RESUMO
BACKGROUND: Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP3) is currently being tested in phase II clinical trials in premature infants to prevent bronchopulmonary dysplasia, but its impact on the neonatal intestine remains unclear. The aim of this study was to determine whether rhIGF-1/BP3 protects against necrotizing enterocolitis (NEC) in mice and to investigate the mechanisms involved. METHODS: Neonatal mice were dam fed or injected intraperitoneally with rhIGF-1/BP3 (or vehicle) and submitted to an experimental NEC model. Serum IGF-1 was assessed by ELISA and intestinal vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression by Western blot. Intestinal endothelial cell proliferation, and enterocyte proliferation and migration were examined by immunofluorescence. Pup survival and histological intestinal injury were determined. RESULTS: In pups exposed to experimental NEC, serum IBP3-bound IGF-1 level was decreased. Exogenous rhIGF-1/BP3 preserved VEGF and VEGFR2 protein expression, decreased vascular permeability, and preserved endothelial cell proliferation in the small intestine. Furthermore, rhIGF-1/BP3 promoted enterocyte proliferation and migration, which effects were attenuated by inhibiting VEGFR2 signaling, decreased enterocyte apoptosis and decreased systemic and intestinal inflammation. rhIGF-1/BP3 improved survival and reduced the incidence of severe intestinal injury in experimental NEC. CONCLUSIONS: Exogenous rhIGF-1/BP3 protects neonatal mice against experimental NEC via multiple mechanisms. IMPACT: Exogenous rhIGF-1/BP3 preserves intestinal microvascular development and integrity, promotes enterocyte proliferation and migration, decreases local and systemic inflammation, and protects neonatal mice against NEC. The article adds pre-clinical evidence of a protective role for rhIGF-1/BP3 on the premature gut. It provides evidence supporting the use of rhIGF1/BP3 in premature neonates to protect against NEC.
Assuntos
Animais Recém-Nascidos , Proliferação de Células , Modelos Animais de Doenças , Enterocolite Necrosante , Fator de Crescimento Insulin-Like I , Proteínas Recombinantes , Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Animais , Enterocolite Necrosante/prevenção & controle , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Camundongos , Fator de Crescimento Insulin-Like I/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Enterócitos/metabolismo , Humanos , Intestinos/patologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos Endogâmicos C57BL , Movimento Celular , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células Endoteliais/metabolismo , FemininoRESUMO
OBJECTIVE: To test the hypothesis that nailfold capillaroscopy can noninvasively detect dysregulated retinal angiogenesis and predict retinopathy of prematurity (ROP) in infants born premature before its development. METHODS: In a cohort of 32 infants born <33 weeks of gestation, 1386 nailfold capillary network images of the 3 middle fingers of each hand were taken during the first month of life. From these, 25 infants had paired data taken 2 weeks apart during the first month of life. Images were analyzed for metrics of peripheral microvascular density using a machine learning-based segmentation approach and a previously validated microvascular quantification platform (REAVER vascular analysis). Results were correlated with subsequent development of ROP based on a published consensus ROP severity scale. RESULTS: In total, 18 of 32 (56%) (entire cohort) and 13 of 25 (52%) (2-time point subgroup) developed ROP. Peripheral vascular density decreased significantly during the first month of life. In the paired time point analysis, vessel length density, a key metric of peripheral vascular density, was significantly greater at both time points among infants who later developed ROP (15â563 and 11â996 µm/mm2, respectively) compared with infants who did not (12â252 and 8845 µm/mm2, respectively) (P < .001, both time points). A vessel length density cutoff of >15â100 at T1 or at T2 correctly detected 3 of 3 infants requiring ROP therapy. In a mixed-effects linear regression model, peripheral vascular density metrics were significantly correlated with ROP severity. CONCLUSIONS: Nailfold microvascular density assessed during the first month of life is a promising, noninvasive biomarker to identify premature infants at highest risk for ROP before detection on eye exam.
