RESUMO
We recently demonstrated a codominant role of C5aR and FcgammaRIII in the initiation of IgG immune complex-mediated inflammation in mice. In this study, we investigated the relative contribution of FcgammaRIII in the generation of several cytokines during experimental hypersensitivity pneumonitis/alveolitis in vivo. Induction of immune complex-alveolitis in C57BL/6 mice resulted in strong accumulation of neutrophils into the lung and enhanced chemotactic activity within bronchoalveolar lavage fluid accompanied by an increased production of the proinflammatory cytokines TNF-alpha and IL-1beta as well as the ELR-CXC chemokines macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC). FcgammaRIII-deficient C57BL/6 mice (FcgammaRIII(-/-)) showed a marked reduction of the inflammatory response due to decreased production of TNF-alpha, IL-1beta, and MIP-2. Results obtained in C57BL/6 mice either lacking the TNF-alpha class I receptor (TNF-alphaRI(-/-)) or treated with neutralizing anti-TNF-alpha mAb demonstrated an essential contribution of TNF-alpha for mediating IL-1beta release, neutrophil influx, and hemorrhage. Surprisingly, MIP-2 and KC chemokine levels remained largely unaffected in TNF-alphaRI(-/-) mice or after functional inhibition of TNF-alpha. These data suggest that in immune complex alveolitis, the activation of FcgammaRIII may induce divergent downstream effector pathways with TNF-alpha acting independently of CXC chemokines to trigger the inflammatory response in C57BL/6 mice.
Assuntos
Alveolite Alérgica Extrínseca/imunologia , Quimiocinas CXC/biossíntese , Doenças do Complexo Imune/imunologia , Receptores de IgG/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Alveolite Alérgica Extrínseca/patologia , Animais , Complexo Antígeno-Anticorpo/administração & dosagem , Líquido da Lavagem Broncoalveolar/imunologia , Movimento Celular/imunologia , Quimiocina CXCL2 , Quimiocinas/biossíntese , Quimiocinas/fisiologia , Quimiocinas CXC/fisiologia , Quimiotaxia de Leucócito/imunologia , Citocinas/metabolismo , Doenças do Complexo Imune/patologia , Imunoglobulina G/administração & dosagem , Injeções Intravenosas , Interleucina-1/biossíntese , Interleucina-1/metabolismo , Intubação Intratraqueal , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/patologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Receptores de IgG/deficiência , Receptores de IgG/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
NK cells reject non-self hematopoietic bone marrow (BM) grafts via Ly49 receptor-mediated MHC class I-specific recognition and calibration of receptor expression levels. In this paper we investigated how Ly49+ subset frequencies were regulated dependent on MHC class I expression. The development of donor and host Ly49A+ (recognizes H-2Dd and H-2Dk ligands) and Ly49C/I+ (Ly49CBALB/c recognizes H-2Kb, H-2Kd, and H-2Dd, and Ly49CB6 recognizes only H-2Kb) NK cell frequencies were monitored for 120 days in murine-mixed allogeneic BM chimeras. C57BL/6 (H-2b) BM was transplanted into BALB/c (H-2d) mice and vice versa. Peripheral NK cell populations were examined every 5 days. Chimerism was found to be stable with 80-90% donor NK cells. In contrast to syngeneic controls reexpressing pretransplant patterns, donor and host NK cells revealed new and mainly reduced subset frequencies 55 days after allogeneic transplantation. Recipient NK cells acquired these later than donor NK cells. In H-2d --> H-2b chimeras Ly49A+, Ly49C/I+, and Ly49A+/Ly49C/I+ proportions were mainly diminished upon interaction with cognate ligands. Also in H-2b --> H-2d chimeras, Ly49A+ and Ly49A+/Ly49C/I+ subsets were reduced, but there was a transient normalization of Ly49C/I+ proportions in the noncognate host. After 120 days all subsets were reduced. Therefore, down-regulation of developing Ly49A+ and Ly49C/I+ chimeric NK cell frequencies by cognate ligands within 7-8 wk after BM transplantation may be important for successful engraftment.
