RESUMO
Crescentic glomerulonephritis is a life-threatening renal disease that has been extensively studied by the experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM-GN) model. Although T cells have a significant role in this model, athymic/nude mice and rats still develop severe renal disease. Here we further explored the contribution of intrinsic renal cells in the development of T-cell-independent GN lesions. Anti-GBM-GN was induced in three strains of immune-deficient mice (Rag2-/-, Rag2-/-Il2rg-/-, and Rag2-/-Il2rb-/-) that are devoid of either T/B cells or T/B/NK cells. The Rag2-/-Il2rg-/- or Rag2-/-Il2rb-/- mice harbor an additional deletion of either the common gamma chain (γC) or the interleukin-2 receptor ß subunit (IL-2Rß), respectively, impairing IL-15 signaling in particular. As expected, all these strains developed severe anti-GBM-GN. Additionally, bone marrow replenishment experiments allowed us to deduce a protective role for the glomerular-expressed γC during anti-GBM-GN. Given that IL-15 has been found highly expressed in nephritic kidneys despite the absence of lymphocytes, we then studied this cytokine in vitro on primary cultured podocytes from immune-deficient mice (Rag2-/-Il2rg-/- and Rag2-/-Il2rb-/-) compared to controls. IL-15 induced downstream activation of JAK1/3 and SYK in primary cultured podocytes. IL-15-dependent JAK/SYK induction was impaired in the absence of γC or IL-2Rß. We found γC largely induced on podocytes during human glomerulonephritis. Thus, renal lesions are indeed modulated by intrinsic glomerular cells through the γC/IL-2Rß receptor response, to date classically described only in immune cells.
Assuntos
Proteínas de Ligação a DNA/imunologia , Glomerulonefrite/imunologia , Subunidade gama Comum de Receptores de Interleucina/imunologia , Subunidade beta de Receptor de Interleucina-2/imunologia , Glomérulos Renais/imunologia , Podócitos/imunologia , Animais , Autoanticorpos/toxicidade , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-15/imunologia , Interleucina-15/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/metabolismo , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Células Matadoras Naturais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Podócitos/metabolismo , Cultura Primária de Células , Transdução de Sinais , Quinase Syk/metabolismoRESUMO
Because genetic background plays a pivotal role in humans and in various experimental models, we carefully monitored its impact on glomerular pathological characteristics during experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM-GN), using two leading mouse strains, 129S2/SvPas (129Sv) and C57bl/6J (B6J). These mice exhibited different severities of renal failure, hypertension, and glomerular lesions, according to their genetic background. In addition to the classic glomerular proliferative lesions, glomerular thrombotic microangiopathy (TMA) was found as a common genetic background-dependent histopathological hallmark of anti-GBM-GN, combined with hemolytic anemia and thrombocytopenia. Glomerular expression profiling, using microarrays and Western blot analysis in B6J TMA-resistant and 129Sv TMA-prone mice, demonstrated major differences in vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) 2 pathways, despite similar Vegfa expression levels. Further analysis revealed a lower basal glomerular endothelial Vegfr2 expression level in 129Sv TMA-prone mice compared with B6J TMA-resistant mice. This difference was even more pronounced during anti-GBM-GN, explaining why an exogenous VEGFA supply failed to rescue any 129Sv TMA lesions. Conversely, the systemic blocking of Vegfr2 amplified TMA lesions only in B6J mice. Herein, we specified the role that genetic background plays in determining, in particular, the level of Vegfr2 expression. We also demonstrated that glomerular Vegfr2-dependent TMA lesions are an underevaluated common hallmark of anti-GBM-GN in mice.
Assuntos
Doença Antimembrana Basal Glomerular/genética , Doença Antimembrana Basal Glomerular/patologia , Transdução de Sinais/fisiologia , Microangiopatias Trombóticas/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Doença Antimembrana Basal Glomerular/metabolismo , Western Blotting , Modelos Animais de Doenças , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Análise Serial de Tecidos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Circulating levels of soluble forms of urokinase-type plasminogen activator receptor (suPAR) are generally elevated in sera from children and adults with FSGS compared with levels in healthy persons or those with other types of kidney disease. In mice lacking the gene encoding uPAR, forced increases in suPAR concentration result in FSGS-like glomerular lesions and proteinuria. However, whether overexpression of suPAR, per se, contributes to the pathogenesis of FSGS in humans remains controversial. We conducted an independent set of animal experiments in which two different and well characterized forms of recombinant suPAR produced by eukaryotic cells were administered over the short or long term to wild-type (WT) mice. In accordance with the previous study, the delivered suPARs are deposited in the glomeruli. However, such deposition of either form of suPAR in the kidney did not result in increased glomerular proteinuria or altered podocyte architecture. Our findings suggest that glomerular deposits of suPAR caused by elevated plasma levels are not sufficient to engender albuminuria.