Assuntos
Retinopatia da Prematuridade , Recém-Nascido , Lactente , Humanos , Retinopatia da Prematuridade/diagnóstico , Retinopatia da Prematuridade/terapia , Angioscopia Microscópica , Recém-Nascido Prematuro , Retina , Idade Gestacional , Fatores de RiscoRESUMO
Necrotizing enterocolitis (NEC) is a deadly bowel necrotic disease of premature infants. Low levels of plasma IGF-1 predispose premature infants to NEC. While increasing evidence suggests that defective perinatal intestinal microvascular development plays a role in NEC, the involved mechanism remains incompletely understood. We report here that serum and intestinal IGF-1 are developmentally regulated during the perinatal period in mice and decrease during experimental NEC. Neonatal intestinal macrophages produce IGF-1 and promote endothelial cell sprouting in vitro via IGF-1 signaling. In vivo, in the neonatal intestine, macrophage-derived IGF-1 promotes VEGF expression and endothelial cell proliferation and protects against experimental NEC. Exogenous IGF-1 preserves intestinal microvascular density and protects against experimental NEC. In human NEC tissues, villous endothelial cell proliferation and IGF-1- producing macrophages are decreased compared to controls. Together, our results suggest that defective IGF-1-production by neonatal macrophages impairs neonatal intestinal microvascular development and predisposes the intestine to necrotizing enterocolitis.
Assuntos
Enterocolite Necrosante , Animais , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/prevenção & controle , Feminino , Humanos , Recém-Nascido , Fator de Crescimento Insulin-Like I/metabolismo , Intestinos , Macrófagos/metabolismo , Camundongos , Gravidez , Transdução de SinaisRESUMO
Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17. Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development.
Assuntos
Enterocolite Necrosante/metabolismo , Inflamação/metabolismo , Intestino Delgado/irrigação sanguínea , Microvasos/metabolismo , Neovascularização Fisiológica , Efeitos Tardios da Exposição Pré-Natal , Fator de Necrose Tumoral alfa/metabolismo , Animais , Permeabilidade Capilar , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/patologia , Enterocolite Necrosante/fisiopatologia , Feminino , Idade Gestacional , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/complicações , Inflamação/patologia , Inflamação/fisiopatologia , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Microvasos/fisiopatologia , Gravidez , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants with intestinal inflammation and necrosis. The neonatal intestinal inflammatory response is rich in macrophages, and blood monocyte count is low in human NEC. We previously found that NF-κB mediates the intestinal injury in experimental NEC. However, the role of NF-κB in myeloid cells during NEC remains unclear. Herein, inhibitor of kappaB kinase ß (IKKß), a critical kinase mediating NF-κB activation, was deleted in lysozyme M (Lysm)-expressing cells, which were found to be Cd11b+Ly6c+ monocytes but not Cd11b+Ly6c- macrophages in the dam-fed neonatal mouse intestine. NEC induced differentiation of monocytes into intestinal macrophages and up-regulation of monocyte recruitment genes (eg, L-selectin) in the macrophage compartment in wild-type mice, but not in pups with IKKß deletion in Lysm+ cells. Thus, NF-κB is required for NEC-induced monocyte activation, recruitment, and differentiation in neonatal intestines. Furthermore, pups with Lysm-IKKß deletion had improved survival and decreased incidence of severe NEC compared with littermate controls. Decreased NEC severity was not associated with an improved intestinal barrier. In contrast, NEC was unabated in mice with IKKß deletion in intestinal epithelial cells. Together, these data suggest that recruitment of Ly6c+ monocytes into the intestine, NF-κB activation in these cells, and differentiation of Ly6c+ monocytes into macrophages are critical cellular and molecular events in NEC development to promote disease.
