RESUMO
Radiotherapy (RT) initiates a local and systemic immune response which can induce anti-tumor immunity and improve immunotherapy efficacy. Neutrophils are among the first immune cells that infiltrate tumors after RT and are suggested to be essential for the initial anti-tumor immune response. However, neutrophils in tumors are associated with poor outcomes and RT induced neutrophil infiltration could also change the composition of the tumor microenvironment (TME) in favor of tumor progression. To improve RT efficacy for cancer patients it is important to understand the interplay between RT and neutrophils. Here, we review the literature on how RT affects the infiltration and function of neutrophils in the TME of solid tumors, using both patients studies and preclinical murine in vivo models. In general, it was found that neutrophil levels increase and reach maximal levels in the first days after RT and can remain elevated up to three weeks. Most studies report an immunosuppressive role of neutrophils in the TME after RT, caused by upregulated expression of neutrophil indoleamine 2,3-dioxygenase 1 (IDO1) and arginase 1 (ARG1), as well as neutrophil extracellular trap (NET) formation. RT was also associated with increased reactive oxygen species (ROS) production by neutrophils, which can both improve and inhibit anti-tumor immunity. In addition, multiple murine models showed improved RT efficacy when depleting neutrophils, suggesting that neutrophils have a pro-tumor phenotype after RT. We conclude that the role of neutrophils should not be overlooked when developing RT strategies and requires further investigation in specific tumor types. In addition, neutrophils can possibly be exploited to enhance RT efficacy by combining RT with neutrophil-targeting therapies.
RESUMO
Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, R. W. Erickson, T. Muhlebach, H. Messner, R. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence of cytosol (Koshkin, V., and E. Pick. 1993. FEBS [Fed. Eur. Biochem. Soc.] Lett. 327:57), the rate of NADPH-dependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47-phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome b558 is possible.
Assuntos
Grupo dos Citocromos b/metabolismo , Glutamina , Doença Granulomatosa Crônica/sangue , Proteínas de Membrana Transportadoras , NADH NADPH Oxirredutases/metabolismo , NADPH Desidrogenase/metabolismo , Fosfoproteínas/metabolismo , Prolina , Western Blotting , Grupo dos Citocromos b/biossíntese , Citosol/metabolismo , Humanos , Substâncias Macromoleculares , NADH NADPH Oxirredutases/biossíntese , NADPH Desidrogenase/biossíntese , NADPH Oxidases , Neutrófilos/metabolismo , Consumo de Oxigênio , Fosfoproteínas/biossíntese , Mutação Puntual , Processamento de Proteína Pós-Traducional , Valores de ReferênciaRESUMO
Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to generate superoxide, needed for the intracellular killing of microorganisms. This is caused by mutations in any one of the four subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of this enzyme (p67-phox) is missing. We here report on a patient with a mutation in the p67-phox gene that leads to expression of a nonfunctional p67-phox protein. The purified granulocytes of this patient failed to produce superoxide and contained about half of the normal amount of p67-phox. Analysis of the cDNA and genomic DNA of this patient showed that the patient is a compound heterozygote for a triplet nucleotide deletion in the p67-phox gene, predicting an in-frame deletion of lysine 58 in the p67-phox protein and a larger deletion of 11-13 kb in the other allele. Interestingly, the 58Lys deletion in p67-phox disrupts the interaction with p21-rac1, a ras-related protein involved in the activation of the NADPH oxidase. In contrast to normal neutrophils, in which p47-phox and p67-phox translocate to the plasma membrane upon cell activation, the cells of the patient did not show this translocation, indicating that an interaction between p67-phox and p21-rac1 is essential for translocation of these cytosolic proteins and activation of the NADPH oxidase. Moreover, this CGD patient represents the first case of disease caused by a disturbed binding of a ras-related protein to its target protein.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Doença Granulomatosa Crônica/etiologia , Doença Granulomatosa Crônica/genética , Mutação , Fosfoproteínas/genética , Transporte Biológico , Compartimento Celular , Criança , Chile/etnologia , Feminino , Doença Granulomatosa Crônica/classificação , Humanos , NADPH Oxidases , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Proteínas rac de Ligação ao GTPRESUMO
Leukocytes are involved in the pathogenesis of idiopathic inflammatory myopathies (IIMs). Immunoglobulin G (IgG) receptors (FcgammaR) link the specificity of IgG to the effector functions of leukocytes. Several FcgammaR subclasses display functional polymorphisms that determine in part the vigour of the inflammatory response. FcgammaRIIIa genotypes were differentially distributed among 100 IIM patients compared with 514 healthy controls with a significant increase of the homozygous FcgammaRIIIa-V-158 genotype (3 x 2 contingency table, chi(2) = 6.3, P = 0.04). Odds ratios (ORs) increased at the addition of each FcgammaRIIIa-V-158 allele, in particular among patients with non-specific myositis and dermatomyositis {OR 2.1 [95% confidence interval (CI) 1.1-4.3] and 2.7 (95% CI 1.1-6.4) for FcgammaRIIIa-V/F158 and FcgammaRIIIa-V/V158 genotypes, respectively, using FcgammaRIIIa-F/F158 as a reference group}. These data suggest that the FcgammaRIIIa-V-158 allele may constitute a genetic risk marker for IIM.
