RESUMEN
We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.
Asunto(s)
Anticuerpos/análisis , Antígenos CD40/análisis , Colon/química , Membrana Mucosa/química , Animales , Anticuerpos/inmunología , Antígenos CD40/inmunología , Cromatografía de Afinidad/métodos , Humanos , Macaca fascicularis , Ratas , Espectrometría de Masas en Tándem/métodosRESUMEN
Active episodes of the inflammatory bowel diseases are associated with the infiltration of large numbers of myeloid cells including neutrophils, monocytes, and macrophages. The objective of this study was to systematically characterize and define the different populations of myeloid cells generated in a mouse model of chronic gut inflammation. Using the T cell transfer model of chronic colitis, we found that induction of disease was associated with enhanced production of myelopoietic cytokines (IL-17 and G-CSF), increased production of neutrophils and monocytes, and infiltration of large numbers of myeloid cells into the mesenteric lymph nodes (MLNs) and colon. Detailed characterization of these myeloid cells revealed three major populations including Mac-1(+)Ly6C(high)Gr-1(low/neg) cells (monocytes), Mac-1(+)Ly6C(int)Gr-1(+) cells (neutrophils), and Mac-1(+)Ly6C(low/neg)Gr-1(low/neg) leukocytes (macrophages, dendritic cells, and eosinophils). In addition, we observed enhanced surface expression of MHC class II and CD86 on neutrophils isolated from the inflamed colon when compared with neutrophils obtained from the blood, the MLNs, and the spleen of colitic mice. Furthermore, we found that colonic neutrophils had acquired APC function that enabled these granulocytes to induce proliferation of OVA-specific CD4(+) T cells in an Ag- and MHC class II-dependent manner. Finally, we observed a synergistic increase in proinflammatory cytokine and chemokine production following coculture of T cells with neutrophils in vitro. Taken together, our data suggest that extravasated neutrophils acquire APC function within the inflamed bowel where they may perpetuate chronic gut inflammation by inducing T cell activation and proliferation as well as by enhancing production of proinflammatory mediators.
Asunto(s)
Presentación de Antígeno/inmunología , Colitis/inmunología , Neutrófilos/inmunología , Animales , Proliferación Celular , Enfermedad Crónica , Colitis/patología , Mediadores de Inflamación , Activación de Linfocitos/inmunología , Ratones , Neutrófilos/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
BACKGROUND: Inflammatory arthritis is a chronic disease, resulting in synovitis and subchondral and bone area destruction, which can severely affect a patient's quality of life. The most common form of inflammatory arthritis is rheumatoid arthritis (RA) in which many of the disease mechanisms are not well understood. The collagen-induced arthritis (CIA) mouse model is similar to RA as it exhibits joint space narrowing and bone erosion as well as involves inflammatory factors and cellular players that have been implicated in RA pathogenesis. Quantitative data for disease progression in RA models is difficult to obtain as serum blood markers may not always reflect disease state and physical disease indexes are subjective. Thus, it is important to develop tools to objectively assess disease progression in CIA. RESULTS: Micro-CT (Computed Tomography) is a relatively mature technology that has been used to track a variety of anatomical changes in small animals. In this study, micro-CT scans of several joints of control and CIA mice were acquired at 0, 4, 7, and 9 weeks after the immunization with collagen type II. Each micro-CT scan was analyzed by applying a segmentation algorithm to individual slices in each image set to provide 3-dimensional representations of specific bones including the humerus, femur, and tibia. From these representations, the volume and mean density of these bones were measured and compared. This analysis showed that both the volume and the density of each measured bone of the CIA mice were significantly smaller than those of the controls at week 7. CONCLUSIONS: This study demonstrates that micro-CT can be used to quantify bone changes in the CIA mouse model as an alternative to disease index assessments. In conclusion, micro-CT could be useful as a non-invasive method to monitor the efficacy of new treatments for RA tested in small animals.