Assuntos
Antígenos Ly/metabolismo , Enterocolite Necrosante/metabolismo , Células Epiteliais/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Animais , Antígenos Ly/genética , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Células Epiteliais/patologia , Deleção de Genes , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Selectina L/genética , Selectina L/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Regulação para CimaRESUMO
Necrotizing enterocolitis (NEC), a leading cause of morbidity and mortality in preterm neonates, is a devastating disease characterized by intestinal tissue inflammation and necrosis. NEC pathogenesis is multifactorial but remains unclear. Translocation of bacteria and/or bacterial products across a weak intestinal barrier in the setting of impaired mucosal immunity leads to an exaggerated inflammatory response and secondary mucosal epithelial injury. In addition to prematurity, other risk factors for NEC include congenital heart disease, maternal pre-eclampsia with placental vascular insufficiency, severe anemia and blood transfusion - all conditions that predispose the intestine to ischemia. We recently found that maldevelopment of the intestinal microvasculature plays an important role in NEC pathogenesis. Here we review the evidence supporting a role for defective development of the intestinal mucosal microvasculature and perturbations of intestinal blood flow in NEC, emphasizing the importance of vascular endothelial growth factor (VEGF) and the VEGF receptor-2 signaling pathway.
Assuntos
Enterocolite Necrosante/metabolismo , Mucosa Intestinal/irrigação sanguínea , Intestinos/irrigação sanguínea , Microcirculação/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Humanos , Recém-Nascido , Recém-Nascido PrematuroRESUMO
BackgroundNecrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by intestinal necrosis. Hypoxia-inducible factor-1α (HIF-1α) has a critical role in cellular oxygen homeostasis. Here, we hypothesized that prolyl hydroxylase (PHD) inhibition, which stabilizes HIF-1α, protects against NEC by promoting intestinal endothelial cell proliferation and improving intestinal microvascular integrity via vascular endothelial growth factor (VEGF) signaling.MethodsTo assess the role of PHD inhibition in a neonatal mouse NEC model, we administered dimethyloxalylglycine (DMOG) or vehicle to pups before or during the NEC protocol, and determined mortality and incidence of severe intestinal injury. We assessed intestinal VEGF by western blot analysis and quantified endothelial cell and epithelial cell proliferation following immunofluorescence.ResultsDMOG decreased mortality and incidence of severe NEC, increased intestinal VEGF expression, and increased intestinal villus endothelial and epithelial cell proliferation in experimental NEC. Inhibiting VEGFR2 signaling eliminated DMOG's protective effect on intestinal injury severity, survival, and endothelial cell proliferation while sparing DMOG's protective effect on intestinal epithelial cell proliferation.ConclusionDMOG upregulates intestinal VEGF, promotes endothelial cell proliferation, and protects against intestinal injury and mortality in experimental NEC in a VEGFR2 dependent manner. DMOG's protective effect on the neonatal intestinal mucosa may be mediated via VEGFR2 dependent improvement of the intestinal microvasculature.
Assuntos
Aminoácidos Dicarboxílicos/farmacologia , Enterocolite Necrosante/patologia , Intestinos/patologia , Microcirculação , Animais , Animais Recém-Nascidos , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Intestinos/irrigação sanguínea , Intestinos/lesões , Camundongos , Camundongos Endogâmicos C57BL , Prolil Hidroxilases/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The pathogenesis of necrotizing enterocolitis (NEC), a common gastrointestinal disease affecting premature infants, remains poorly understood. We previously found that intestinal VEGF-A expression is decreased in human NEC samples and in a neonatal mouse NEC model prior to detectable histological injury. Therefore, we hypothesized that lack of VEGF receptor 2 (VEGFR2) signaling facilitates neonatal intestinal injury by impairing intestinal microvasculature development. Here, we found that intestinal VEGF-A and its receptor, VEGFR2, were highly expressed at the end of fetal life and significantly decreased after birth in mice. Furthermore, selective inhibition of VEGFR2 kinase activity and exposure to a neonatal NEC protocol significantly decreased the density of the intestinal microvascular network, which was further reduced when both interventions were provided together. Furthermore, VEGFR2 inhibition resulted in greater mortality and incidence of severe injury in pups submitted to the NEC model. The percentage of lamina propria endothelial cells was decreased during NEC induction, and further decreased when VEGFR2 signaling was inhibited. This was associated with decreased endothelial cell proliferation rather than apoptosis. In conclusion, we found that VEGF-A and VEGFR2 proteins are highly expressed in the intestine before birth, and are significantly downregulated in the immediate neonatal period. Furthermore, VEGFR2 signaling is necessary to maintain the integrity of the intestinal mucosal microvasculature during the postnatal period and lack of VEGFR2 signaling predisposes to NEC in neonatal mice.