Assuntos
Predisposição Genética para Doença , Miosite/genética , Receptores de IgG/genética , Adulto , Idoso , Feminino , Proteínas Ligadas por GPI , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Miosite/epidemiologia , Países Baixos/epidemiologia , Polimorfismo GenéticoRESUMO
γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA-restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA-directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Animais , Humanos , CamundongosRESUMO
INTRODUCTION: In more recent years, anti-CD20 monoclonal antibodies have become the backbone in the treatment of chronic lymphocytic leukemia (CLL). Historically, these antibodies were typically combined with chemotherapeutic agents. At present, therapeutic outcomes are significantly improving with the introduction of several novel targeted drugs to the clinical repertoire. AREAS COVERED: In this review, we summarize the current clinical standard together with the latest developments in the field of anti-CD20 antibodies against CLL. In addition, novel promising drugs against CLL are discussed, as well as their potential for combination with anti-CD20 monoclonal antibodies. EXPERT OPINION: At present, there are three different anti-CD20 monoclonal antibodies approved for the treatment of CLL with diverse effector mechanisms. These antibodies provide a robust foundation for combination therapy with novel molecules. Current research should be focused on reducing toxicity and reaching long-lasting remissions. There is still much to gain regarding the optimization of treatment combinations and dosing schedules for CLL. Overcoming the limitations of currently used anti-CD20 antibodies will be critical to further improve the efficacy of CLL therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Rituximab/uso terapêutico , Terapia Combinada , HumanosRESUMO
The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.
Assuntos
Grupo dos Citocromos b/genética , Doença Granulomatosa Crônica/genética , NADH NADPH Oxirredutases/genética , Mutação Puntual , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Pré-Escolar , Citosol/metabolismo , Feminino , Doença Granulomatosa Crônica/enzimologia , Humanos , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , NADPH Desidrogenase/metabolismo , NADPH Oxidases , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Homologia de Sequência , Superóxidos/metabolismoRESUMO
When microorganisms invade the body, they encounter a large asssortment of defense mechanisms. Among these, phagocytes play an important role in the process of killing pathogens. This event is mediated by two important processes, viz. activation of the NADPH oxidase enzyme, which leads to the production of toxic oxygen metabolites, and fusion of intracellular granules with the phagosome (the vesicle that contains the ingested micro-organisms), which causes release of the toxic granule contents into this vesicle. The human NADPH oxidase is a very complex enzyme, in two ways: 1. it exists of at least 6 components: cytochrome b558 (a heterodimer comprised of gp91-phox and p22-phox), p47-phox, p67-phox, p40-phox, rac and Rap1A, and 2. there are multiple signal transduction pathways leading to activation of the NADPH oxidase. The most likely reason for this complexity is the toxicity of the oxygen radicals produced by the active NADPH oxidase; these compounds are not only harmful to the invading pathogens, but also to the surrounding tissues. This latter effect is enforced by the activation of metalloproteases released by neutrophils and by oxidation of protease inhibitors by oxygen metabolites. Therefore, an improper activation of the NADPH oxidase must be prevented at all costs and, when the infection has been cleared, a rapid deactivation mechanism is imperative. In this review, the interaction between the different components of the NADPH oxidase and the activation of these proteins will be discussed.