Assuntos
Nefrite/etiologia , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/etiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Nefrite/metabolismo , Nefrite/patologia , Proteinúria/metabolismo , Proteinúria/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Proteínas Recombinantes/administração & dosagemRESUMO
Toll-like receptors (TLRs) play a crucial role in the innate and adaptive immune responses against microbial infection, tissue injury and cancer. Ligands of TLR9 have been developed as therapy in non-small-cell lung carcinoma (NSCLC). However, phase III clinical trials in metastatic NSCLC were negative. Our objective was to determine whether TLR9 affects tumor growth. We generated a mouse model of lung adenocarcinoma (ADC) mutated for K-ras (K-ras(LA1) ), with and without TLR9 inactivation (K-ras(LA1) TLR9(-/-) and K-ras(LA1) TLR9(+/+) , respectively). TLR9 was functionally expressed only in mononuclear cells of K-ras(LA1) TLR9(+/+) mice. These mice had significantly worse survival and a higher tumor burden than K-ras(LA1) TLR9(-/-) mice. Lung tumors were analyzed for 24 cytokines/growth factors using Bio-Plex multiplex bead-based assays. Factor VIII was assessed by immunochemistry. Tumors from K-ras(LA1) TLR9(+/+) mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K-ras(LA1) TLR9(-/-) mice. LKR13 cells, an ADC cell line derived from K-ras(LA1) mice, were subcutaneously injected into TLR9(-/-) and TLR9(+/+) mice. Syngeneic tumors regressed in TLR9(-/-) but not in TLR9(+/+) mice. Peripheral blood mononuclear cells from TLR9(-/-) mice released less VEGF than those from TLR9(+/+) mice. In 61 patients with early-stage NSCLC, TLR9 was expressed in mononuclear cells that infiltrated tumors, as assessed by immunochemistry, and contributed to worse survival. Our results suggest that TLR9 expression in mononuclear cells was associated with an angiogenic phenotype and promoted lung cancer progression. These findings may aid clinical development of TLR9 ligands to treat cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/mortalidade , Inflamação/imunologia , Leucócitos Mononucleares/patologia , Neoplasias Pulmonares/mortalidade , Neovascularização Patológica , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Receptor Toll-Like 9/fisiologia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Animais , Apoptose , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Carga Tumoral , Fator A de Crescimento do Endotélio VascularRESUMO
Excessive recruitment of monocytes and progression of fibrosis are hallmarks of chronic kidney disease (CKD). Recently we reported that the expression of connexin 43 (Cx43) was upregulated in the kidney during experimental nephropathy. To investigate the role of Cx43 in the progression of CKD, we interbred RenTg mice, a genetic model of hypertension-induced CKD, with Cx43+/- mice. The renal cortex of 5-month-old RenTgCx43+/- mice showed a marked decrease of cell adhesion markers leading to reduced monocyte infiltration and interstitial renal fibrosis compared with their littermates. In addition, functional and histological parameters such as albuminuria and glomerulosclerosis were ameliorated in RenTgCx43+/- mice. Interestingly, treatment with Cx43 antisense produced remarkable improvement of renal function and structure in 1-year-old RenTg mice. Similar results were found in Cx43+/- or wild-type mice treated with Cx43 antisense after obstructive nephropathy. Furthermore, in these mice, Cx43 antisense attenuated E-cadherin downregulation and phosphorylation of the transcription factor Sp1 by the ERK pathway resulting in decreased transcription of type I collagen gene. Interestingly, Cx43-specific blocking peptide inhibited monocyte adhesion in activated endothelium and profibrotic pathways in tubular cells. Cx43 was highly increased in biopsies of patients with CKD. Thus, Cx43 may represent a new therapeutic target against the progression of CKD.