RESUMEN
OBJECTIVE: Cytomegalovirus has been implicated in cardiovascular disease, possibly through the induction of inflammatory processes. P-selectin and L-selectin are adhesion molecules that mediate early microvascular responses to inflammatory stimuli. This study examined the role of these selectins in the microvascular dysfunction that occurs during persistent CMV infection. METHODS: C57Bl/6, P- or L-selectin-deficient mice were mock-inoculated or infected with murine CMV, and five weeks later placed on normal diet or high cholesterol diet for six weeks. P-selectin expression was measured or intravital microscopy was performed to determine arteriolar vasodilation and venular blood cell recruitment. RESULTS: P-selectin expression was significantly increased in the heart, lung, and spleen of mCMV-ND, but not mCMV-HC C57Bl/6. mCMV-ND and mCMV-HC exhibited impaired arteriolar function, which was reversed by treatment with an anti-P-selectin antibody, but not L-selectin deficiency. mCMV-HC also showed elevated leukocyte and platelet recruitment. P-selectin inhibition abrogated, whereas L-selectin deficiency partially reduced these responses. CONCLUSIONS: We provide the first evidence for P-selectin upregulation by persistent mCMV infection and implicate this adhesion molecule in the associated arteriolar dysfunction. P-selectin, and to a lesser extent L-selectin, mediates the leukocyte and platelet recruitment induced by CMV infection combined with hypercholesterolemia.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Hipercolesterolemia/metabolismo , Muromegalovirus/metabolismo , Selectina-P/biosíntesis , Regulación hacia Arriba , Animales , Anticuerpos/farmacología , Arteriolas/metabolismo , Arteriolas/virología , Plaquetas/metabolismo , Infecciones por Herpesviridae/genética , Hipercolesterolemia/genética , Hipercolesterolemia/virología , Selectina L/genética , Selectina L/metabolismo , Leucocitos/metabolismo , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Selectina-P/antagonistas & inhibidores , Selectina-P/genética , Factores de TiempoRESUMEN
One of the best characterized mouse models of the inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) is the CD4+CD45RBhigh T cell transfer model of chronic colitis. Following our relocation to Texas Tech University Health Sciences Center (TTUHSC), we observed a dramatic reduction in the incidence of moderate-to-severe colitis from a 16-year historical average of 90% at Louisiana State University Health Sciences Center (LSUHSC) to <30% at TTUHSC. We hypothesized that differences in the commensal microbiota at the 2 institutions may account for the differences in susceptibility to T cell-induced colitis. Using bioinformatic analyses of 16S rRNA amplicon sequence data, we quantified and compared the major microbial populations in feces from healthy and colitic mice housed at the 2 institutions. We found that the bacterial composition differed greatly between mice housed at LSUHSC vs TTUHSC. We identified several genera strongly associated with, and signficantly overrepresented in high responding RAG-/- mice housed at LSUHSC. In addition, we found that colonization of healthy TTUHSC RAG-/- mice with feces obtained from healthy or colitic RAG-/- mice housed at LSUHSC transferred susceptibility to T cell-induced colitis such that the recipients developed chronic colitis with incidence and severity similar to mice generated at LSUHSC. Finally, we found that the treatment of mice with preexisting colitis with antibiotics remarkably attenuated disease. Taken together, our data demonstrate that specific microbial communities determine disease susceptibility and that manipulation of the intestinal microbiota alters the induction and/or perpetuation of chronic colitis.
Asunto(s)
Antibacterianos/farmacología , Colitis/inmunología , Colitis/microbiología , Colon/patología , Microbioma Gastrointestinal/efectos de los fármacos , Traslado Adoptivo , Animales , Bacterias/clasificación , Colon/efectos de los fármacos , Modelos Animales de Enfermedad , Heces/microbiología , Microbioma Gastrointestinal/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Ribosómico 16S/genética , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Inflammation-associated lymphangiogenesis (IAL) is frequently observed in inflammatory bowel diseases. IAL is believed to limit inflammation by enhancing fluid and immune cell clearance. Although monocytes/macrophages (MΦ) are known to contribute to intestinal pathology in inflammatory bowel disease, their role in intestinal IAL has never been studied mechanistically. We investigated contributions of monocytes/MΦ to the development of intestinal inflammation and IAL. METHODS: Because inflammatory monocytes express CC chemokine receptor 2 (CCR2), we used CCR2 diphtheria toxin receptor transgenic (CCR2.DTR) mice, in which monocytes can be depleted by diphtheria toxin injection, and CCR2 mice, which have reduced circulating monocytes. Acute or chronic colitis was induced by dextran sodium sulfate or adoptive transfer of CD4CD45RB T cells, respectively. Intestinal inflammation was assessed by flow cytometry, immunofluorescence, disease activity, and histopathology, whereas IAL was assessed by lymphatic vessel morphology and density. RESULTS: We demonstrated that intestinal MΦ expressed vascular endothelial growth factor-C/D. In acute colitis, monocyte-depleted mice were protected from intestinal injury and showed reduced IAL, which was reversed after transfer of wild-type monocytes into CCR2 mice. In chronic colitis, CCR2 deficiency did not attenuate inflammation but reduced IAL. CONCLUSIONS: We propose a dual role of MΦ in (1) promoting acute inflammation and (2) contributing to IAL. Our data suggest that intestinal inflammation and IAL could occur independently, because IAL was reduced in the absence of monocytes/MΦ, even when inflammation was present. Future inflammatory bowel disease therapies might exploit promotion of IAL and suppression of MΦ independently, to restore lymphatic clearance and reduce inflammation.