Assuntos
Antígenos Ly , Transplante de Medula Óssea/imunologia , Antígenos H-2/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Glicoproteínas de Membrana , Quimeras de Transplante/imunologia , Animais , Tolerância Imunológica , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK , Receptores Semelhantes a Lectina de Células NK , Transplante HomólogoRESUMO
The generation of autoantibodies and deposition of immune complexes (ICs) in tissue play a primary role in autoimmune diseases. However, the IC-triggered response consists of complex mechanisms that make it difficult to identify the pathogenesis and develop specific therapy. We clarified here a sequential mechanism for the induction of hypersensitivity angiitis by analyzing the responsible Fc receptor (FcR), effector cells, and mediators in an animal model using FcR-deficient mice. In this model, rheumatoid factor-mediated skin vasculitis was induced in wild-type mice, whereas FcRgamma-deficient mice did not develop the vasculitis. Adoptive transfer of various FcR(+) cells into FcRgamma-deficient mice showed that mast cells but not macrophages derived from wild-type mice triggered skin vasculitis. Mast cells derived from either FcgammaRIII-deficient or tumor necrosis factor (TNF)-deficient mice did not possess the inducibility of skin vasculitis. These results indicate that triggering of vascular inflammation was induced by mast cells through IC binding on FcgammaRIII. TNF produced by such activated mast cells was mainly responsible for the pathogenesis of autoantibody-mediated vasculitis. These findings illustrate the clinical significance of mast cells, Fcgamma receptors, and TNF in IC-induced vasculitis syndrome.
Assuntos
Autoanticorpos/imunologia , Mastócitos/imunologia , Receptores Fc/imunologia , Fator de Necrose Tumoral alfa/imunologia , Vasculite/imunologia , Animais , Mastócitos/patologia , Camundongos , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Vasculite/etiologia , Vasculite/patologiaRESUMO
Shock is frequently accompanied by thrombocytopenia. To investigate the pathogenic role of platelets in shock, we examined the in vivo effects of monoclonal antibodies (MoAbs) against mouse platelet membrane proteins. Injection of the platelet-specific MoAb MWReg30 to the fibrinogen receptor (gpIIb/IIIa) rendered mice severely hypothermic within minutes. Isotype-matched control antibodies, even if they also recognized platelet surface antigens, did not induce comparable signs. MWReg30 induced early signs of acute lung injury with increased cellularity in the lung interstitium and rapid engorgement of alveolar septal vessels. Despite this in vivo activity, MWReg30 inhibited rather than stimulated platelet aggregation in vitro. MWReg30-binding to platelets led to phosphorylation of gpIIIa, but did not induce morphological signs of platelet activation. The MWReg30-induced reaction was abolished after treatment with MoAbs 2.4G2 to FcgammaRII/III and was absent in FcgammaRIII-deficient mice, clearly demonstrating the requirement for FcgammaRIII on involved leukocytes. Simultaneous administration of tumor necrosis factor exacerbated, whereas a tolerizing regimen of tumor necrosis factor or bacterial lipopolysaccharide completely prevented the reaction. These data suggest that platelet surface-deposited MWReg30-immune complexes lead to an acute Fc-mediated reaction with pulmonary congestion and life-threatening potential that could serve as an in vivo model of acute lung injury.
