RESUMEN
Efficient formation of early GCs depends on the close interaction between GC B cells and antigen-primed CD4(+) follicular helper T cells (TFH ). A tight and stable formation of TFH /B cell conjugates is required for cytokine-driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of TFH /B cell conjugates is crucial for B-cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for TFH -cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin(+) DC subsets in vivo, we show that the functionality of TFH /B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC-depleted mice show a reduction in somatic hypermutation in B cells isolated from TFH /B cell conjugates and markedly reduced GC reactions within skin-draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B-cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.
Asunto(s)
Antígenos de Protozoos/inmunología , Centro Germinal/inmunología , Células de Langerhans/inmunología , Leishmaniasis Cutánea/inmunología , Activación de Linfocitos/inmunología , Animales , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Femenino , Citometría de Flujo , Inmunohistoquímica , Leishmania/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
Bacterial infection with group B Streptococcus (GBS) represents a prominent threat to neonates and fetuses in the Western world, causing severe organ damage and even death. To improve current therapeutic strategies and to investigate new approaches, an appropriate in vivo model to study the immune response of a human immune system is needed. Therefore, we introduced humanized mice as a new model for GBS-induced sepsis. Humanized mice feature deficiencies similar to those found in neonates, such as lower immunoglobulin levels and myeloid cell dysfunction. Due to the husbandry in specific-pathogen-free (SPF) facilities, the human immune cells in these mice also exhibit a naive phenotype which mimics the conditions in fetuses/neonates. Following infection, cytokine release and leukocyte trafficking from the bone marrow to the lymphoid organ (spleen) and into the peritoneum (site of infection) as well as bacterial spreading and clearance were traceable in the humanized mice. Furthermore, we investigated the effects of betamethasone and indomethacin treatment using this novel sepsis model. Although both drugs are commonly used in perinatal care, little is known about their effects on the neonatal immune system. Treatment of infected humanized mice not only induced the reduction of human leukocytes in the spleen but also increased the bacterial load in all analyzed organs, including the brain, which did not show infiltration of live GBS in untreated controls. These studies demonstrate the utility of the humanized mice as a new model to study an immature human immune response during bacterial infection and allow the investigation of side effects induced by various treatments.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Betametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Indometacina/uso terapéutico , Infecciones Estreptocócicas/tratamiento farmacológico , Streptococcus agalactiae , Análisis de Varianza , Animales , Carga Bacteriana/efectos de los fármacos , Médula Ósea , Células de la Médula Ósea/citología , Movimiento Celular/efectos de los fármacos , Recuento de Colonia Microbiana , Citocinas/metabolismo , Modelos Animales de Enfermedad , Leucocitos/efectos de los fármacos , Ratones , Sepsis/tratamiento farmacológico , Bazo/citología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/metabolismoRESUMEN
Immunosuppression, impaired cytokine production and high susceptibility to secondary infections are characteristic for septic patients, and for mice after induction of polymicrobial septic peritonitis by sublethal cecal ligation and puncture (CLP). Here, we demonstrate that CLP markedly altered subsequent B-cell responses. Total IgG and IgM levels, as well as the memory B-cell response, were increased in septic mice, but antigen-specific primary antibody production was strongly impaired. We found that two days after CLP, CD11b(+) splenocytes were activated as demonstrated by the increased expression of activation markers, expression of arginase and production of NO by immature myeloid cells. The in vivo clearance of a bacterial infection was not impaired. DCs demonstrated reduced IL-12 production and altered antigen presentation, resulting in decreased proliferation but enhanced IFN-γ production by CD4(+) cells. CD4(+) T cells from mice immunized on day 2 after CLP showed reduced Th1 and Th2 cytokine production. In addition, there was an increase in Treg cells. Interestingly, levels of immature B cells decreased but levels of mature B cells increased two days after CLP. However, adoptive transfer of naïve CD4(+) T cells, naïve B cells, or naïve DCs did not rescue the antigen-specific antibody response.