Asunto(s)
Colitis/inmunología , Colitis/patología , Linfangiogénesis , Vasos Linfáticos/patología , Macrófagos/inmunología , Monocitos/inmunología , Enfermedad Aguda , Traslado Adoptivo , Animales , Enfermedad Crónica , Colitis/inducido químicamente , Sulfato de Dextran , Femenino , Recuento de Leucocitos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/metabolismo , Receptores CCR2/genética , Factor C de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Chronic colitis is accompanied by extensive myelopoiesis and accumulation of CD11b+Gr-1+ cells in spleens and secondary lymphoid tissues. Although cells with similar phenotype have been described in cancer, chronic infection, or autoimmunity, where they were associated with suppression of T cell responses, little is known regarding how these cells affect CD4 T cell responses in the context of chronic intestinal inflammation. Therefore, we undertook this study to characterize the interplay between colitis-induced myeloid cells and CD4 T cell. Within the CD11b+Gr-1+ population, only monocytes (Ly6G(neg)Ly6C(high)) but not other myeloid cell subsets suppressed proliferation and production of cytokines by CD4 T cells. Suppression was mediated by cell-contact, NO and partially by IFN-γ and PGs. Interestingly, Ly6C(high) MDCs, isolated from colitic colons, showed up-regulation of iNOS and arginase-1 and were more potent suppressors than those isolated from spleen. On a single-cell level, MDCs inhibited Th1 responses but enhanced generation of foxp3+ T cells. MDCs, cocultured with activated/Teffs, isolated from inflamed colons under hypoxic (1% O2) conditions typical for the inflamed intestine, suppressed proliferation but not their production of proinflammatory cytokines and chemokines. Taken together, expansion of monocytes and MDCs and activation of their suppressive properties may represent a homeostatic mechanism aimed at restraining excessive T cell activation during chronic inflammatory settings. The contribution of immunosuppressive monocytes/MDCs to chronic colitis and their role in shaping T cell responses in vivo require further investigation.
Asunto(s)
Colitis/inmunología , Monocitos/inmunología , Linfocitos T/inmunología , Traslado Adoptivo/efectos adversos , Animales , Proliferación Celular , Células Cultivadas , Quimiotaxis de Leucocito , Enfermedad Crónica , Colitis/sangre , Colitis/etiología , Colitis/patología , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Femenino , Genes RAG-1 , Homeostasis , Inmunofenotipificación , Activación de Linfocitos , Tejido Linfoide/inmunología , Tejido Linfoide/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/clasificación , Linfocitos T/trasplanteRESUMEN
BACKGROUND: Myeloid cells are the most abundant and heterogeneous population of leukocytes. They are rapidly recruited from the blood to areas of inflammation and perform a number of important biological functions. Chronic inflammatory conditions contribute to generation of myeloid-derived suppressor cells (MDSCs). These pathologically activated cells are increasingly recognized as important players in cancer, transplantation, and autoimmunity for their abilities to modulate innate and adaptive immune responses. METHODS: Since clinical data on MDSC accumulation in human patients affected with inflammatory bowel diseases (IBD) are relatively scarce, most of the information described in this review came from studies using experimental mouse models of IBD. RESULTS: In this review, we discuss possible roles of these cells in chronic immune-mediated disorders focusing on studies conducted in IBD. We will review the available evidence on how MDSCs are involved in modulating T cell responses and look into the complex relationship between Th1, Th17 cells, and myeloid cells. Finally, we will review some recent successes and failures resulted from therapies aimed at manipulating myeloid cell numbers and/or their function. CONCLUSIONS: Although MDSCs have been described in animal models of experimental colitis and in patients with IBD, their exact role in IBD pathogenesis is unclear and needs to be studied further. Information obtained from these studies will be useful to better understand the cross talk between myeloid cells in T cells during chronic inflammation and may identify novel pathways to be targeted therapeutically.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Células Mieloides/inmunología , Células Mieloides/patología , Animales , Humanos , RatonesRESUMEN