Assuntos
Conexina 43/genética , Oligonucleotídeos Antissenso/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Albuminúria/tratamento farmacológico , Albuminúria/genética , Animais , Caderinas/metabolismo , Adesão Celular , Colágeno Tipo I/genética , Conexina 43/análise , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Fibrose , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/genética , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Monócitos/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Fosforilação/efeitos dos fármacos , Insuficiência Renal Crônica/genética , Fator de Transcrição Sp1/metabolismo , Transcrição GênicaRESUMO
Although cells of the myeloid lineages, including tissue macrophages and conventional dendritic cells, were rapidly recognized, in addition to CD4+ T lymphocytes, as target cells of HIV-1, their specific roles in the pathophysiology of infection were initially largely neglected. However, numerous studies performed over the past decade, both in vitro in cell culture systems and in vivo in monkey and humanized mouse animal models, led to growing evidence that macrophages play important direct and indirect roles as HIV-1 target cells and in pathogenesis. It has been recently proposed that macrophages are likely involved in all stages of HIV-1 pathogenesis, including virus transmission and dissemination, but above all, in viral persistence through the establishment, together with latently infected CD4+ T cells, of virus reservoirs in many host tissues, the major obstacle to virus eradication in people living with HIV. Infected macrophages are indeed found, very often as multinucleated giant cells expressing viral antigens, in almost all lymphoid and non-lymphoid tissues of HIV-1-infected patients, where they can probably persist for long period of time. In addition, macrophages also likely participate, directly as HIV-1 targets or indirectly as key regulators of innate immunity and inflammation, in the chronic inflammation and associated clinical disorders observed in people living with HIV, even in patients receiving effective antiretroviral therapy. The main objective of this review is therefore to summarize the recent findings, and also to revisit older data, regarding the critical functions of tissue macrophages in the pathophysiology of HIV-1 infection, both as major HIV-1-infected target cells likely found in almost all tissues, as well as regulators of innate immunity and inflammation during the different stages of HIV-1 pathogenesis.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Vírus da Imunodeficiência Símia , Humanos , Animais , Camundongos , Macrófagos , HIV-1/fisiologia , Inflamação , Linfócitos T CD4-Positivos , Latência Viral , Replicação ViralRESUMO
BACKGROUND: The maturation cocktail composed of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α and prostaglandin E2 is considered the "gold standard" for inducing the maturation of dendritic cells (DCs) for use in cancer immunotherapy. Nevertheless, although this maturation cocktail induces increased expression of several activation markers, such as CD83, the co-stimulation molecules CD80, CD86 and CD40 and the chemokine receptor involved in DC homing in lymph nodes CCR7, the DC immune stimulatory function in vivo contrasts with this mature phenotype, and good clinical outcomes in patients with cancer treated with DC-based vaccines remain rare. METHODS: Phenotypic characterization of the immunosuppressive status of DCs differentiated from peripheral blood mononuclear cells of healthy volunteers and matured with the "gold standard" cocktail was performed. Glucocorticoid-induced leucine zipper (GILZ) targeting small interfering RNA (siRNA) was electroporated into DCs after maturation to increase their immunogenicity. RESULTS: The mature phenotype of DCs treated for 48 h with this cocktail was associated with the expression of several immunosuppressive regulators, including programmed cell death 1 ligand 1 (PD-L1), IL-10 and GILZ. Electroporation is a very efficient and safe way to deliver siRNA into DCs (80% of DCs receive at least one molecule of siRNA). Silencing GILZ in clinical-grade DCs by siRNA leads to a decrease of the PD-L1 expression associated with an increase in their IL-12 secretion and T-cell induction capability. CONCLUSIONS: GILZ silencing is a promising approach to achieving complete clinical-grade DC maturation and avoiding the immunosuppressive effects of the maturation cocktail on DCs intended for clinical use.