Asunto(s)
Linfocitos B/metabolismo , Ciego/inmunología , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Sepsis/inmunología , Traslado Adoptivo , Animales , Formación de Anticuerpos , Presentación de Antígeno , Linfocitos B/inmunología , Linfocitos B/patología , Antígeno CD11b/metabolismo , Antígenos CD4/metabolismo , Ciego/microbiología , Ciego/patología , Ciego/cirugía , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Modelos Animales de Enfermedad , Epítopos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/patología , Infecciones por Escherichia coli/fisiopatología , Humanos , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patología , Peritonitis , Sepsis/complicaciones , Sepsis/patología , Sepsis/fisiopatología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patologíaRESUMEN
PURPOSE: There is evidence that open as well as minimally invasive abdominal surgery impair post-operative innate and acquired immune function. To compare the impact of these approaches as well as the one of different peritoneal gas exposures on immune function, we investigated cellular as well as cytokine-based immune parameters in mesenteric lymph nodes and the spleen postoperatively. METHODS: Mice (n = 26) were randomly assigned to the 4 study groups: (1) sham controls undergoing anesthesia alone, (2) laparotomy, and (3) air, or (4) carbon dioxide pneumoperitoneum. Mice were sacrificed 48 h after the intervention, and their spleens and mesenteric lymph nodes were harvested. Cytokine production (TNF-α, IL-6, IL-10, and IFN-γ), splenic T cell subpopulations (cytotoxic T cells, T helper cells, and regulatory T cells) were analyzed. RESULTS: TNF-α production of splenocytes 16 h after ex vivo lipopolysaccharides (LPS) stimulation was significantly increased in the laparotomy group compared to all other groups. In contrast, TNF-α production of lymph node cells and IL-6 production of splenocytes after ex vivo LPS stimulation did not differ significantly between the groups. The numbers of regulatory T cells (Treg) in the spleen differed between groups. A significant reduction in Treg cell frequency was detected in the CO(2) insufflation group compared to the laparotomy and the air insufflation group. CONCLUSION: Our findings demonstrate a distinct difference in immune effector functions and cellular composition of the spleen with regard to splenic TNF-α production and increased numbers of Treg cells in the spleen. These findings are in line with a higher peritoneal inflammatory status consequent to peritoneal air rather than CO(2) exposure. Treg turned out to be key modulators of postoperative dysfunction of acquired immunity.
Asunto(s)
Citocinas/inmunología , Laparoscopía , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Mesenterio/citología , Mesenterio/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunología , Análisis de Varianza , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Laparotomía , Ratones , Neumoperitoneo Artificial , Distribución AleatoriaRESUMEN
The immunological impact on antibody-based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD-scid IL2Rγ(null) mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor-cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T-cell maturation and tumor cell-specific T-cell activation. In addition, Natural-Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody-dependent cellular cytotoxicity-based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system-related target cancer therapy and resistance.
Asunto(s)
Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Modelos Animales de Enfermedad , Ratones , Animales , Línea Celular Tumoral , Femenino , Rechazo de Injerto/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Interleucina-15/inmunología , Interleucina-15/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Linfocitos T/inmunologíaRESUMEN
Lymphotoxin beta receptor (LTßR) activation on mouse fibrosarcoma cells (BFS-1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro-angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTßR, namely LTα(1) ß(2) and LIGHT, are involved in the activation of LTßR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTßR in these fibrosarcoma tumors, we used conditional LTß-deficient mice that specifically lack LTß expression either on T cells (T-LTß(-/-)) or on B cells (B-LTß(-/-)). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild-type mice, indicating the participation of both T and B host lymphocytes in the activation of LTßR in these tumors. Tumor growth was also reduced in LIGHT-deficient mice, suggesting a contribution of this ligand to the activation of LTßR in BFS-1 fibrosarcomas. LTßR signaling can involve IκBα and/or NFκB-inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro-angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro-angiogenic signaling after LTßR activation. Our data support the idea that the ablation of LTßR signaling should be considered for cancer treatment.