BACKGROUND: L-selectin (CD62L) and ß(7) integrins are important for trafficking of naive T cells under steady-state conditions. The objectives of this study were to dissect the requirements for T cell-associated CD62L and ß(7) integrins during initiation, progression, and regulation of chronic colitis. METHODS: Using the T-cell transfer model, we compared colitogenic potential between T cells lacking one or both of these molecules with wild-type T cells. To assess trafficking of cells to the secondary lymphoid tissue and the gut, we performed co-homing experiments. RESULTS: Adoptive transfer of wild-type, CD62L(-/-) or ß(7)(-/-) single-deficient T cells induced moderate to severe disease with slightly different kinetics. However, transfer of CD62L(-/-) ß(7)(-/-) double-deficient (DKO) T cells produced significantly attenuated gut inflammation, which correlated with fewer T cells and reduced levels of proinflammatory cytokines in the colon lamina propria. Our subsequent experiments established that lack of colitogenic potential of these cells was due to inability of DKO T cells to home to the secondary lymphoid tissue. Furthermore, homing of in vitro-generated effector DKO T cells to the inflamed intestine was significantly impaired. Lastly, DKO regulatory T cells were ineffective at suppressing colitis induced by wild-type T cells. CONCLUSIONS: We established that T cells can use either CD62L(-/-) or ß(7)(-/-) integrins to induce chronic colitis, but lack of both abrogates their colitogenic potential. Effector T cells critically rely on ß(7) integrin during their recruitment to the inflamed intestinal mucosa. Finally, regulation of intestinal inflammation by regulatory T cells requires one or both of these adhesion molecules.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Colitis/metabolismo , Tracto Gastrointestinal/metabolismo , Cadenas beta de Integrinas/fisiología , Selectina L/fisiología , Animales , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Enfermedad Crónica , Citocinas/metabolismo , Femenino , Citometría de Flujo , Tracto Gastrointestinal/patología , Proteínas de Homeodominio/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
INTRODUCTION: We have previously demonstrated that adoptive transfer of naïve CD4(+) T cells devoid of lymphocyte function-associated antigen-1-deficient (LFA-1; CD11a/CD18) into recombination activating gene-1 (RAG-1) deficient (RAG(-/-) ) mice fails to induce chronic colitis whereas transfer of wild type (WT) T-cells induces unrelenting and chronic disease. METHODS: The objectives of this study were to assess the role of lymphocyte function-associated antigen-1 (LFA-1) in enteric antigen (EAg)-induced activation of T cells in vitro and in vivo and to define the importance of this integrin in promoting trafficking of T cells to the mesenteric lymph nodes (MLNs) and colon. RESULTS: We found that EAg-pulsed dendritic cells (DCs) induced proliferation of LFA-1-deficient (CD11a(-/-) ) CD4(+) T cells that was very similar to that induced using WT T cells, suggesting that LFA-1 is not required for activation/proliferation of T cells in vitro. Coculture of WT or CD11a(-/-) T cells with EAg-pulsed DCs induced the generation of similar amounts of interferon-gamma, interleukin (IL)-4, and IL-10, whereas IL-17A production was reduced ≈ 2-fold in cocultures with CD11a(-/-) T cells. Short-term (20-22 hours) trafficking studies demonstrated that while both WT and CD11a(-/-) T cells migrated equally well into the spleen, liver, lungs, small intestine, cecum, and colon, trafficking of CD11a(-/-) T cells to the MLNs was reduced by 50% when compared to WT T cells. When the observation period was extended to 3-7 days posttransfer, we observed ≈ 2-3-fold more WT T cells within the MLNs and colon than CD11a(-/-) T cells, whereas T-cell proliferation (as measured by CFSE dilution) was comparable in both populations. CONCLUSIONS: Taken together, our data suggest that LFA-1 is not required for EAg-induced activation of CD4(+) T cells in vitro or in vivo but is required for trafficking of T cells to the MLNs and homing of colitogenic effector cells to the colon where they initiate chronic gut inflammation.
