Renal cytokine responses in acute Escherichia coli pyelonephritis in IL-6-deficient mice

The aim of this study was to investigate the influence of IL-6 on mortality, bacterial growth and cytokine expression in experimental acute pyelonephritis. Female IL-6-deficient mice and their wild-type counterparts, 8-10 weeks old, were infected with Escherichia coli CFT 073 or injected with NaCl 0.9% (w/v) via the urethra and thereafter obstructed for 6 h. Animals were killed at 48 h, 6 days or 8 weeks and cytokine and bacterial renal levels were assessed at each time point. We found that IL-6-deficient mice had increased mortality and extensive renal bacterial growth on day 6, compared with wild-type mice (P < 0.05) and the histopathological changes were generally more severe and widespread in the IL-6-deficient mice. Peak mRNA expression of IL-1beta, IL-4, IL-10, IL-12 and interferon-gamma (IFN-gamma) occurred 48 h after infection in both IL-6 knock out and wild-type mice. Transforming growth factor-beta (TGF-beta) levels also peaked at 48 h in E. coli-infected wild-type mice, while in the IL-6-deficient strain both TGF-beta mRNA and protein levels were significantly lower at 48 h than wild-type levels (P < 0.0008 and P < 0.03, respectively) and remained stationary throughout the study period. Animals injected with NaCl 0.9% (w/v) displayed a similar decrease in TGF-beta expression (P < 0.02). When splenocytes from the IL-6-deficient mice were incubated with murine recombinant IL-6, TGF-beta levels increased to those of wild-type mice. No increase was observed when splenocytes from wild-type mice were incubated with the same doses of rIL-6. We therefore conclude that IL-6 plays an important role in bacterial clearance and directly influences the TGF-beta levels in experimental acute pyelonephritis. We also demonstrate that urethral obstruction per se induces an increase in TGF-beta the magnitude of which is decreased in IL-6-deficient mice.     

IL-4 and IL-10 modulate autoimmune haemolytic anaemia in NZB mice

New Zealand Black (NZB) mice spontaneously develop autoimmune haemolytic anaemia (AIHA). Here the effect of injecting NZB mice with plasmids encoding IL-4 (pIL-4) or IL-10 (pIL-10) on NZB disease was tested. Both constructs delayed the development of anaemia as judged by increased haematocrit values as compared with controls, but neither altered the IgG1 to IgG2 red blood cell (RBC) bound autoantibody levels. The increased haematocrit value was associated temporally with increased RBC bound IgG in NZB mice treated with pIL-10, but not pIL-4. By contrast, up-regulation of splenic macrophage FcgammaRIIb2 mRNA was associated temporally with increased haematocrit values in NZB mice given pIL-4. However, no such increase occurred in NZB mice that inhaled a peptide containing a dominant T-cell epitope, although this treatment is known to bias the autoimmune response towards Th2 and to reduce the severity of anaemia. It is considered that IL-4 treatment, in part, ameliorates NZB anaemia by increasing the expression of the inhibitory FcgammaRIIb2 and thereby reducing the capacity of splenic macrophages to phagocytose autoantibody coated RBC, but that this mechanism does not explain the beneficial effects of the inhaled peptide.     

应用基因敲除鼠研究动脉粥样硬化

ApoE和LDL-R基因敲除鼠可自发形成动脉粥样硬化斑块,是动脉粥样硬化研究的常用模型。在此基础上应用基因打靶技术建立的白介素-1、C反应蛋白、清道夫受体、IgGFc、补体C1q及CD44等模型对研究动脉粥样硬化中炎症和免疫因子的作用机制起了重要作用。     

Eliminating the antilipolytic adenosine A1 receptor does not lead to compensatory changes in the antilipolytic actions of PGE2 and nicotinic acid

Aim: We examined whether compensatory changes after adenosine A₁ receptor knockout [A₁R (−/−)] eliminate the antilipolytic actions mediated by this receptor. Methods: Lipolysis experiments were performed on adipocytes prepared from the wild type A₁R (+/+), A₁R (−/−) and heterozygous mice. Gene expression was assayed with cDNA microarray technique and real‐time PCR; protein expression with immunoblotting. Results: The A₁R was the only adenosine receptor involved in lipolysis. The effects of adenosine deaminase and 2‐chloroadenosine were abolished in A₁R (−/−) mice. The IC₅₀ value of 2‐chloroadenosine doubled from 16.6 to 33.6 nm when half of the A₁Rs were eliminated. Adrenergic α₂ agonists had no effects on lipolysis. Prostaglandin E₂ (PGE₂) inhibited lipolysis with an IC₅₀ value of 5.8 nm (4.7–7.2 nm ) in the A₁R (+/+) mice and 10.6 nm (9.0–12.6 nm ) in the A₁R (−/−) mice. Nicotinic acid inhibited lipolysis with an IC₅₀ value of 0.30 μm (0.19–0.46 μm ) in the A₁R (+/+) mice and 0.24 μm (0.16–0.37 μm ) in the A₁R (−/−) mice. G_iα₁ mRNA was significantly up‐regulated in adipose tissue from A₁R (−/−) mice. However, immunoblotting showed that G_iα1 was not up‐regulated at the protein level. Conclusion: The A₁R mediates the antilipolytic actions of adenosine. Deletion of the A₁R in mice does not result in compensatory increases in G‐protein‐mediated antilipolytic actions of PGE₂ or nicotinic acid.     