Asunto(s)
Fibrosarcoma/patología , Heterotrímero de Linfotoxina alfa1 y beta2/metabolismo , Receptor beta de Linfotoxina/metabolismo , Linfotoxina beta/fisiología , FN-kappa B/metabolismo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/fisiología , Animales , Apoptosis , Linfocitos B/metabolismo , Linfocitos B/patología , Western Blotting , Proliferación Celular , Células Cultivadas , Quimiocina CXCL2/metabolismo , Femenino , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The mitogen-activated protein kinase phosphatase Dusp1 (also known as MKP-1) is essential for control of the inflammatory response to systemic challenge with the lipopolysaccharide of Gram-negative bacteria. Here, we have investigated the consequences of Dusp1-deficiency in colon ascendens stent peritonitis (CASP) and caecal ligation and puncture (CLP), two mouse models of septic peritonitis. Following CASP, Dusp1(-/-) mice had increased serum levels of CCL4, interleukin-10 (IL-10) and IL-6, with differences from wild-type mice being dependent on severity of sepsis. These cytokines, along with inducible nitric oxide synthase messenger RNA, were also expressed at higher levels in spleen and liver. Similar over-production of these cytokines was detected in the CLP model, with even larger differences from wild-type mice. Despite the increased inflammatory response, bacterial clearance was impaired in Dusp1(-/-) mice subjected to CASP and CLP. Dusp1(-/-) mice suffered increased lethality in both peritonitis models. Together our data indicate that exaggerated inflammatory responses to gut bacteria introduced into the peritoneum in the absence of Dusp1 do not help to control bacterial replication but are detrimental for the host.
Asunto(s)
Citocinas/biosíntesis , Fosfatasa 1 de Especificidad Dual/metabolismo , Bacterias Grampositivas/fisiología , Infecciones por Bacterias Grampositivas/inmunología , Peritonitis/inmunología , Animales , Carga Bacteriana/genética , Ciego/inmunología , Ciego/microbiología , Ciego/cirugía , Colon Ascendente/inmunología , Colon Ascendente/microbiología , Colon Ascendente/cirugía , Citocinas/sangre , Citocinas/genética , Modelos Animales de Enfermedad , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/inmunología , Bacterias Grampositivas/patogenicidad , Infecciones por Bacterias Grampositivas/sangre , Infecciones por Bacterias Grampositivas/complicaciones , Infecciones por Bacterias Grampositivas/genética , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Inflamación/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peritonitis/sangre , Peritonitis/complicaciones , Peritonitis/genética , Peritonitis/microbiología , Sepsis , Stents/estadística & datos numéricosRESUMEN
Recent studies indicate that chronic psychosocial stress favors the development of generalized immune dysfunction. During stressor exposure neuroendocrine factors affect numbers and functionality of leukocytes. However, the exact mechanisms leading to systemic changes in immune functions during stress are still not clear. During chronic subordinate colony housing, a model of chronic psychosocial stress, mice developed spontaneous colonic inflammation. Decreased glucocorticoid signaling, induced by a combination of adrenal insufficiency and glucocorticoid resistance, was thought to prevent tempering of local immune cells, and to promote tissue inflammation. In this study we investigated changes in the systemic immune status after chronic subordinate colony housing and analyzed potential mechanisms underlying those alterations. Analysis of T helper cell subsets in peripheral lymph nodes revealed a reduction of regulatory T cells, accompanied by increased T cell effector functions. Generalized activation of T cells was shown by elevated cytokine production upon stimulation. In addition, we observed no apparent shift towards T helper type 2 responses. It is likely, that the previously reported hypocorticism in this stress model led to a steady production of inflammatory Th1, Th2, and Th17 cytokines and obstructed the shift towards an anti-inflammatory response. In conclusion, we established chronic subordinate colony housing as a model to investigate the outcome of stress on the systemic immune status. We also provide evidence that distinct T helper cell subtypes react differentially to the suppressive effect of glucocorticoids.