Assuntos
NADPH Oxidases/metabolismo , Sequência de Aminoácidos , Animais , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Ativação Enzimática , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/patologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , NADPH Oxidases/genética , Fagocitose , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
GVHD remains a major problem in allo-SCT. We explored the presence of APC in skin biopsies of GVHD patients, using the IgG receptor CD64 expression as a hallmark for activated APC. By immunohistochemistry we demonstrated CD64 to be upregulated on host APC in skin biopsies of patients with acute GVHD and, less prominently, in chronic GVHD. Double staining for CD32 polymorphism revealed CD64-positive cells to be mainly of host origin. The majority of CD64-positive cells coexpressed CD68, indicating a macrophage phenotype. Given its very restricted cellular distribution, CD64 may represent an excellent target for APC-directed therapies in GVHD.
Assuntos
Regulação da Expressão Gênica , Doença Enxerto-Hospedeiro/metabolismo , Receptores de IgG/biossíntese , Dermatopatias/metabolismo , Pele/metabolismo , Doença Aguda , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/patologia , Doença Crônica , Feminino , Doença Enxerto-Hospedeiro/patologia , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Masculino , Pele/patologia , Dermatopatias/patologia , Transplante de Células-Tronco , Transplante HomólogoRESUMO
The human NADPH oxidase is a very intriguing enzyme; although its catalytic unit is retained within cytochrome b558, various additional proteins are required for activity of the NADPH oxidase. In the past few years substantial progress has been made to elucidate the protein-protein interactions and the activation events involved. The following facts have become evident: (1) activation of rac and subsequent interaction with p67-phox is crucial for the interaction of p67-phox with cytochrome b558, and probably with gp91-phox; (2) p47-phox interacts with p22-phox, and phosphorylation of 379Ser of p47-phox is obligatory for this event; (3) p47-phox and p67-phox regulate each other's translocation in a positive sense (see also reference 71). To put it differently: it is vital to gain insight in the intrigues within the phox family and associated characters to fully understand NADPH oxidase activation.
Assuntos
NADPH Oxidases/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sistema Livre de Células , Ativação Enzimática , Doença Granulomatosa Crônica/enzimologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , NADPH Oxidases/química , NADPH Oxidases/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Conformação ProteicaRESUMO
We have identified a rare polymorphism (G to C at nucleotide 1102) in CYBB, which codes for gp91-phox, a component of NADPH oxidase. Polymorphonuclear leukocytes with this enzyme produced normal amounts of superoxide anion.
Assuntos
Doença Granulomatosa Crônica/genética , Glicoproteínas de Membrana/genética , NADH NADPH Oxirredutases/genética , Polimorfismo Genético , Cromossomo X , Sequência de Aminoácidos , Sequência de Bases , DNA/sangue , DNA/química , DNA/isolamento & purificação , Primers do DNA , Eletroforese em Gel de Ágar , Éxons , Feminino , Doença Granulomatosa Crônica/sangue , Humanos , Leucócitos/fisiologia , Masculino , Dados de Sequência Molecular , NADPH Oxidase 2 , NADPH Oxidases , Mutação Puntual , Reação em Cadeia da PolimeraseRESUMO
A 12 year old boy suffering from p67-phox deficient chronic granulomatous disease presented with a bullous skin disease and a lung infection with paecilomyces species. The histopathology of a bullous lesion showed subepidermal blister formation and microabcesses containing eosinophils in the dermal papillae. By direct immunofluorescence, linear staining of IgA at the dermal-epidermal junction was detected which confirmed the clinical diagnosis of chronic bullous disease of childhood (linear IgA dermatosis).
Assuntos
Doenças Autoimunes/complicações , Doença Granulomatosa Crônica/complicações , Pneumopatias Fúngicas/complicações , Paecilomyces , Dermatopatias Vesiculobolhosas/complicações , Anfotericina B/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Antifúngicos/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Criança , Quimioterapia Combinada , Doença Granulomatosa Crônica/tratamento farmacológico , Doença Granulomatosa Crônica/metabolismo , Humanos , Itraconazol/uso terapêutico , Pneumopatias Fúngicas/tratamento farmacológico , Pneumopatias Fúngicas/metabolismo , Masculino , Fosfoproteínas/deficiência , Prednisolona/uso terapêutico , Dermatopatias Vesiculobolhosas/tratamento farmacológico , Dermatopatias Vesiculobolhosas/metabolismoRESUMO
Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane-bound heterodimeric protein. Src homology 3 (SH3) domains and their proline-rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67phox, p47phox, and p40phox reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique. Wild-type and mutated 35S-labeled p67phox and p47phox were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67phox to blotted p47phox and blotted p40phox was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227-231) in p67phox. We also found a specific association of native p47phox with blotted p67phox. Deletions in a putative SH3-binding region of p47phox completely abrogated the interaction with p67phox. Other results suggest that the C terminus of p47phox exposes this SH3-binding domain for interaction with p67phox. Similar results were obtained when the binding of cytosolic p67phox to wild-type or mutated p47phox were studied in solution. Interestingly, mutants of p47phox unable to bind to p67phox were fully capable of supporting superoxide production under cell-free activation conditions. We conclude that an interaction between the C-terminal proline-rich region of p47phox and the second SH3 domain of p67phox is not required for oxidase activity in the cell-free assay.