Assuntos
Enterocolite Necrosante/metabolismo , Mucosa Intestinal/metabolismo , Microvasos/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Células Cultivadas , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/crescimento & desenvolvimento , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Decreased intestinal perfusion may contribute to the development of necrotizing enterocolitis (NEC). Vascular endothelial growth factor (VEGF) is an angiogenic protein necessary for the development and maintenance of capillary networks. Whether VEGF is dysregulated in NEC remains unknown. OBJECTIVES: The objective of this study was to determine whether intestinal VEGF expression is altered in a neonatal mouse model of NEC and in human NEC patients. METHODS: We first assessed changes of intestinal VEGF mRNA and protein in a neonatal mouse NEC model before significant injury occurs. We then examined whether exposure to formula feeding, bacterial inoculation, cold stress and/or intermittent hypoxia affected intestinal VEGF expression. Last, we visualized VEGF protein in intestinal tissues of murine and human NEC and control cases by immunohistochemistry. RESULTS: Intestinal VEGF protein and mRNA were significantly decreased in pups exposed to the NEC protocol compared to controls. Hypoxia, cold stress and commensal bacteria, when administered together, significantly downregulated intestinal VEGF expression, while they had no significant effect when given alone. VEGF was localized to a few single intestinal epithelial cells and some cells of the lamina propria and myenteric plexus. VEGF staining was decreased in murine and human NEC intestines when compared to control tissues. CONCLUSION: Intestinal VEGF protein is reduced in human and experimental NEC. Decreased VEGF production might contribute to NEC pathogenesis.
Assuntos
Enterocolite Necrosante/genética , Mucosa Intestinal/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Resposta ao Choque Frio , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Hipóxia/fisiopatologia , Imuno-Histoquímica , Lactente , Recém-Nascido , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The goal of the current study is to determine the effects of blocking phosphatidylserine (PS) on the growth of neuroblastoma in mice. PS, an anionic phospholipid restricted to the cytoplasmic surface of plasma membranes in most cells, is externalized to the surface of apoptotic cells. PS has been shown to induce immune tolerance to self-antigens. PS can also be found on the surface of live cells and in particular tumor cells. Annexin-V (AnV) is a protein that specifically binds and blocks PS. To determine the effects of blocking PS with AnV on tumor growth and immunogenicity, mice were inoculated with AGN2a, a poorly immunogenic murine neuroblastoma that expresses high level of PS on the cell surface. Survival and anti-tumor T cell response were determined. AGN2a were engineered to secrete AnV. Secreted protein effectively blocked tumor PS. 40 % of mice inoculated with AnV-expressing AGN2a cells survived free of tumor, whereas none of the mice inoculated with control cells survived (p = 0.0062). The benefits of AnV were lost when mice were depleted of T cells. The findings suggest that AnV could protect mice from tumor challenge through an immune mediated mechanism. Mice were then immunized with irradiated AnV-secreting or control cells, and challenged with wild-type AGN2a cells. AnV-secreting cell vaccine protected 80 % of mice from AGN2a challenge, while control cell vaccine prevented tumor growth in only 30 % of animals (p = 0.012). ELISPOT analysis demonstrated that AnV-secreting cell vaccine induced a greater frequency of interferon-gamma producing splenic T cells. T cells isolated from mice immunized with AnV-secreting but not control vaccine lysed AGN2a. In summary, AnV blocked PS, enhanced T cell mediated tumor immunity, and inhibited tumor growth.