Assuntos
Anticorpos Monoclonais/toxicidade , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Edema Pulmonar/etiologia , Choque/etiologia , Trombocitopenia/etiologia , Animais , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Eritema/etiologia , Eritema/imunologia , Eritema/fisiopatologia , Hipotermia/etiologia , Hipotermia/imunologia , Hipotermia/fisiopatologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/uso terapêutico , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Edema Pulmonar/imunologia , Edema Pulmonar/fisiopatologia , Receptores de IgG/deficiência , Receptores de IgG/genética , Receptores de IgG/imunologia , Choque/complicações , Choque/fisiopatologia , Choque/prevenção & controle , Organismos Livres de Patógenos Específicos , Trombocitopenia/imunologia , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/uso terapêutico , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
The contributions of Fc receptors (FcRs) for IgG (FcgammaRs) and complement to immune complex (IC)-mediated peritonitis were evaluated in BALB/c-, C57BL/6-, FcRgamma chain-, and FcR type III for IgG (FcgammaRIII)-deficient mice, backcrossed to the C57BL/6 background. In BALB/c mice, but not in C57BL/6 mice, neutrophil migration was markedly attenuated after complement depletion. In mice lacking FcRgamma chain, neutrophil migration was abolished, whereas it was unaffected in FcgammaRIII-deficient mice. Huge amounts of TNF-alpha (TNF) were found in the peritoneal exudate of BALB/c and C57BL/6 mice but were absent in mice lacking FcRgamma chain or FcgammaRIII. Surprisingly, a functional inhibition of TNF in BALB/c and C57BL/6 mice had no effect on neutrophil infiltration. These data provide evidence that in IC peritonitis, the activation of FcR type I for IgG on peritoneal macrophages and the activation of the complement cascade, but not the interaction of ICs with FcgammaRIII and the subsequent release of TNF, initiate the inflammatory response in BALB/c and C57BL/6 mice.
Assuntos
Reação de Arthus/imunologia , Proteínas do Sistema Complemento/imunologia , Macrófagos Peritoneais/imunologia , Peritonite/imunologia , Receptores de IgG/imunologia , Animais , Reação de Arthus/genética , Líquido Ascítico/química , Líquido Ascítico/citologia , Quimiotaxia de Leucócito , Cruzamentos Genéticos , Citotoxicidade Imunológica , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neutrófilos/imunologia , Peritonite/genética , Especificidade da Espécie , Fator de Necrose Tumoral alfa/análise , Antígenos HLA-ERESUMO
In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcgammaR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcgammaRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti-red blood cell antibodies in mice defective for FcgammaRIII, and comparing the clinical outcome to those in wild-type mice. FcgammaRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcgammaRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcgammaRIII reflects an activation of FcgammaRI that is normally coexpressed with FcgammaRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcgammaRIII and further suggest that FcgammaRI, in addition to FcgammaRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.
Assuntos
Anemia Hemolítica Autoimune/imunologia , Eritrócitos/imunologia , Imunoglobulina G/imunologia , Receptores de IgG/deficiência , Animais , Autoanticorpos/imunologia , Citotoxicidade Imunológica , Humanos , Isotipos de Imunoglobulinas/imunologia , Camundongos , Camundongos Knockout , Fagocitose , Receptores de IgG/genética , Receptores de IgG/imunologiaRESUMO
Previously, we have demonstrated that phagocytosis of IgG1-coated particles by macrophages in vitro is impaired by deletion of Fc gamma RIII in mice, suggesting that IgG1 may interact preferentially with Fc gamma RIII. In the present study, the biologic relevance of this observation was addressed by triggering various effector functions of the immune system in Fc gamma RIII(-/-) mice, using panels of mAbs of different IgG subclasses. Both binding and phagocytosis of IgG1-coated sheep or human erythrocytes by Fc gamma RIII(-/-) macrophages in vitro were strongly impaired, indicating that the impaired ingestion of complexed IgG1 by Fc gamma RIII(-/-) macrophages is due to a defect in binding. An in vivo consequence of the defective phagocytosis was observed by resistance of Fc gamma RIII-deficient mice to experimental autoimmune hemolytic anemia, as shown by a lack of IgG1-mediated erythrophagocytosis in vivo by liver macrophages. Furthermore, trapping of soluble IgG1-containing immune complexes by follicular dendritic cells in mesenteric lymph nodes from Fc gamma RIII(-/-) mice was abolished. Whole blood from Fc gamma RIII(-/-) mice was unable to induce lysis of tumor cells in the presence of IgG1 antitumor Abs. Finally, IgG1 mAbs proved unable to mount a passive cutaneous anaphylaxis in Fc gamma RIII(-/-) mice. Together, these results demonstrate that IgG1 complexes, either in particulate or in soluble form, trigger in vitro and in vivo immune effector functions in mice predominantly via Fc gamma RIII.