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Glucocorticoides/genética , Zíper de Leucina/genética , Diferenciação Celular/imunologia , Células Dendríticas/metabolismo , Dinoprostona/administração & dosagem , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucocorticoides/metabolismo , Interleucina-1beta/administração & dosagem , Interleucina-6/administração & dosagem , Zíper de Leucina/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , RNA Interferente Pequeno/genética , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Semen is an important vector for sexual HIV-1 transmission. Although CXCR4-tropic (X4) HIV-1 may be present in semen, almost exclusively CCR5-tropic (R5) HIV-1 causes systemic infection after sexual intercourse. To identify factors that may limit sexual X4-HIV-1 transmission, we generated a seminal fluid-derived compound library and screened it for antiviral agents. We identified four adjacent fractions that blocked X4-HIV-1 but not R5-HIV-1 and found that they all contained spermine and spermidine, abundant polyamines in semen. We showed that spermine, which is present in semen at concentrations up to 14 mM, binds CXCR4 and selectively inhibits cell-free and cell-associated X4-HIV-1 infection of cell lines and primary target cells at micromolar concentrations. Our findings suggest that seminal spermine restricts sexual X4-HIV-1 transmission.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Espermidina/farmacologia , Espermina/farmacologia , Infecções por HIV/tratamento farmacológico , Linhagem Celular , Receptores CXCR4RESUMO
During human glomerulonephritis, the severity of injuries correlates with glomerular fibrin deposits, which are tightly regulated by the intraglomerular fibrinolytic system. Here, we evaluated the role of vitronectin (VTN; also known as complement S protein), the principal cofactor of the plasminogen activator inhibitor-1 (PAI-1), in a mouse model of acute glomerulonephritis. We found that in mice subjected to nephrotoxic serum, the absence of VTN resulted in a lower glomerular PAI-1 activity and a higher glomerular fibrinolytic activity. Challenged VTN(-/-) mice displayed significantly less fibrin deposits, proteinuria, and renal failure than their wild-type counterparts. Notably, this protective effect afforded by VTN deficiency was still observed after a C3 depletion. Finally, the injection of VTN(+/+) serum in VTN(-/-) mice induced the glomerular deposition of VTN, increased PAI-1 deposition, decreased glomerular fibrinolytic activity, and aggravated glomerular injury. As in mice, abundant glomerular VTN deposits were also observed in patients with severe glomerulonephritis. Here, we show that plasma-exchange therapy, admittedly beneficial in this clinical context, induces a significant depletion in circulating VTN, which might modulate PAI-1 activity locally and accelerate the clearance of fibrin deposits in the glomeruli. Collectively, these results demonstrate that VTN exerts a deleterious role independently from complement, by directing PAI-dependent fibrinolysis in the glomerular compartment.
Assuntos
Fibrinólise/fisiologia , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Vitronectina/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Membrana Basal Glomerular/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/toxicidade , Camundongos , Camundongos Knockout , Insuficiência Renal/metabolismo , Serpina E2/genética , Serpina E2/metabolismo , Vitronectina/genéticaRESUMO
Dendritic cells (DCs), essential for the initiation and regulation of adaptive immune responses, have been used as anticancer vaccines. DCs may also directly trigger tumor cell death. In the current study, we have investigated the tumoricidal and immunostimulatory activities of mouse bone marrow-derived DCs. Our results indicate that these cells acquire killing capabilities toward tumor cells only when activated with LPS or Pam3Cys-SK4. Using different transgenic mouse models including inducible NO synthase or GP91 knockout mice, we have further established that LPS- or Pam3Cys-SK4-activated DC killing activity involves peroxynitrites. Importantly, after killing of cancer cells, DCs are capable of engulfing dead tumor cell fragments and of presenting tumor Ags to specific T lymphocytes. Thus, upon specific stimulation, mouse bone marrow-derived DCs can directly kill tumor cells through a novel peroxynitrite-dependent mechanism and participate at virtually all levels of antitumor immune responses, which reinforces their interest in immunotherapy.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Ácido Peroxinitroso/toxicidade , Animais , Morte Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HeLa , Humanos , Melanoma Experimental , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Experimentais/metabolismoRESUMO
Dendritic cells (DC) are professional antigen-presenting cells currently being used as a cellular adjuvant in cancer immunotherapy strategies. Unfortunately, DC-based vaccines have not demonstrated spectacular clinical results. DC loading with tumor antigens and DC differentiation and activation still require optimization. An alternative technique for providing antigens to DC consists of the direct fusion of dendritic cells with tumor cells. These resulting hybrid cells may express both major histocompatibility complex (MHC) class I and II molecules associated with tumor antigens and the appropriate co-stimulatory molecules required for T-cell activation. Initially tested in animal models, this approach has now been evaluated in clinical trials, although with limited success. We summarize and discuss the results from the animal studies and first clinical trials. We also present a new approach to inducing hybrid formation by expression of viral fusogenic membrane glycoproteins.