Asunto(s)
Colitis/etiología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Linfocitos T/fisiología , Animales , Movimiento Celular/inmunología , Movimiento Celular/fisiología , Colitis/inmunología , Citocinas/inmunología , Citocinas/fisiología , Células Dendríticas/inmunología , Células Dendríticas/fisiología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Activación de Linfocitos/fisiología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Mesenterio/inmunología , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunologíaRESUMEN
Clinical trials and animal studies have revealed a role for the renin-angiotensin system in the enhanced thrombus development that is associated with hypertension. Because T lymphocytes have been implicated in the vascular dysfunction and blood pressure elevation associated with increased angiotensin II (Ang II) levels, we evaluated the role of the adaptive immune system in mediating the enhanced thrombosis during Ang II-induced hypertension. Light/dye-induced thrombosis was induced in cremaster arterioles of wild-type, immunodeficient Rag-1(-/-), CD8(+), or CD4(+) lymphocyte-deficient and NADPH oxidase (gp91(phox))-deficient mice implanted with an Ang II-loaded pump for 2 weeks. Chronic Ang II infusion enhanced arteriolar thrombosis in wild-type mice but not in Rag-1(-/-), CD4(+) T-cell-deficient, or gp91(phox-/-) mice. CD8(+) T-cell(-/-) mice exhibited partial protection. Adoptive transfer of T cells derived from wild-type or gp91(phox-/-) mice into Rag-1(-/-) restored the prothrombotic phenotype induced by Ang II. T lymphocytes (CD4(+) and, to a lesser extent, CD8(+)) play a major role in mediating the accelerated microvascular thrombosis associated with Ang II-induced hypertension. NADPH oxidase-derived reactive oxygen species, produced by cells other than T lymphocytes, also appear critical for the Ang II-enhanced, T cell-dependent thrombosis response.
Asunto(s)
NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/inmunología , Trombosis/inmunología , Traslado Adoptivo , Angiotensina II , Animales , Desoxicorticosterona/farmacología , Modelos Animales de Enfermedad , Femenino , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación , Distribución Aleatoria , Sensibilidad y Especificidad , Trombosis/inducido químicamenteRESUMEN
BACKGROUND: It is well known that enteric bacterial antigens drive the development of chronic colitis in a variety of different mouse models of the inflammatory bowel diseases (IBD). The objective of this study was to evaluate the role of gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes (MLNs) and spleen in the pathogenesis of chronic colitis in mice. METHODS: Surgical as well as genetic approaches were used to generate lymphopenic mice devoid of one or more of these lymphoid tissues. For the first series of studies, we subjected recombinase activating gene-1-deficient mice (RAG(-/-) ) to sham surgery (Sham), mesenteric lymphadenectomy (MLNx), splenectomy (Splx) or both (MLNx/Splx). In a second series of studies we intercrossed lymphotoxinß-deficient (LTß(-/-) ) mice with RAG(-/-) animals to generate LTß(-/-) x RAG(-/-) offspring that were anticipated to contain functional MLNs but be devoid of GALT and most peripheral lymph nodes. Flow purified naïve (CD4(+) CD45RB(high) ) T-cells were adoptively transferred into the different groups of RAG(-/-) recipients to induce chronic colitis. RESULTS: We found that at 3-5 wks following T-cell transfer, all four of the surgically-manipulated RAG(-/-) groups (Sham, MLNx, Splx and MLNx/Splx) developed chronic colitis that was similar in onset and severity. Flow cytometric analysis revealed no differences among the different groups with respect to surface expression of different gut-homing markers nor were there any differences noted in IFN-γ and IL-17 generation by mononuclear cells isolated among these surgically-manipulated mice. Although we anticipated that LTß(-/-) x RAG(-/-) mice would contain functional MLNs but be devoid of GALT and peripheral lymph nodes (PLNs), we found that LTß(-/-) x RAG(-/-) mice were in fact devoid of MLNs as well as GALT and PLNs. Adoptive transfer of CD45RB(high) T-cells into LTß(-/-) x RAG(-/-) mice or their littermate controls (LTß(+/+) x RAG(-/-) ) induced rapid and severe colitis in both groups. CONCLUSIONS: Taken together, our data demonstrate that: a) neither the GALT, MLNs nor PLNs are required for induction of chronic gut inflammation in this model of IBD and b) T-and/or B-cells may be required for the development of MLNs in LTß(-/-) mice.