Assuntos
Complexo Antígeno-Anticorpo/fisiologia , Imunoglobulina G/fisiologia , Receptores de IgG/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/metabolismo , Antígenos de Grupos Sanguíneos/imunologia , Neoplasias da Mama , Células Dendríticas/imunologia , Eritrócitos/imunologia , Eritrócitos/metabolismo , Humanos , Soros Imunes/fisiologia , Imunoglobulina G/sangue , Imunoglobulina G/farmacologia , Fígado/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/imunologia , Anafilaxia Cutânea Passiva , Fagocitose/imunologia , Formação de Roseta , Células Tumorais CultivadasRESUMO
Fc gamma receptors for the Fc part of IgG are the mediators for antibody effector functions. FcgammaRIII and FcgammaRII are low affinity receptors that, through the interaction with immune complexes, initiate a variety of immunological responses, such as phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators. We set out to define the IgG binding site on human FcgammaRIII. We assumed that potential beta-turns in Ig-like domains are the most probable determinants for ligand binding, and chimeric FcgammaRIIIB/FcepsilonRI receptors as well as single residue mutants were constructed in these regions of FcgammaRIIIB. Substitution of four amino acids in the membrane-proximal domain (Gln126, Arg156, Lys162, Val164) resulted in decreased binding of human IgG1. Lys162 and Val164 were found also to be crucial for the interaction with the IgG-binding inhibitory monoclonal antibody 3G8. In a putative three-dimensional model constructed in this study, these residues map on the CC loop (Gln126), on F beta-sheet (Arg156), and on the FG loop (Lys162, Val164). Our data are consistent with the study about human FcgammaRII (Hulett, M. D., Witort, E., Brinkworth, R. I., McKenzie, I. F. C., and Hogarth, P. M. (1994) J. Biol. Chem. 269, 15287-15293), suggesting that common structural determinants, i.e. FG loop or the GFC surface of the membrane-proximal domain, can be involved in interactions with IgG by both low affinity receptor classes FcgammaRII and FcgammaRIII.
Assuntos
Imunoglobulina G/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Receptores de IgG/química , Receptores de IgG/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Sítios de Ligação , Linhagem Celular , Membrana Celular/imunologia , DNA Complementar , Humanos , Imunoglobulina G/química , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Modelos Estruturais , Dados de Sequência Molecular , Mieloma Múltiplo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
Immune complexes localize to the glomerular mesangium in many forms of human glomerulonephritis. In this investigation, we determined whether primary cultured human glomerular mesangial cells (HMC) express mRNA and functional protein of FcR for IgG. Performing reverse transcription-PCR with subsequent control Southern blots, we showed that in contrast to human monocytes or granulocytes, mRNA for Fc gamma RI and Fc gamma RII is not detectable in either growth-arrested (48 h serum free) or unstimulated proliferating (medium with 10% FCS) HMC. However, IFN-gamma in combination with LPSE.coli (LPS), but not LPS alone elicited a significant transcription of hFc gamma RIII mRNA in resting HMC, whereas cytokines, such as IL-1 beta or TGF-beta, failed to induce any of the three Fc gamma Rs in resting HMC. Proliferating HMC showed a basal transcription of Fc gamma RIII mRNA, which was enhanced by IFN-gamma in combination with LPS. Slot-blot analysis indicated that only the Fc gamma RIII-A gene encoding the transmembrane isoform of Fc gamma RIII was expressed by HMC. For the first time, transcripts for the gamma-chain of Fc epsilon RI were found in HMC. Fc gamma RIII protein expression was detected after LPS/IFN-gamma stimulation both by specific staining of paraformaldehyde-fixed HMC and immunoprecipitation of Fc gamma RIII protein from HMC membranes. Fc gamma RIII-A receptors are functionally active, as HMC IL-6 mRNA synthesis was stimulated by heat-aggregated IgG or F(ab')2 fragments of CLBGran1 mAb only after induction of Fc gamma RIII-A. These in vitro data suggest that HMC that are basically negative for Fc gamma RIII can be stimulated to express low affinity Fc gamma Rs, and thus may participate actively in human glomerulonephritis involving immune complexes.