Assuntos
Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Células Híbridas , Imunoterapia/tendências , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismoRESUMO
Clinicians are well aware of existing pharmacologically-induced immune deficient status in kidney-transplanted patients that will favor their susceptibility to bacterial or viral infections. Previous studies indicated that advanced Stage 4-5 Chronic Kidney Disease might also be regarded as an immune deficiency-like status as well, even though the mechanisms are not fully understood. Here, we analyzed the ex vivo frequency and the functional properties of both conventional and innate-like T (ILT) lymphocyte subsets in the peripheral blood of 35 patients on hemodialysis, 29 kidney transplanted patients and 38 healthy donors. We found that peripheral blood cell count of ILT cells, as iNKT (invariant Natural Killer T) and MAIT (mucosal-associated invariant T), were significantly decreased in hemodialyzed patients compared to healthy controls. This deficiency was also observed regarding conventional T cells, including the IL-17-producing CD4(+) Th17 cells. Pertaining to regulatory T cells, we also noticed major modifications in the global frequency of CD4(+)CD25(+)Foxp3(+) T lymphocytes, including the resting suppressive CD45RA(+)Foxp3lo and activated suppressive CD45RA-Foxp3hi T cell subpopulations. We found no significant differences between the immune status of hemodialyzed and kidney-transplanted subjects. In conclusion, we demonstrated that both ILT and conventional T cell numbers are equally impaired in hemodialyzed and kidney-transplanted patients.
Assuntos
Imunidade Inata , Transplante de Rim , Diálise Renal , Subpopulações de Linfócitos T/imunologia , Adulto , Citocinas/metabolismo , Feminino , Humanos , Hospedeiro Imunocomprometido , Imunofenotipagem , Falência Renal Crônica/etiologia , Falência Renal Crônica/imunologia , Falência Renal Crônica/terapia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Fenótipo , Subpopulações de Linfócitos T/metabolismo , Adulto JovemRESUMO
Cancer chemotherapy can induce tumor regression followed, in many cases, by relapse in the long-term. Thus this study was performed to assess the determinants of such phenomenon using an in vivo cancer model and in vitro approaches. When animals bearing an established tumor are treated by cisplatin, the tumor initially undergoes a dramatic shrinkage and is characterized by giant tumor cells that do not proliferate but maintain DNA synthesis. After several weeks of latency, the tumor resumes its progression and consists of small proliferating cells. Similarly, when tumor cells are exposed in vitro to pharmacological concentrations of cisplatin, mitotic activity stops initially but cells maintain DNA duplication. This DNA endoreduplication generates giant polyploid cells that then initiate abortive mitoses and can die through mitotic catastrophe. However, many polyploid cells survive for weeks as non-proliferating mono- or multi-nucleated giant cells which acquire a senescence phenotype. Prolonged observation of these cells sheds light on the delayed emergence of a limited number of extensive colonies which originate from polyploid cells, as demonstrated by cell sorting analysis. Theses colonies are made of small diploid cells which differ from parental cells by stereotyped chromosomal aberrations and an increased resistance to cytotoxic drugs. These data suggest that a multistep pathway, including DNA endoreduplication, polyploidy, then depolyploidization and generation of clonogenic escape cells can account for tumor relapse after initial efficient chemotherapy.
Assuntos
Cisplatino/farmacologia , Neoplasias do Colo/patologia , Dano ao DNA , Duplicação Gênica/efeitos dos fármacos , Poliploidia , Evasão Tumoral/efeitos dos fármacos , Animais , Bromodesoxiuridina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Diploide , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Fenótipo , Ratos , Fatores de TempoRESUMO
Dendritic cells (DCs) are well known for their capacity to induce adaptive antitumor immune response through Ag presentation and tumor-specific T cell activation. Recent findings reveal that besides this role, DCs may display additional antitumor effects. In this study, we provide evidence that LPS- or IFN-gamma-activated rat bone marrow-derived dendritic cells (BMDCs) display killing properties against tumor cells. These cytotoxic BMDCs exhibit a mature DC phenotype, produce high amounts of IL-12, IL-6, and TNF-alpha, and retain their phagocytic properties. BMDC-mediated tumor cell killing requires cell-cell contact and depends on NO production, but not on perforin/granzyme or on death receptors. Furthermore, dead tumor cells do not exhibit characteristics of apoptosis. Thus, intratumoral LPS injections induce an increase of inducible NO synthase expression in tumor-infiltrating DCs associated with a significant arrest of tumor growth. Altogether, these results suggest that LPS-activated BMDCs represent powerful tumoricidal cells which enforce their potential as anticancer cellular vaccines.