Intercellular adhesion molecule-1 (ICAM-1) deficiency protects mice against severe forms of experimentally induced colitis

ICAM-1 (CD54), the ligand for LFA-1 and Mac-1, is up-regulated during inflammatory reaction on the activated vascular endothelium. To determine its role in intestinal inflammation, we induced acute experimental colitis in mice with a deleted ICAM-1 gene, by feeding them with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. Chronic colitis was elicited by DSS similarly, followed by 2 weeks with water. In the acute phase of inflammation, ICAM-1-deficient mice exhibited a significantly lower mortality rate (5%) than control C57Bl/6J mice (35%). Control animals, but not the ICAM-1-deficient mice, exhibited diarrhoea and rectal bleeding. Histological examination of large-bowel samples evaluated the intensity of inflammatory changes, and type and extent of mucosal lesions. In the acute phase, 33.3% of samples from ICAM-1-deficient mice exhibited mucosal defects (flat and fissural ulcers), predominantly mild to moderate inflammatory infiltrate within the lamina propria mucosae and lower grades of mucosal lesions. Much stronger inflammatory changes were present in control animals, flat ulcers (sometimes multiple) and fissural ulcers being observed in 62.5% of samples. Mucosal inflammatory infiltrate was moderate to severe, typically with higher grades of mucosal lesions. In chronic colitis, smaller inflammatory changes were found in the large bowel. The two mouse strains differed, the chronic colitis being accompanied by an increased serum level of anti-epithelial IgA autoantibodies in C57Bl/6 control mice but not in ICAM-1-deficient mice. These findings provide direct evidence of the participation of ICAM-1 molecule in the development of experimentally induced intestinal inflammation.     

Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non‐obese diabetic mice

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of β cells and perforin deficiency effectively reduces diabetes in non‐obese diabetic (NOD) mice, it can be deduced that β cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non‐obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB^–/– mice developed diabetes spontaneously with kinetics similar to those of wild‐type NOD (wt‐NOD) mice. Adoptive transfer study with regulatory T cell (T_reg)‐depleted splenocytes (SPCs) into NOD‐SCID mice or in‐vivo T_reg depletion by anti‐CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB^–/– mice and wt‐NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre‐diabetic NOD.GzmB^–/– mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide‐promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4⁺, CD8⁺ and CD4⁺CD25⁺ T cells in SPCs from NOD.GzmB^–/– mice than those from wt‐NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for β cell destruction in NOD mice.     

IL-2 and IL-10 serum levels in HIV-1-infected patients with or without active antiretroviral therapy

We analyzed IL-2 and IL-10 serum levels in 26 HIV-1-infected patients naive of antiretroviral treatment and in 34 patients receiving highly active antiretroviral therapy (HAART). All patients without treatment were asymptomatic. When they were stratified according to levels of CD4+ T cells, IL-2 levels were significantly increased in patients with > or =200 CD4+/microl and IL-10 levels were significantly increased in patients with <200 CD4+/microl compared to controls. A significant negative correlation was observed between IL10 levels and CD4+ T-cell counts. No correlation was observed between IL-2 and IL-10 levels and viral load due to the wide range of variability in the number of HIV copies/ml present in the different patients. However, IL-2 levels were higher in patients with high viral load than in patients with low viral load. In patients with HAART, IL-2 and IL-10 levels were similar to the control group and no differences were detected respecting CD4+ T cells counts and viral load. Our findings show that the modifications in IL-2 and IL-10 serum levels in HIV-1-infected patients naive of antiretroviral treatment are associated with the progression of immunological damage. Furthermore, they show a dysbalance of type-1/type-2 cytokines with an involvement of type-2 cytokines in later stages of HIV infection. Cytokine dysregulation can be reversed by HAART in the context of immune restoration and viral suppression.     

