imunologia , Animais , Biotecnologia/métodos , Citometria de Fluxo , Humanos , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/imunologia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Immunization against the human platelet alloantigen (HPA)-3a residing on alphaIIbbeta3 integrin accounts for approximately 2 percent of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Anti-HPA-3a alloantibodies are sometimes difficult to detect and can be overlooked by standard antigen capture assays. STUDY DESIGN AND METHODS: The reactivity of 12 anti-HPA-3a and 2 anti-HPA-3b alloantibodies from patients with FNAIT and posttransfusion purpura was analyzed by serologic (monoclonal antibody-specific immobilization of platelet antigens [MAIPA] assay, flow cytometry) and immunochemical (immunoprecipitation, immunoblotting) techniques. The influence of platelet (PLT) age, storage conditions, recombinant antigens from Chinese hamster ovary (CHO) cells, and sialic acids (treatment with neuraminidase) were analyzed. RESULTS: The most sensitive anti-HPA-3 alloantibody detection in MAIPA assay could be achieved with fresh homozygous PLTs. During a PLT storage period of 14 days before use, three types of anti-HPA-3 alloantibodies were found: 1) complete loss of reactivity (n = 6), 2) considerably weakened reaction (> or =50% reduction; n = 3), and 3) minor reduction of reactivity (< or =40% decrease; n = 5). When cryopreserved PLTs were used, 10 of 12 anti-HPA-3a and all anti-HPA-3b alloantibodies reacted positive. Only 6 of 10 serum samples reacted with recombinant HPA-3a on CHO cells. Neuraminidase treatment of PLTs showed that some anti-HPA-3a alloantibodies require the presence of sialic acids. The storage lesion seems to be related to cleavage of sialic acids. Immunochemical analysis revealed evidence that most anti-HPA-3a alloantibodies require an intact three-dimensional alphaIIbbeta3 integrin structure. CONCLUSIONS: Anti-HPA-3 alloantibodies show considerable heterogeneity, which may hamper the serologic diagnosis of FNAIT. Preservation of the alphaIIbbeta3 integrin and protection from enzymatic degradation seem to be important during PLT storage.
Assuntos
Antígenos de Plaquetas Humanas/imunologia , Isoanticorpos/imunologia , Trombocitopenia Neonatal Aloimune/diagnóstico , Animais , Especificidade de Anticorpos/imunologia , Antígenos de Plaquetas Humanas/genética , Células CHO , Cricetinae , Cricetulus , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Recém-Nascido , Proteínas Recombinantes/imunologia , Síndrome , Trombocitopenia Neonatal Aloimune/imunologia , TransfecçãoRESUMO
The neonatal Fc receptor, FcRn, plays a central role in immunoglobulin G (IgG) transport across placental barriers. Genetic variations of FcRn-dependent transport across the placenta may influence antibody-mediated pathologies of the fetus and the newborn. Sequencing analysis of 20 unrelated individuals demonstrated no missense mutation within the five exons of the FcRn gene. However, a variable number of tandem repeats (VNTR) region within the FcRn promoter was observed, consisting of five different alleles (VNTR1-VNTR5). Alleles with two (VNTR2) and three (VNTR3) repeats were found to be most common in Caucasians (7.5 and 92.0%, respectively). Real-time polymerase chain reaction revealed that monocytes from VNTR3 homozygous individuals express 1.66-fold more FcRn transcript than do monocytes from VNTR2/VNTR3 heterozygous individuals (P = 0.002). In reporter plasmid assays, the VNTR3 allele supported the transcription of a reporter gene twice as effectively as did the VNTR2 allele (P = 0.003). Finally, under acidic conditions, monocytes from VNTR3 homozygous individuals showed an increased binding to polyvalent human IgG when compared with monocytes from VNTR2/VNTR3 heterozygous individuals (P = 0.021). These data indicate that a VNTR promoter polymorphism influences the expression of the FcRn receptor, leading to different IgG-binding capacities.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Regiões Promotoras Genéticas/genética , Receptores Fc/genética , Sequências de Repetição em Tandem , Alelos , Células Cultivadas , Expressão Gênica , Variação Genética , Heterozigoto , Homozigoto , Humanos , Imunoglobulina G/metabolismo , Recém-Nascido , Monócitos/metabolismo , Ligação Proteica , Estatísticas não Paramétricas , Transcrição GênicaRESUMO
BACKGROUND: Single-amino-acid substitution Leu33Pro in the beta3-integrin is responsible for the formation of the human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (beta3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura. STUDY DESIGN AND METHODS: While HPA-1 genotyping a large cohort of patients with thromboembolic disease with a thermal cycler (LightCycler), one patient was identified with a unique HPA-1a melting curve. RESULTS: Sequence analysis revealed a C-to-G transversion at nucleotide 175 in the beta3-integrin (ITGB3) gene that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n = 2950) identified one nonrelated Pro33Val33-positive individual. To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, transfectants were generated expressing recombinant beta3-Leu33 or beta3-Val33. Interestingly, differences in the reactivity of anti-HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with beta3-Val33 compared to beta3-Leu33 and others reacting equally with both types. Similar findings were observed with recombinant human HPA-1a antibodies, with one of the three not binding to beta3-Val33. CONCLUSIONS: Our results demonstrate that the naturally occurring Leu33Val mutation in the beta3-integrin can disrupt some HPA-1a epitopes. These findings provide evidence for a heterogeneous humoral response against HPA-1a that may have potential clinical implications for alloimmune thrombocytopenia disorders.