Asunto(s)
Citocinas/inmunología , Vivienda para Animales , Ganglios Linfáticos/patología , Estrés Psicológico/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interleucina-10/inmunología , Interleucina-4/inmunología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T Colaboradores-Inductores/clasificación , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Factores de TiempoRESUMEN
Emerging data indicate that alterations in the expression of tumour necrosis factor (TNF) superfamily members play a crucial role in the pathogenesis of intestinal inflammation. Recent results demonstrated that sustained transgenic expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT; TNFSF14) induced severe intestinal inflammation, suggesting a specific role of LIGHT-mediated signalling to the intestinal compartment. In order to dissect the role of LIGHT in intestinal inflammation, we used LIGHT-deficient mice in the mouse model of acute dextran sodium sulphate-induced colitis. Interestingly, LIGHT-deficient mice were characterized by strongly reduced signs of intestinal inflammation compared with wild-type mice in this experimental model. Determination of mouse LIGHT mRNA expression in colon tissues of wild-type mice revealed a strong induction of mouse LIGHT mRNA expression during acute DSS-induced colitis. We therefore generated anti-mouse LIGHT monoclonal antibodies in LIGHT-deficient mice which bind specifically to LIGHT and are capable of neutralizing the activity of LIGHT in vitro and in vivo. With these antibodies, we demonstrated that neutralization of LIGHT during acute DSS-induced colitis resulted in reduced signs of intestinal inflammation. These data suggest that LIGHT is an important mediator in intestinal inflammation and may serve as a new target for therapeutic intervention.
Asunto(s)
Colitis Ulcerosa/metabolismo , Colon/metabolismo , ARN Mensajero/análisis , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Anticuerpos Bloqueadores/metabolismo , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/genética , Colitis Ulcerosa/inmunología , Colon/inmunología , Sulfato de Dextran/administración & dosificación , Epítopos , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunologíaRESUMEN
TNF is a pleiotropic cytokine involved in a variety of inflammatory processes and immune responses. TNF effects are mediated via two distinct membrane receptors: TNFR1 and TNFR2. Investigations concerning regulation and function of TNFR2 revealed a novel TNFR2 isoform in human and mouse cells, termed icp75TNFR, with mainly intracellular localization. As human icp75TNFR is capable of functional interaction with mouse TNF, mouse lines transgenic for the human icp75TNFR were generated and characterized. Transgenic expression was identified in several organs, and soluble human (sh)TNFR2 was detected in serum. shTNFR2 released from transfected cells or peritoneal macrophages of transgenic mice protected from TNF-induced cytotoxicity. Although in vivo, no change in inflammatory reactions was observed in models of septic peritonitis, of colitis, or after stimulation with bacterial LPS, liver injury was strongly enhanced in transgenic mice after Con A challenge. Thus, the functional properties of human icp75TNFR seem to be similar to that of TNFR2, resulting in exacerbation of inflammatory tissue damage, thus revealing the functional importance of TNFR2 in pathophysiological processes.
Asunto(s)
Espacio Intracelular/metabolismo , Hepatopatías/patología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas , Concanavalina A , Susceptibilidad a Enfermedades , Galactosamina/farmacología , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Transgénicos , Pruebas de Neutralización , Isoformas de Proteínas/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Solubilidad/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The lymphotoxin beta receptor (LTbetaR) signalling pathway is involved in the development of secondary lymphoid organs and the maintenance of organized lymphoid tissues. Additionally, previous studies clearly demonstrated the involvement of the LTbetaR interaction with its ligands in promoting intestinal inflammation. In order to dissect the role of LTbetaR activation in the mouse model of acute DSS-induced colitis we treated mice with a functional inhibitor of LTbetaR activation (LTbetaR:Ig) and compared it to disease in LTbetaR-deficient and LTalphabeta-deficient mice. All these modes of LTbetaR signalling ablation resulted in significant aggravation of the disease and in release of inflammatory cytokines such as TNF, IL-6, and IFNgamma. Finally, using mice with conditionally ablated expression of membrane bound LTbeta on T or B cells, respectively, distinct and opposite contributions of surface LTbeta expressed on T or B cells was found. Thus, activation of LTbetaR by LTalphabeta mainly expressed on T lymphocytes is crucial for the down regulation of the inflammatory response in this experimental model.