Assuntos
Morte Celular/fisiologia , Citotoxicidade Imunológica , Células Dendríticas/metabolismo , Neoplasias/imunologia , Óxido Nítrico/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Western Blotting , Células da Medula Óssea/imunologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Humanos , Lipopolissacarídeos/farmacologia , RatosRESUMO
Tumor necrosis factor (TNF) antagonists represent a milestone in the therapy of autoimmune conditions. Anti-TNF antibodies have been approved for clinical use and during the last eight years thousands of patients have been treated. However, the long-term sequelae of anti-TNF agents in promoting carcinogenesis remain unclear. This study sought to define the role of intra-tumor TNF-alpha production on cancer cell progression and to determine whether TNF-alpha antibodies can suppress anti-tumoral immunity. Using an experimental animal tumor model we demonstrate that anti-TNF-alpha antibodies hinder anti-tumor immune responses and promote growth of immunogenic rat colon tumors (REG) that are always rejected by immunocompetent untreated rats. The major role of TNF-alpha in the anti-tumoral immune response was confirmed by transfecting progressive and tolerogenic rat colon tumor cells (PRO) with the TNF-alpha gene. PRO tumor cells secreting TNF-alpha induce tumor-infiltrating dendritic cell (DC) activation. This triggers a potent immune response leading to tumor rejection and long-lasting immunity. Therefore, the prominent role of TNF-alpha in anti-tumoral immune responses underscores the need for caution and close surveillance following the administration of TNF inhibitors.
Assuntos
Anticorpos Bloqueadores/efeitos adversos , Neoplasias do Colo/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Bloqueadores/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Tolerância Imunológica , Neovascularização Patológica/imunologia , Ratos , Ratos Nus , Linfócitos T/imunologiaRESUMO
The identification of the most efficient strategy for tumor antigen loading of dendritic cells (DCs) remains a challenge in cancer immunotherapy protocols. Autologous dead tumor cells have been demonstrated to constitute an acceptable source of multiple tumor-associated antigens (TAA) to pulse DCs. However the optimal approach for inducing cell death that would lead to effective endocytosis and activation of DCs remains controversial. In this study we have induced and defined 3 distinct mechanisms of tumor cell death (apoptosis, necrosis and fusion-mediated cell death), and investigated their differential effects on DCs. Bone marrow-derived DCs demonstrated comparable uptake of primary apoptotic, necrotic, or fused dead tumor cells. Furthermore, the distinct modes of cancer cell death had analogous potential in activating the transcription factors NF-kappaB and STAT1 and in maturing DCs, resulting in an equally effective stimulation of immune T cells. The current study therefore provides further informations on the use of dead whole tumor cells as antigen sources for effective active anti-cancer immunotherapy.
Assuntos
Apresentação de Antígeno/imunologia , Apoptose/imunologia , Fusão Celular , Células Dendríticas/imunologia , Necrose/imunologia , Animais , Neoplasias do Colo/imunologia , Interleucina-12/biossíntese , Ativação Linfocitária , NF-kappa B/metabolismo , Fenótipo , Ratos , Fator de Transcrição STAT1/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Células Tumorais CultivadasRESUMO
We investigated the mechanisms of immune tolerance raised by tumors by comparing immunogenic and tolerogenic tumor cell clones isolated from a rat colon carcinoma. When injected into syngeneichosts, the immunogenic REGb cells yield tumors that are rejected, while the tolerogenic PROb cells yield progressive tumors and inhibit the regression of REGb tumors. We show here that PROb tumor volume is correlated with an expansion of CD4(+)CD25(+) regulatory T lymphocytes in lymphoid tissues. These cells delay in vivo the rejection of REGb tumors and inhibit in vitro T cell-mediated immune responses against REGb cells through a mechanism that requires cell contact between effector and regulatory T cells and involves TGF-beta. While total T cells fromPROb tumor-bearing rats yield no apparent anti-tumor immune response, depletion of CD25(+) T cells restores this reactivity. A single administration of cyclophosphamide depletes CD4(+)CD25(+) T cells in PROb tumor-bearing animals, delays the growth of PROb tumors, and cures rats bearing established PROb tumors when followed by an immunotherapy which has no curative effect when administered alone. These results demonstrate the role of CD4(+)CD25(+) regulatory T cells in tumor-induced immune tolerance and the interest of regulatory T cell depletion to sensitize established tumors to immunotherapy.