Asunto(s)
Colitis/etiología , Proteínas de Homeodominio/fisiología , Tejido Linfoide , Linfotoxina beta/fisiología , Ganglios Linfáticos Agregados , Animales , Enfermedad Crónica , Femenino , Interleucina-17/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Noqueados , Bazo/inmunología , Bazo/patología , Linfocitos T/inmunología , Células TH1/inmunologíaRESUMEN
BACKGROUND: Donor-specific blood transfusion (DST) prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx). METHODOLOGY/PRINCIPAL FINDINGS: Tolerance to Dark Agouti (DA; RT1(a)) rat liver allografts was induced by injection (iv) of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1(l)) rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4(+) T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg) transcription factor Foxp3 nor did they suppress alloantigen (DA)-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone) induced the time-dependent formation of CD4(+)Foxp3(+) Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4(+)CD45RC(-) population whereas in which approximately 50% of these Tregs express CD25. CONCLUSIONS/SIGNIFICANCE: We conclude that preoperative DST, in the absence of liver allograft transplantation, induces the formation of CD4(+) T-cells that are not themselves Tregs but give rise directly or indirectly to fully functional CD4(+)CD45RC(-)Foxp3(+)Tregs when transferred into MHC mismatched recipients prior to LTx. These Tregs possess potent suppressive activity and are capable of suppressing acute liver allograft rejection. Understanding the mechanisms by which preoperative DST induces the generation of tolerogenic Tregs in the presence of alloantigens may lead to the development of novel antigen-specific immunological therapies for the treatment of solid organ rejection.
Asunto(s)
Factores de Transcripción Forkhead/fisiología , Trasplante de Hígado/métodos , Linfocitos T Reguladores/citología , Animales , Transfusión Sanguínea , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Citocinas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Rechazo de Injerto , Inmunosupresores/uso terapéutico , Isoantígenos/química , Cinética , Masculino , Ratas , Ratas Endogámicas LewRESUMEN
The inflammatory bowel diseases (Crohn's disease; ulcerative colitis) are idiopathic chronic inflammatory disorders of the intestine and/or colon. A major advancement in our understanding of the pathogenesis of these diseases has been the development of mouse models of chronic gut inflammation. One model that has been instrumental in delineating the immunological mechanisms responsible for the induction as well as regulation of intestinal inflammation is the T cell transfer model of chronic colitis. This paper presents a detailed protocol describing the methods used to induce chronic colitis in mice. Special attention is given to the immunological concepts that explain disease pathogenesis in this model, considerations and potential pitfalls in using this model, and finally different "tricks" that we have learned over the past 12 years that have allowed us to develop a more simplified version of this model of experimental IBD.
Asunto(s)
Traslado Adoptivo , Linfocitos T CD4-Positivos/trasplante , Colitis/inmunología , Colon/inmunología , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/inmunología , Animales , Antígeno CD11a/genética , Antígeno CD11a/metabolismo , Linfocitos T CD4-Positivos/inmunología , Separación Celular , Células Cultivadas , Enfermedad Crónica , Colitis/patología , Colon/patología , Progresión de la Enfermedad , Citometría de Flujo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Interleucina-10/genética , Interleucina-10/metabolismo , Antígenos Comunes de Leucocito/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Índice de Severidad de la Enfermedad , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Factores de TiempoRESUMEN
The adaptive immune system plays an important role in host defense against invading micro-organisms. Yet, mice deficient in T- and B-cells are surprisingly healthy and develop few spontaneous infections when raised under specific pathogen-free conditions (SPF). The objective of this study was to ascertain what role phagocyte-associated NADPH oxidase or myeloperoxidase (MPO) plays in host defense in mice lacking both T- and B-cells. To do this, we generated lymphopenic mice deficient in either NADPH oxidase or MPO by crossing gp91(phox)-deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko). We found that neither gp91 ko, MPO ko mice nor lymphocyte-deficient RAG ko mice developed spontaneous infections when raised under SPF conditions and all mice had life spans similar to wild-type (WT) animals. In contrast, gp91xRAG double-deficient (DKO) but not MPOxRAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Infections in the gp91xRAG DKO mice were characterized by granulomatous inflammation of the skin, liver, heart, brain, kidney, and lung. Addition of antibiotics to the drinking water attenuated the spontaneous infections and increased survival of the mice. Oyster glycogen-elicited polymorphonuclear neutrophils (PMNs) and macrophages obtained from gp91 ko and gp91xRAG DKO mice had no detectable NADPH oxidase activity whereas WT, RAG ko, and MPOxRAG DKO PMNs and macrophages produced large and similar amounts of superoxide in response to phorbol myristate acetate. The enhanced mortality of the gp91xRAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase activity (iNOS) as total numbers of elicited PMNs and macrophages as well as PMN- and macrophage-derived production of nitric oxide-derived metabolites in these mice were similar and not reduced when compared to that of WT mice. Taken together, our data suggest that that NADPH oxidase but not MPO (nor iNOS) is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each other's deficiency in providing resistance to spontaneous bacterial infections.