Assuntos
NADH NADPH Oxirredutases/sangue , NADPH Desidrogenase/sangue , Neutrófilos/enzimologia , Fosfoproteínas/sangue , Sequência de Bases , Sistema Livre de Células , Citosol/enzimologia , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Mutagênese , NADPH Desidrogenase/biossíntese , NADPH Desidrogenase/isolamento & purificação , NADPH Oxidases , Oligodesoxirribonucleotídeos , Fosfoproteínas/biossíntese , Fosfoproteínas/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Superóxidos/metabolismoRESUMO
Three highly homologous genes (A, B and C) and six transcripts have been identified for the class I human IgG receptor (CD64). The hFcgammaRIa1 isoform encodes the prototypic high-affinity receptor for IgG. The alternatively spliced hFcgammaRIb2 transcript was postulated to exist as a second surface-expressed CD64 isoform on myeloid cells. In this report we assessed this proposed role for hFcgammaRIb2 in detail. As CD64 monoclonal antibodies might not recognize hFcgammaRIb2, we tagged the receptor with an hemagglutinin tag and transfected hFcgammaRIb2tag in the presence of FcR gamma-chain into IIA1.6 cells. Both transcript and protein of hFcgammaRIb2tag were clearly present in transfectants. However, in contrast to the (control) hFcgammaRIa1tag, no surface expression of hFcgammaRIb2tag was detectable with a tag-specific monoclonal antibody. Confocal scan laser microscopy revealed hFcgammaRIb2tag to be retained in the endoplasmic reticulum, resulting in absent plasma membrane expression. These results show hFcgammaRIb2 neither to be surface expressed, nor to represent a separate CD64 isoform. This finding, furthermore, implicates that other FcR transcripts defined at the mRNA level may not represent true FcR isoforms either.
Assuntos
Receptores de IgG/genética , Receptores de IgG/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/imunologia , Primers do DNA/genética , Hemaglutininas/genética , Humanos , Técnicas In Vitro , Camundongos , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/imunologia , TransfecçãoRESUMO
To further define the role played by protein kinase C (PKC) in the activation of the neutrophil NADPH oxidase, we have utilized a pseudosubstrate of PKC which was myristoylated at the N terminus. In electropermeabilized neutrophils, the myristoylated pseudosubstrate Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-psi PKC) inhibited PMA-induced protein phosphorylations and activation of the NADPH oxidase, induced either by PMA or by the receptor agonist formyl-methionyl-leucyl-phenylalanine. Both the pseudosubstrate lacking the N-terminal myristate (psi PKC) and a myristoylated control peptide (Phe-Ala-Glu-Asp-Gly-Ala-Leu-Glu-Gln, myr-CP) were without effect on these responses. The myristoylated pseudosubstrate was also tested in a cell-free system, in which NADPH oxidase activation can be achieved by addition of SDS and guanosine 5'-3-O-(thio)triphosphate in a staurosporine-insensitive manner. Myr-psi PKC, but not psi PKC or myr-CP, proved to be a potent inhibitor of NADPH oxidase activity in the cell-free system, indicating that the inhibition observed in permeabilized neutrophils may have been caused by an effect other than PKC inhibition. In the presence of myr-psi PKC, translocation in the cell-free system of the cytosolic oxidase components p47-phox and p67-phox to the plasma membrane was inhibited. From these results we conclude that myristoylation profoundly increases the ability of pseudosubstrates of PKC to inhibit not only PKC-mediated phosphorylations, but also NADPH oxidase activation. The latter effect, however, is most probably not related to PKC inhibition but may indicate a critical role of the membrane surface charge in the translocation of the cytosolic oxidase components p47-phox and p67-phox.
Assuntos
Miristatos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Neutrófilos/enzimologia , Oligopeptídeos/farmacologia , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Dados de Sequência Molecular , Miristatos/química , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Desidrogenase/metabolismo , NADPH Oxidases , Oligopeptídeos/química , Fosfoproteínas/metabolismo , Fosforilação , Dodecilsulfato de Sódio/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.