Asunto(s)
Linfocitos B/metabolismo , Colitis/metabolismo , Receptor beta de Linfotoxina/antagonistas & inhibidores , Linfotoxina beta/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Enfermedad Aguda , Animales , Linfocitos B/efectos de los fármacos , Colitis/inducido químicamente , Colitis/enzimología , Colitis/patología , Modelos Animales de Enfermedad , Femenino , Inmunoglobulinas/farmacología , Inflamación/metabolismo , Ligandos , Receptor beta de Linfotoxina/deficiencia , Receptor beta de Linfotoxina/metabolismo , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Pérdida de Peso/efectos de los fármacosRESUMEN
Tumor necrosis factor (TNF) and its receptors TNF receptor type 1 (TNFR1) and type 2 (TNFR2) have a central role in chronic inflammatory diseases. While TNFR1 mainly confers inflammation, activation of TNFR2 elicits not only pro-inflammatory but also anti-inflammatory effects. In this study, we wanted to investigate the anti-inflammatory therapeutic potential of selective activation of TNFR2 in mice with established collagen-induced arthritis. Mice with established arthritis induced by immunization with bovine collagen type II were treated with six injections of the TNFR2-specific agonist TNCscTNF80, given every second day. Two days after treatment cessation, the cell compositions of bone marrow, spleen and lymph nodes were analyzed. Mice were visually scored until day 30 after the start of therapy and the degree of joint inflammation was determined by histology. Treatment with TNCscTNF80 increased arthritis-induced myelopoiesis. Little effect was seen on the infiltration rate of inflammatory immature myeloid cells and on the reduction of lymphoid cells in secondary lymphoid organs. Upon treatment, frequency of regulatory T (Treg) cells in the CD4+ T-cell population was increased in both spleen and inguinal lymph nodes. In addition, the expression of TNFR2 on Treg cells was enhanced. The clinical score started to improve 1 week after cessation treatment and remained lower 30 days after initiation of therapy. The histological score also revealed amelioration of joint inflammation in TNCscTNF80-treated versus control mice. Activation of TNFR2 might provide a suitable therapeutic strategy in autoimmune arthritis by increasing the numbers of regulatory cell types, in particular Treg cells, and by attenuation of arthritis.
Asunto(s)
Artritis Experimental/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Linfocitos B/metabolismo , Peso Corporal , Bovinos , Proliferación Celular , Citocinas/biosíntesis , Cinética , Ganglios Linfáticos/metabolismo , Masculino , Ratones Endogámicos DBA , Células Mieloides/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Bazo/metabolismoRESUMEN
The Cre-loxP technology allows the introduction of somatic gene alterations in a tissue and/or cell type specific manner. The development of transgenes that target Cre expression to specific cell types is a critical component in this system. Here, we describe the generation and characterization of transgenic mouse lines expressing Cre recombinase under the control of the baboon alpha-chymase promoter, designated Chm:Cre, in order to direct Cre expression specifically to mouse mast cells. Chm:Cre expression was detected in mast cells in lung and colon tissue. Cre-mediated recombination in these mice identified a population of mature tissue resident mast cells using ROSA26R reporter mice. No Cre-expression and Cre-mediated recombination was induced in in vitro generated bone marrow derived mast cells or mast cells isolated from the peritoneal cavity indicating that Cre-expression under the control of the alpha-chymase promoter is solely activated in tissue resident mast cells. These Chm:Cre transgenic mice represent a useful tool to specifically inactivate genes of interest in mast cells of these tissues.
Asunto(s)
Quimasas/genética , Integrasas/genética , Ratones Transgénicos , Animales , Células Cultivadas , Quimasas/metabolismo , Clonación Molecular , Colon/metabolismo , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Papio/genética , Proteínas/genética , ARN no TraducidoRESUMEN
The remarkable ability of TNF, especially in combination with Interferon-gamma or melphalan, to inhibit the growth of malignant tumor cells is so far unmatched. Unfortunately, its high systemic toxicity and hepatotoxicity prevent its systemic use in cancer patients. An elegant manner to circumvent this problem is the isolated limb and liver perfusion for the treatment of melanoma, soft tissue sarcoma and liver tumors, respectively, although the latter method can lead to a reversible hepatotoxicity. In order to allow also the treatment of other cancers with TNF, new strategies have to be developed that aim at sensitizing tumor cells to TNF and at reducing its systemic and liver toxicity, without losing its antitumor efficiency. Moreover, the lectin-like domain of TNF, which is spatially distinct from the receptor binding sites, could be useful in reducing cancer treatment-related pulmonary edema formation. This review will discuss some recent developments in these areas, which can lead to a renewed interest in TNF for the systemic treatment of cancer.