Assuntos
Anexina A5/imunologia , Neuroblastoma/imunologia , Neuroblastoma/patologia , Fosfatidilserinas/antagonistas & inibidores , Tolerância a Antígenos Próprios , Animais , Anexina A5/genética , Sobrevivência Celular/imunologia , Imunoterapia , Interferon gama/biossíntese , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos , Neuroblastoma/terapia , Fosfatidilserinas/imunologia , Baço/imunologia , Linfócitos T/imunologiaRESUMO
R-Ras is a member of the RAS superfamily of small GTP-binding proteins. The physiologic function of R-Ras has not been fully elucidated. We found that R-Ras is expressed by lymphoid and nonlymphoid tissues and drastically up-regulated when bone marrow progenitors are induced to differentiate into dendritic cells (DCs). To address the role of R-Ras in DC functions, we generated a R-Ras-deficient mouse strain. We found that tumors induced in Rras(-/-) mice formed with shorter latency and attained greater tumor volumes. This finding has prompted the investigation of a role for R-Ras in the immune system. Indeed, Rras(-/-) mice were impaired in their ability to prime allogeneic and antigen-specific T-cell responses. Rras(-/-) DCs expressed lower levels of surface MHC class II and CD86 in response to lipopolysaccharide compared with wild-type DCs. This was correlated with a reduced phosphorylation of p38 and Akt. Consistently, R-Ras-GTP level was increased within 10 minutes of lipopolysaccharide stimulation. Furthermore, Rras(-/-) DCs have attenuated capacity to spread on fibronectin and form stable immunologic synapses with T cells. Altogether, these findings provide the first demonstration of a role for R-Ras in cell-mediated immunity and further expand on the complexity of small G-protein signaling in DCs.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/genética , Células Dendríticas/fisiologia , Ativação Linfocitária/genética , Proteínas ras/fisiologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Feminino , Imunidade Celular/genética , Imunidade Celular/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Neuroblastoma is a pediatric solid tumor that can be stratified into stroma-rich and stroma-poor histological subgroups. The stromal compartment of neuroblastoma is composed mostly of Schwann cells, and they play critical roles in the differentiation, survival, and angiogenic responses of tumor cells. In certain neuroblastoma cell lines, the coexistence of neuroblastic N-type and substrate-adherent S-type is frequently observed. One such cell line, SK-N-SH, harbors a F1174L oncogenic mutation in the anaplastic lymphoma kinase (ALK) gene. Treatment of SK-N-SH with an ALK chemical inhibitor, TAE684, resulted in the outgrowth of S-type cells that expressed the Schwann cell marker, S100α6. Nucleotide sequencing analysis of these TAE684-resistant (TR) sublines revealed the presence of the ALK F1174L mutation, suggesting their tumor origin, although ALK protein was not detected. Consistent with these findings, TR cells displayed approximately 9-fold higher IC(50) values than N-type cells. Also, unlike N-type cells, TR cells have readily detectable phosphorylated STAT3 but weaker phosphorylated AKT. Under coculture conditions, TR cells conferred survival to N-type cells against the apoptotic effect of TAE684. Cocultivation also greatly enhanced the overall phosphorylation of STAT3 and its transcriptional activity in N-type cells. Finally, conditioned medium from TR clones enhanced cell viability of N-type cells, and this effect was phosphatidylinositol 3-kinase dependent. Taken together, these results demonstrate the ability of tumor-derived S-type cells in protecting N-type cells against the apoptotic effect of an ALK kinase inhibitor through upregulating prosurvival signaling.
RESUMO
A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4⺠T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4⺠T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25⺠Treg cells was similar to that achieved by in vivo depletion of all CD4⺠T cells. Depletion of CD25⺠Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4⺠T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8⺠T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.