Assuntos
Antígenos CD18/fisiologia , Citotoxicidade Imunológica , Antígeno de Macrófago 1/fisiologia , Proteínas de Membrana Transportadoras , Neutrófilos/fisiologia , Receptores Fc/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Neoplasias da Mama/patologia , Candida albicans , Carcinoma/patologia , Adesão Celular , Cruzamentos Genéticos , Exocitose , Feminino , Glucuronidase/metabolismo , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Lactoferrina/metabolismo , Fusão de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NADPH Desidrogenase/metabolismo , NADPH Oxidases , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Proteínas Opsonizantes/imunologia , Fagocitose , Fosfoproteínas/metabolismo , Transporte Proteico , Explosão Respiratória , Células Tumorais CultivadasRESUMO
Circulating CD8+ T cells with a CD45RA+CD27- phenotype resemble cytolytic effector cells because they express various cytolytic mediators and are able to execute cytotoxicity without prior stimulation in vitro. We here demonstrate that CD8+CD45RA+CD27- T cells can use both granule exocytosis and Fas/Fas ligand pathways to induce apoptosis in target cells. The availability of these cytolytic mechanisms in circulating T cells suggests that the activity of these cells must be carefully controlled to prevent unwanted tissue damage. For this reason, we analyzed the expression of surface receptors that either enhance or inhibit T cell function. Compared with memory-type cells, effector cells were found to express normal levels of CD3epsilon and TCRzeta and relatively high levels of CD8. CTLA-4 was absent from freshly isolated effector cells, whereas a limited number of unstimulated memory cells expressed this molecule. In line with recent findings on CD8+CD28- T cells, CD45RA+CD27- T cells were unique in the abundant expression of NK cell-inhibitory receptors, both of Ig superfamily and C-type lectin classes. Binding of NK cell-inhibitory receptors to classical and nonclassical MHC class I molecules may inhibit the activation of the cytolytic machinery induced by either Ag receptor-specific or nonspecific signals in CD8+CD45RA+CD27- T cells.
Assuntos
Citotoxicidade Imunológica/imunologia , Imunoconjugados , Antígenos Comuns de Leucócito/biossíntese , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/biossíntese , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Abatacepte , Antígenos CD , Antígenos de Diferenciação/biossíntese , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4 , Células Cultivadas , Exocitose/imunologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Antígenos Comuns de Leucócito/sangue , Proteínas de Membrana/biossíntese , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Imunológicos/biossíntese , Receptores KIR , Transdução de Sinais/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/sangueRESUMO
Within multi-subunit Ig receptors, the FcR gamma-chain immunoreceptor tyrosine-based activation motif (ITAM) plays a crucial role in enabling antigen presentation. This process involves antigen-capture and targeting to specific degradation and major histocompatibility complex (MHC) class II loading compartments. Antigenic epitopes are then presented by MHC class II molecules to specific T cells. The high-affinity receptor for IgG, hFcgammaRIa, is exclusively expressed on myeloid lineage cells and depends on the FcR gamma-chain for surface expression, efficient ligand binding, and most phagocytic effector functions. However, we show in this report, using the IIA1.6 cell model, that hFcgammaRIa can potentiate MHC class II antigen presentation, independently of a functional FcR gamma-chain ITAM. Immunoelectron microscopic analyses documented hFcgammaRIa alpha-chain/rabbit IgG-Ovalbumin complexes to be internalized and to migrate via sorting endosomes to MHC class II-containing late endosomes. Radical deletion of the hFcgammaRIa alpha-chain cytoplasmic tail did not affect internalization of rabbit IgG-Ovalbumin complexes. Importantly, however, this resulted in diversion of receptor-ligand complexes to the recycling pathway and decreased antigen presentation. These results show the hFcgammaRIa cytoplasmic tail to contain autonomous targeting information for intracellular trafficking of receptor-antigen complexes, although deficient in canonical tyrosine- or dileucine-targeting motifs. This is the first documentation of autonomous targeting by a member of the multichain FcR family that may critically impact the immunoregulatory role proposed for hFcgammaRIa (CD64).
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulina G/imunologia , Receptores de IgG/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Endocitose , Peroxidase do Rábano Silvestre , Humanos , Camundongos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Ovalbumina/imunologia , Coelhos , Receptores de IgG/química , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Frações Subcelulares/química , TransfecçãoRESUMO
The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)