Asunto(s)
Infecciones/fisiopatología , Neoplasias/fisiopatología , Factor de Necrosis Tumoral alfa/fisiología , Adenosina Trifosfato/metabolismo , Antineoplásicos/uso terapéutico , Endotelio Vascular/fisiopatología , Humanos , Inflamación/fisiopatología , Leucocitos/fisiología , Hígado/metabolismo , Necrosis , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Factor de Necrosis Tumoral alfa/uso terapéutico , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Ficolins are members of the collectin family of proteins which are able to recognize pathogen-associated molecular pattern (PAMP) on microbial surfaces. Upon binding to their specific PAMP, ficolins may trigger activation of the immune system by either binding to cellular receptors for collectins or by initiating activation of complement via the lectin pathway. For the latter, the human ficolins (i.e. L-, H- and M-ficolin) and murine ficolin-A were shown to associate with the lectin pathway-specific serine protease MBL-associated serine protease-2 (MASP-2) and catalyse its activation which in turn activates C4 and C4b-bound C2 to generate the C3 convertase C4b2a. There is mounting evidence underlining the lectin nature of ficolins with a wide range of carbohydrate moieties recognized on microbial surfaces. However, not all members of the ficolin family appear to act as lectin pathway recognition components. For example, murine ficolin-B does not associate with MASP-2 and appears to be absent in plasma and other humoral fluids. Its stringent cellular localization points to other functions within the immune response, possibly acting as an intracellular scavenger to target and facilitate clearance of PAMP-bearing debris. When comparing ficolin orthologues from different species, it appears evident that human, murine, and porcine ficolins differ in many aspects, a specific point that we aim to address in this review.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Sistema Inmunológico , Inmunidad Innata , Lectinas/metabolismo , Animales , Humanos , Lectinas/genética , Lectinas/fisiología , Macrófagos/microbiología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/microbiología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Ratones , Modelos Biológicos , Polimorfismo Genético , Especificidad de la Especie , Porcinos , FicolinasRESUMEN
In this report a TNF receptor type 2 (TNFR2) isoform in the mouse, termed micp75TNFR, was compared to its human orthologue hicp75TNFR and to the known mouse TNFR2. The micp75TNFR is generated by the use of an alternative transcriptional start site within the mouse TNFR2 gene. This receptor isoform was found to be expressed in different macrophage-like and endothelial tumor cell lines and mouse spleen cells after stimulation. Lacking the leader sequence and part of the amino-terminal end of the mature TNFR2, the micp75TNFR was characterized by mainly intracellular expression. In contrast to its human paralogue, the micp75TNFR lacked the capacity to bind TNF. Therefore, micp75TNFR expression did not mediate protection from TNF-induced cytotoxicity but may interfere with TNFR2 signalling.
Asunto(s)
Receptores Tipo II del Factor de Necrosis Tumoral/genética , Animales , Secuencia de Bases , Citotoxicidad Inmunológica/efectos de los fármacos , Exones/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
TNF receptor type 2 (TNFR2) has gained attention as a costimulatory receptor for T cells and as critical factor for the development of regulatory T cells (Treg) and myeloid suppressor cells. Using the TNFR2-specific agonist TNCscTNF80, direct effects of TNFR2 activation on myeloid cells and T cells were investigated in mice. In vitro, TNCscTNF80 induced T cell proliferation in a costimulatory fashion, and also supported in vitro expansion of Treg cells. In addition, activation of TNFR2 retarded differentiation of bone marrow-derived immature myeloid cells in culture and reduced their suppressor function. In vivo application of TNCscTNF80-induced mild myelopoiesis in naïve mice without affecting the immune cell composition. Already a single application expanded Treg cells and improved suppression of CD4 T cells in mice with chronic inflammation. By contrast, multiple applications of the TNFR2 agonist were required to expand Treg cells in naïve mice. Improved suppression of T cell proliferation depended on expression of TNFR2 by T cells in mice repeatedly treated with TNCscTNF80, without a major contribution of TNFR2 on myeloid cells. Thus, TNFR2 activation on T cells in naïve mice can lead to immune suppression in vivo. These findings support the important role of TNFR2 for Treg cells in immune regulation.