Asunto(s)
Infecciones/genética , Infecciones/inmunología , Linfopenia/inmunología , Glicoproteínas de Membrana/fisiología , NADPH Oxidasas/fisiología , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Linfopenia/complicaciones , Linfopenia/enzimología , Glicoproteínas de Membrana/genética , Ratones , Ratones Mutantes , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Peroxidasa/genética , Linfocitos T/enzimología , Linfocitos T/inmunologíaRESUMEN
The induction and perpetuation of chronic colitis are thought to involve a complex set of adhesive interactions between T cells and endothelial cells located on the vasculature within secondary lymphoid tissue and the intestine. The objective of this study was to assess the roles of T cell-associated CD18, CD62L (L-selectin), ICAM-1, and P-selectin glycoprotein ligand-1 (PSGL-1) in the induction of chronic colitis in mice. CD4(+)CD25(-) T cells derived from either wild-type (WT), CD18-deficient [CD18 knockout (KO)], CD62L KO, ICAM-1 KO, or PSGL-1 KO mice were adoptively transferred into recombinase activating gene-1 (RAG-1)-deficient mice (RAG KO mice) to assess the potential of these T cells to induce chronic colitis. At 8-10 wk following T cell transfer, we observed moderate to severe colitis as assessed by increases in colon weight-to-length ratios and by blinded histopathological analysis. In contrast, we found that transfer of CD18 KO T cells into RAG KO recipients resulted in the significant attenuation of colonic inflammation in these mice. Furthermore, we observed fewer infiltrating CD4(+) T cells in the colonic lamina propria in the CD18 KO-->RAG KO group compared with the WT-->RAG KO group. Finally, message levels of colonic TNF-alpha, IL-1beta, and IFN-gamma were significantly reduced in CD18 KO-->RAG KO mice compared with colitic control animals. We conclude that T cell-associated CD18, but not CD62L, ICAM-1, or PSGL-1, is required for the development of chronic colitis.
Asunto(s)
Antígenos CD18/inmunología , Linfocitos T CD4-Positivos/inmunología , Colitis/inmunología , Colon/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Selectina L/inmunología , Glicoproteínas de Membrana/inmunología , Traslado Adoptivo , Animales , Antígenos CD18/genética , Antígenos CD18/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Colitis/metabolismo , Colitis/patología , Colon/metabolismo , Colon/patología , Modelos Animales de Enfermedad , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Proteínas de Homeodominio/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Subunidad alfa del Receptor de Interleucina-2/análisis , Selectina L/genética , Selectina L/metabolismo , Activación de Linfocitos , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
It is well known that transfer of CD4+CD45RBhigh (naïve) T cells into syngeneic lymphocyte-deficient mice induces chronic colitis. However, no studies have reported the presence of small bowel inflammation in this T cell-dependent model. Therefore, the objective of this study was to evaluate and compare small and large bowel inflammation induced by transfer of naïve T cells into two different immunodeficient recipient mice. T and B cell-deficient recombinase activating gene 1-deficient [RAG knockout (KO)] and T cell-deficient T cell receptor-beta x T cell receptor-delta double-deficient (TCR KO) mice were reconstituted with wild-type naïve T cells and observed for signs of disease. We found that reconstituted RAG KO mice developed moderate to severe colitis and inflammation of the entire small intestine at 6-8 wk after T cell transfer. Adoptive transfer of naïve T cells into TCR KO mice induced a milder form of chronic colitis and small bowel inflammation that was confined primarily to the duodenum at 10-12 wk after T cell transfer. T helper cell 1 and macrophage-derived proinflammatory cytokine mRNA levels correlated well with the localization and severity of the chronic large and small bowel inflammation. In addition, we observed comparable homing and expansion of donor lymphocytes in the gut and secondary lymphoid tissues of both recipients. Taken together, our data demonstrate that transfer of naïve T cells into immunodeficient recipient mice induces both chronic small and large bowel inflammation and that the presence of B cells in the TCR KO recipients may play a role in regulating chronic intestinal inflammation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Síndromes de Inmunodeficiencia/genética , Intestino Grueso/inmunología , Intestino Grueso/patología , Intestino Delgado/inmunología , Intestino Delgado/patología , Traslado Adoptivo , Animales , Femenino , Genes RAG-1/genética , Síndromes de Inmunodeficiencia/inmunología , Masculino , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Trasplante IsogénicoRESUMEN