Assuntos
Mesângio Glomerular/imunologia , Receptores de IgE/metabolismo , Receptores de IgG/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular , Membrana Celular/metabolismo , Primers do DNA/química , Expressão Gênica , Mesângio Glomerular/citologia , Humanos , Interleucina-6/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores de IgE/genética , Receptores de IgG/genética , Transcrição GênicaRESUMO
The transmembrane protein CD16 (Fc gamma RIII) is detected on activated macrophages, natural killer (NK) cells and a small subset of T lymphocytes. From CD3-CD56+ CD16+bright NK cells and CD3+ CD56+ CD16+dim non-major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) clones were generated reflecting the stable, but different, CD16 expression of the respective peripheral blood subpopulations. To compare the role of CD16 on NK cells and non-MHC-restricted CTL, Fc gamma RIII activation and its mechanisms were investigated using monoclonal antibodies (mAb). Cross-linking of CD16 induced Ca2+ influx in CD16+bright NK clones. In contrast, there was no Ca2+ mobilization after CD16 activation in CD16+dim CTL, which revealed a good response to cross-linking of CD3 antigen. Pretreatment with CD16 mAb alone or cross-linked CD16 mAb did not block the CD3 response of CD16+dim CTL. Again, CD16 cross-linking induced more interferon-gamma transcription in NK cell clones than in non-MHC-restricted CTL clones. Also a higher tumor necrosis factor-alpha production of NK clones after CD16 cross-linking compared to CD16+dim CTL could be observed. These data suggest that after CD16 activation CD16+dim CTL and CD16+bright NK cells use different second messengers. In addition, signal transduction via CD3 and CD16 appears to function independently in CD16+dim non-MHC-restricted CTL.
Assuntos
Antígenos de Diferenciação/fisiologia , Células Matadoras Naturais/fisiologia , Receptores Fc/fisiologia , Linfócitos T Citotóxicos/fisiologia , Anticorpos Monoclonais , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Northern Blotting , Complexo CD3 , Antígeno CD56 , Cálcio/metabolismo , Células Clonais , Citometria de Fluxo , Humanos , RNA/análise , Radioimunoensaio , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de IgG , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Plasma cells obtained from bone marrow samples of 45 patients with MM, eight patients with MGUS, eight patients with Waldenström's macroglobulinaemia (WM), one patient with immunocytoma, and 12 controls were characterized by immunophenotyping, estimation of DNA content, and labeling index, as well as by morphological analysis. Plasma cells from 37/45 myeloma and 5/8 MGUS patients expressed CD38 and CD56 (N-CAM) on their surface but were negative for other NK cell-associated antigens such as CD16 (Fc gamma RIII) or CD2. All tumor cells of less-differentiated cell type (WM, immunocytoma) and normal polyclonal plasma cells were negative for CD56. CD56-specific mRNA was demonstrated in myeloma cells by northern blot analysis. Another adhesion molecule, ICAM-1 (CD54), was found on plasma cells from all patients and controls examined. Coexpression of CD19 (1/45), CD20 (9/45), or CD33 (3/45) was rare and CD10 with CD14 was expressed by a small tumor cell subpopulation of only one myeloma patient. The individual pattern of surface marker expression was not associated with a special-type myeloma protein isotype, stage or status of disease, LI or histological classification; however, a correlation between CD56 expression or histological classification and DNA content of the tumor cells was found.