RESUMEN
Ficolins are pattern-recognition molecules of the innate immune system able to trigger the lectin pathway of the complement activation upon binding to microbial surfaces. In humans, two plasma ficolins have been identified and characterized, whereas a third cell-associated ficolin (M-ficolin) was found on monocyte surfaces. The mouse homologue of M-ficolin is called ficolin B. Although the spatial-temporal expression patterns of mouse ficolins have been described recently, the subcellular localization of ficolin B protein is so far unknown. By using ficolin B-specific antibodies and confocal microscopy, we show that ficolin B is expressed within mouse peritoneal exudate macrophages and is co-localized with Lamp-1, a marker for lysosomes and late endosomes. In addition, the data indicate that ficolin B expression is up-regulated upon macrophage activation.
Asunto(s)
Lectinas/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Animales , Western Blotting , Médula Ósea/metabolismo , Chinchilla , Femenino , Inmunoprecipitación , Lectinas/biosíntesis , Lectinas/aislamiento & purificación , Proteínas de Membrana de los Lisosomas/metabolismo , Activación de Macrófagos , Macrófagos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Bazo/metabolismo , FicolinasRESUMEN
Growth of solid fibrosarcoma tumors in mice was inhibited by the release of a solublelymphotoxin-beta receptor inhibitor (LTbetaR-immunoglobulin fusion protein) from the tumor cells. Tumor growth arrest in mice deficient in the ligand LTalpha1beta2 demonstrated the requirement for activation of the LTbetaR on the tumor cells by host cell-derived LTalpha1beta2. Activation of the LTbetaR resulted in enhanced release of macrophage inflammatory protein-2. Blocked angiogenesis was revealed in LTbetaR inhibitor-producing tumor nodules by immunohistochemistry and in vivo microscopy. The growth arrest of LTbetaR inhibitor-producing fibrosarcomas was overcome by forced MIP-2 expression in the tumor cells. Thus, LTbetaR activation on tumor cells by activated host lymphocytes can initiate a novel proangiogenic pathway leading to organized tumor tissue development.
Asunto(s)
Fibrosarcoma/irrigación sanguínea , Neovascularización Patológica/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Animales , División Celular/inmunología , División Celular/fisiología , Femenino , Fibrosarcoma/inmunología , Fibrosarcoma/patología , Receptor beta de Linfotoxina , Linfotoxina-alfa/antagonistas & inhibidores , Linfotoxina-alfa/inmunología , Linfotoxina beta , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/patología , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/inmunología , TransfecciónRESUMEN
To study the impact of psychosocial stress on the immune system, male mice were subjected to chronic subordinate colony housing (CSC), a preclinically validated mouse model for chronic psychosocial stress. CSC substantially affected the cell composition of the bone marrow, blood, and spleen by inducing myelopoiesis and enhancing the frequency of regulatory T cells in the CD4 population. Expansion of the myeloid cell compartment was due to cells identified as immature inflammatory myeloid cells having the phenotype of myeloid-derived suppressor cells of either the granulocytic or the monocytic type. Catecholaminergic as well as TNF signaling were implicated in these CSC-induced cellular shifts. Although the frequency of regulatory cells was enhanced following CSC, the high capacity for inflammatory cytokine secretion of total splenocytes indicated an inflammatory immune status in CSC mice. Furthermore, CSC enhanced the suppressive activity of bone marrow-derived myeloid-derived suppressor cells towards proliferating T cells. In line with the occurrence of suppressor cell types such as regulatory T cells and myeloid-derived suppressor cells, transplanted syngeneic fibrosarcoma cells grew better in CSC mice than in controls, a process accompanied by pronounced angiogenesis and clustering of immature myeloid cells in the tumor tissue. In addition, tumor implantation after CSC reinforced the CSC-induced increase in myeloid-derived suppressor cells and regulatory T cell frequencies while the CSC-induced cellular changes eased off in mice without tumor. Together, our data suggest a role for suppressor cells such as regulatory T cells and myeloid-derived suppressor cells in the enhanced tumor growth after chronic psychosocial stress.