Assuntos
Vacinas Anticâncer/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/métodos , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Neuroblastoma/imunologia , Neuroblastoma/terapia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Vacinas Anticâncer/imunologia , Fator 3-gama Nuclear de Hepatócito/imunologia , Camundongos , Linfócitos T/transplante , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplanteRESUMO
In malignancies where no universally expressed dominant Ag exists, the use of tumor cell-based vaccines has been proposed. We have modified a mouse neuroblastoma cell line to express either CD80 (B7.1), CD137L (4-1BBL), or both receptors on the tumor cell surface. Vaccines expressing both induce a strong T cell response that is unique in that among responding CD8 T cells, a T effector memory cell (T(EM)) response arises in which a large number of the T(EM) express the alpha-chain of VLA-2, CD49b. We demonstrate using both in vitro and in vivo assays that the CD49b(+) CD8 T cell population is a far more potent antitumor effector cell population than nonfractionated CD8 or CD49b(-) CD8 T cells and that CD49b on vaccine-induced CD8 T cells mediates invasion of a collagen matrix. In in vivo rechallenge studies, CD49b(+) T cells no longer expanded, indicating that CD49b T(EM) expansion is restricted to the initial response to vaccine. To demonstrate a mechanistic link between the expression of costimulatory molecules on the vaccine and CD49b on responding T cells, we stimulated naive T cells in vitro with artificial APC expressing different combinations of anti-CD3, anti-CD28, and CD137L. Although some mRNA encoding CD49b was induced by combining anti-CD3 with anti-CD28 or CD137L, the highest level was induced when all three signals were present. This indicates that CD49b expression results from additive costimulation and that the level of CD49b message serves as an indicator of the effectiveness of T cell activation by a cell-based vaccine.
Assuntos
Ligante 4-1BB/biossíntese , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Diferenciação Celular/imunologia , Imunoterapia Adotiva , Integrina alfa2/biossíntese , Neuroblastoma/prevenção & controle , Subpopulações de Linfócitos T/imunologia , Ligante 4-1BB/administração & dosagem , Ligante 4-1BB/genética , Ligante 4-1BB/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Vacinas Anticâncer/biossíntese , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Humanos , Integrina alfa2/genética , Células K562 , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos A , Neuroblastoma/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8(+) and CD4(+) T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8(+) T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8(+) T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.
Assuntos
Regulação da Expressão Gênica , Fatores Inibidores da Migração de Macrófagos/metabolismo , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Anexina A5/metabolismo , Vacinas Anticâncer/imunologia , Proliferação de Células , Células Cultivadas , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Transplante de Neoplasias , Ligação Proteica , Interferência de RNARESUMO
Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine that is considered a pro-inflammatory cytokine. However, our studies show that MIF, when produced in super-physiological levels by a murine neuroblastoma cell line (Neuro-2a) exceeding those normally seen during an immune response, inhibits cytokine-, CD3-, and allo-induced T-cell activation. MIF is also able to inhibit T cells that have already received an activation signal. The T-cell inhibitory effects of culture supernatants from neuroblastoma cells were reversed when the cells were transfected with dicer-generated si-RNA to MIF. When T cells were activated in vitro by co-culture with interleukin (IL)-2 and IL-15 and analyzed for cytokine production in the presence or absence of MIF-containing culture supernatant, inhibition of T-cell proliferation and induced cell death were observed even as the treated T cells produced high levels of interferon-gamma (IFN-gamma). The inhibitory effects of MIF were partially reversed when lymphocytes from IFN-gamma knockout mice were tested. We propose that the high levels of MIF produced by neuroblastoma cause activation induced T-cell death through an IFN-gamma pathway and may eliminate activated T cells from the tumor microenvironment and thus contribute to escape from immune surveillance.