Characterization of ST2 transgenic mice with resistance to IL‐33

IL‐33, a member of the IL‐1 family, activates MAPK and NF‐κB through its receptor ST2L and IL‐1RAcP. ST2, a member of the IL‐1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL‐33 and to inhibit the IL‐33/ST2L/IL‐1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL‐33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal MΦ, splenomegaly, accumulation of periodic acid‐Schiff‐positive material in the lung, and high concentrations of serum IL‐5 and IL‐13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal MΦ from IL‐33‐stimulated mice, mRNA expression of M2 MΦ marker genes were increased compared with thioglycollate‐elicited peritoneal MΦ. The IL‐33‐stimulation also increased the secretion of IL‐6 from MΦ. However, when the IL‐33 was preincubated with ST2 prior to its addition to the MΦ cultures, the secretion of IL‐6 was attenuated. These data suggest that, though IL‐33 induced the Th2‐type immune responses and infiltration of M2 type MΦ into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.     

The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice

The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end-plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end-plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild-type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end-plates. Cd59a-deficient mice had very mild disease with some muscle inflammation and essentially undamaged end-plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end-plates. Inhibition of MAC assembly abrogated clinical disease in these double-deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti-C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.     

Reciprocal changes in trefoil 1 and 2 expression in stomachs of mice with gastric unit hypertrophy and inflammation

H+/K+-ATPase beta-subunit-deficient mice (129/Sv background) display numerous pathologies in the stomach. Expression of the mutation in BALB/cCrSlc mice results in the development of an aberrant 'mucus-rich' cell population. 'Mucus-rich' cells have been described in stomachs of mice with autoimmune gastritis, a disease mediated by CD4+ T cells. Other pathological features of autoimmune gastritis are similar to those in H+/K+ beta-deficient mice and include a mononuclear cell infiltrate in the gastric mucosa, non-functional or absent parietal cells, depletion of zymogenic cells, hypergastrinaemia, and gastric unit hypertrophy caused by immature cell hyperplasia. The present study investigates further the aberrant gastric 'mucus-rich' cell lineage and analyses the mRNA expression of mucus cell products TFF1 and TFF2. 'Mucus-rich' cells stained for both acidic and neutral mucins, and with a TFF2-specific antibody. Stomachs from both models expressed decreased TFF1 mRNA and reciprocally increased TFF2 mRNA. The involvement of gastrin in regulating trefoil mRNA expression was also investigated using gastrin-deficient mice. In contrast to previous findings, gastrin did not positively regulate TFF1 mRNA expression, but there was possible augmentation of TFF2. Additionally, a clear role for inflammation was established involving both polymorphonuclear and mononuclear cells in these models, and a link was found between mucosal hypertrophy and increased interleukin-11 (IL-11) expression.     

IL-2Rbeta links IL-2R signaling with Foxp3 expression

Immunological tolerance to self antigens is a tightly regulated process. Recent work has demonstrated that the forkhead family member Foxp3 is a critical element in the differentiation and function of mouse CD4(+)CD25(+) regulatory T cells (Treg). Recent work has suggested an important role for IL-2 in the development and maintenance of Treg. To directly assess the effect of IL-2 signaling on Treg development and function, we analyzed mice that were genetically deficient in components of the IL-2 receptor (IL-2R). Mice lacking CD25 (IL-2Ralpha) displayed a slight decrease in Treg within the thymus, while peripheral numbers are unchanged. In contrast, we found that mice deficient in CD122 (IL-2Rbeta) had a profound reduction in both thymic and peripheral Treg, coinciding with more rapid development of a fatal lymphoproliferative disease. Expression of a Foxp3 transgene restored Treg and protected against the onset of autoimmunity. Thus, a signal mediated by IL-2Rbeta is essential for the development and homeostasis of Foxp3(+) Treg in vivo.     

Lymphocyte infiltration into cerebellum in transgenic mice carrying human IL-2 gene

We created transgenic mice with an intact human genomic interleukin-2 gene (gIL-2) or a mouse metallothionein-I promoter-human IL-2 chimeric gene (MTgIL-2). Nine (2 gIL-2 and 7 MTgIL-2 transgenics) out of 12 transgenic mice which were obtained independently had motor ataxic symptoms. All transgenic offspring of the symptomatic founders showed the same symptoms as their transgenic parents. Morphological examination demonstrated perivascular lymphocyte accumulation in the cerebellar meninx which was followed by increased cell infiltration of neutrophils and monocytes in the destructive cerebellum of all transgenic mice. These findings suggest that the lymphocyte infiltration in the cerebellum is caused by the specific effect of the exogenously introduced human IL-2 gene.     