The beta2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) is important for lymphocyte trafficking and activation as well as recruitment to sites of tissue inflammation. The objective of this study was to assess the role of 'T-cell-associated' LFA-1 in the pathogenesis of chronic colitis in vivo. Transfer of CD4+CD25- T cells isolated from wild-type (wt) mice into immunodeficient recipients [recombinase-activating gene-1-deficient (RAG-1-/-] produced moderate to severe colitis, whereas RAG-1-/- mice injected with CD11a-deficient (CD11a-/-; LFA-1-/-) donor T cells displayed minimal macroscopic and histological evidence of colitis. Surface expression of L-selectin, alpha4, alpha4beta7 and chemokine receptor-7 were similar for wt and CD11a-/- donor T cells. Attenuated disease in the CD11a-/- --> RAG-1-/- animals was associated with decreased numbers of CD4+ T cells in the mesenteric lymph nodes (MLNs), spleen and intestinal lamina propria (LP). In addition, significant reductions in Th1 cytokines were observed following ex vivo stimulation of mononuclear cells obtained from the MLNs and colonic LP. Interestingly, mononuclear cells obtained from the spleens of CD11a-/- --> RAG-1-/- exhibited enhanced pro-inflammatory cytokine production compared with splenocytes obtained from wt --> RAG-1-/- colitic mice. Taken together, our data suggest that T-cell-associated CD11a (LFA-1) expression plays a dual role in the initiation of chronic gut inflammation by facilitating naive T-cell priming/activation and expansion within MLNs and by augmenting pro-inflammatory cytokine production following secondary stimulation by antigen-presenting cells in the colonic interstitium.
Asunto(s)
Colitis/inmunología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Linfocitos T/inmunología , Animales , Biomarcadores , Antígeno CD11a/inmunología , Enfermedad Crónica , Colitis/patología , Colon/citología , Colon/patología , Citocinas/biosíntesis , Femenino , Huésped Inmunocomprometido/inmunología , Ganglios Linfáticos/citología , Mesenterio , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Monocitos/inmunología , Bazo/citología , Bazo/patología , Células TH1/citología , Células TH1/inmunologíaRESUMEN
Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract with unknown multifactorial etiology that, among other things, result in alteration and dysfunction of the intestinal microvasculature. Clinical observations of increased colon microvascular density during IBD have been made. However, there have been no reports investigating the physiological or pathological importance of angiogenic stimulation during the development of intestinal inflammation. Here we report that the dextran sodium sulfate and CD4+CD45RBhigh T-cell transfer models of colitis stimulate angiogenesis that results in increased blood vessel density concomitant with increased histopathology, suggesting that the neovasculature contributes to tissue damage during colitis. We also show that leukocyte infiltration is an obligatory requirement for the stimulation of angiogenesis. The angiogenic response during experimental colitis was differentially regulated in that the production of various angiogenic mediators was diverse between the two models with only a small group of molecules being similarly controlled. Importantly, treatment with the anti-angiogenic agent thalidomide or ATN-161 significantly reduced angiogenic activity and associated tissue histopathology during experimental colitis. Our findings identify a direct pathological link between angiogenesis and the development of experimental colitis, representing a novel therapeutic target for IBD.