Assuntos
Ativação Linfocitária , Fatores Inibidores da Migração de Macrófagos/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Morte Celular , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-15/imunologia , Interleucina-2/imunologia , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Linfócitos T/citologiaRESUMO
The goal of this study was to show that nonviral gene transfection technology can be used to genetically modify neuroblastoma cells with immune stimulatory molecules, and that the modified cells can generate an antitumor immune response. The authors found that an electroporation-based gene transfection method, nucleofection, could be used to modify mouse AGN2a (an aggressive variant of Neuro-2a) neuroblastoma cells to simultaneously express as many as four different immune stimulatory molecules encoded by separate plasmid vectors. Within 18 hours after nucleofection, greater than 60% of the cells typically expressed the transfected gene products, and the percentages of cells expressing the products often exceeded 96%. High levels of plasmid in cell nuclei immediately after nucleofection documented instantaneous availability of gene vectors to the transcriptional machinery. AGN2a cells nucleofected to express the co-stimulatory molecules CD80 and CD86 expressed higher levels of these molecules than cells that had been permanently transfected with these same plasmid vectors, and the nucleofected cells were as effective as the permanently transfected cells at inducing an antitumor response in vivo in a tumor prevention model. AGN2a cells nucleofected with four separate plasmid vectors encoding CD54, CD80, CD86, and CD137L induced a T-cell immune response in vitro and served as a potent tumor vaccine in the tumor prevention model. These data show that transient transfection using a nonviral based method, nucleofection, can be used to rapidly generate novel cell-based tumor vaccines.
Assuntos
Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Vacinas Anticâncer , Molécula 1 de Adesão Intercelular/biossíntese , Neuroblastoma/metabolismo , Receptores de Fator de Crescimento Neural/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Animais , Antineoplásicos/farmacologia , Vacinas Anticâncer/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Eletroporação , Citometria de Fluxo , Vetores Genéticos , Humanos , Técnicas In Vitro , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Linfócitos T/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
The ability to expand tumour-infiltrating lymphocytes in vitro has been greatly enhanced by the use of antigen-independent mechanisms of immune cell costimulation. We have produced human, using the K562 cell line, and murine, using YAC-1 cells, artificial antigen presenting cells (aAPC) and demonstrate that these cell types stimulate murine lymphocyte populations in distinct ways. Using aAPC that have been transfected with CD137L (4-1BBL) and CD32 (FcRgammaII), as a means to bind anti-CD3 and anti-CD28 antibody, we found that CD4 cells preferentially expanded in vitro with K562 aAPC, while CD8 cells expanded with both K562 and YAC-1 aAPC. Co-stimulation mediated by CD137L on aAPC was superior to that mediated by anti-CD28 antibody. This was seen in both long and short-term expansion assays, and by the rapid induction of a CD8+ DX5+ population. DX5 serves, under these in vitro conditions, as a general marker for lymphocyte activation. In vivo, the superiority of CD137L was demonstrated by the induction of T helper 1 effectors seen in freshly isolated splenocytes from mice immunized with CD137L-expressing neuroblastoma tumour vaccines. The ability to stimulate a strong CD8 CTL response in vivo correlated with the induction of a DX5+ cell population in splenocytes with a memory-effector phenotype. The presence of this unique DX5+ cell population, phenotypically distinct with regards to CD69 and CD62L expression from DX5+ cells induced by aAPC in vitro, may be associated with the ability of CD137L to induce strong anti-tumour immunity.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Neuroblastoma/imunologia , Fator de Necrose Tumoral alfa/imunologia , Ligante 4-1BB , Animais , Antígenos CD , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Divisão Celular/imunologia , Citotoxicidade Imunológica , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Células K562 , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos A , Receptores de Fator de Crescimento Neural/imunologia , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Baço/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
A murine model for neuroblastoma, Neuro-2a (N2a), was used to establish a model tumor vaccine. An aggressive subclone of N2a and the less aggressive parental line were transfected with CD80, CD86, or both molecules and stable lines were established. The less aggressive N2a expressing either CD80 or CD86 induced anti-tumor immunity. In contrast, dual expression of CD80 and CD86 was required to initiate a protective anti-tumor immune response against the aggressive subclone. Control of tumor growth was dependent on CD8+ lymphocytes that infiltrated dual-expressing (CD80 and CD86) lesions. These tumor-infiltrating lymphocytes (TIL) exhibited a non-classical mechanism of tumor cell lysis that may require both the up-regulation of cell surface molecules on the tumor and the subsequent lytic activity normally associated with CD8+ TIL. Although Fas was up-regulated by the tumor in the presence of IFN-gamma, N2a and transfected N2a cell lines were not sensitive to Fas-mediated lysis.