Exacerbation of spontaneous autoimmune nephritis following regulatory T cell depletion in B cell lymphoma 2‐interacting mediator knock‐out mice

Regulatory T cells (T_regs) have been recognized as central mediators for maintaining peripheral tolerance and limiting autoimmune diseases. The loss of T_regs or their function has been associated with exacerbation of autoimmune disease. However, the temporary loss of T_regs in the chronic spontaneous disease model has not been investigated. In this study, we evaluated the role of T_regs in a novel chronic spontaneous glomerulonephritis model of B cell lymphoma 2‐interacting mediator (Bim) knock‐out mice by transient depleting T_regs. Bim is a pro‐apoptotic member of the B cell lymphoma 2 (Bcl‐2) family. Bim knock‐out (Bim^–/–) mice fail to delete autoreactive T cells in thymus, leading to chronic spontaneous autoimmune kidney disease. We found that T_reg depletion in Bim^–/– mice exacerbated the kidney injury with increased proteinuria, impaired kidney function, weight loss and greater histological injury compared with wild‐type mice. There was a significant increase in interstitial infiltrate of inflammatory cells, antibody deposition and tubular damage. Furthermore, the serum levels of cytokines interleukin (IL)?2, IL‐4, IL‐6, IL‐10, IL‐17α, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α were increased significantly after T_reg depletion in Bim^–/– mice. This study demonstrates that transient depletion of T_regs leads to enhanced self‐reactive T effector cell function followed by exacerbation of kidney disease in the chronic spontaneous kidney disease model of Bim‐deficient mice.     

The IL-1 receptor 1 is critical for Th2 cell type airway immune responses in a mild but not in a more severe asthma model

IL-1 alpha and IL-1 beta are potent pro-inflammatory cytokines that regulate many physiological systems by binding and signaling to the same receptor termed IL-1 receptor type 1 (IL-1R1). We have investigated the role of IL-1 for pulmonary immune responses in models of allergic asthma using IL-1R1-deficient (IL-1R1(-/-)) mice. In a model of mild asthma, based on repeated sensitization of mice with low doses of ovalbumin in the absence of any adjuvant and multiple intranasal challenges, the pulmonary eosinophilic inflammation and goblet cell hyperplasia were strongly reduced in IL-1R1(-/-) as compared to control BALB/c mice. Moreover, priming of CD4(+) T cells in bronchial lymph nodes and their recruitment to the lung was affected in IL-1R1(-/-) mice associated with impaired antibody responses including IgG, IgE, and IgA. In contrast, sensitization of mice in the presence of alum adjuvant, a more severe asthma model, rendered the IL-1 pathway dispensable for the development of pulmonary allergic Th2 responses, as eosinophilic inflammation, antibody responses, and CD4(+) T cell priming in lymph nodes were comparable between IL-1R1(-/-) and wild-type mice. These results suggest a critical role of IL-1/IL-1R1 for development of allergic Th2 responses, but its requirement can be overcome by using alum as adjuvant for sensitization.     

Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-α)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13

TNF-α is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-α transgene modified with the 3′ untranslated region of β-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-α-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4 weeks old. Control groups of transgenic mice were engrafted with β-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-α transgene, endogenous mouse TNF-α and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3 weeks of treatment (P < 0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-α transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.     

Immunological mechanisms and clinical implications of regulatory T cell deficiency in a systemic autoimmune disorder: roles of IL-2 versus IL-15

Regulatory T cell deficiency is evident in patients with lupus, but the casual [corrected] relationship and underlying mechanism leading to Treg deficiency are unclear. We analyzed the Treg profile, induction and functions of Treg in a lupus mouse model. A characteristic age-dependent biphasic change of Treg frequency was observed in the MRL/lpr mice, which developed a spontaneous lupus-like disease. After an early increase, Treg frequency in the peripheral lymphoid organs rapidly declined with age. Functionally, Treg from both young and old MRL/lpr mice were fully competent in suppressing the wild-type MRL/+ T effector cell (Teff) responses. Adoptive transfer of MRL/+ Treg markedly suppressed clinical disease in the MRL/lpr mice. We demonstrated that the reduced Treg frequency was a result of insufficient peripheral Treg expansion due to defective MRL/lpr Teff in IL-2 production, and the associated defects in dendritic cells, which could be fully restored by exogenous IL-2. In the absence of IL-2, MRL/lpr Teff but not MRL/lpr Treg were highly responsive to IL-15 and could expand rapidly due to enhanced IL-15R expression and IL-15 synthesis. These findings thus provide a clear causal relationship and immunological mechanism underlying Treg deficiency and systemic autoimmunity.     

Dysregulation of IL-2 and IL-8 production in circulating T lymphocytes from young cystic fibrosis patients

It is well documented that patients with cystic fibrosis (CF) are unable to clear persistent airway infections in spite of strong local inflammation, suggesting a dysregulation of immunity in CF. We and others have reported previously that T lymphocytes may play a prominent role in this immune imbalance. In the present work, we compared the reactivity of CD3+ T cells obtained from young CF patients in stable clinical conditions (n = 10, aged 9-16.5 years) to age-matched healthy subjects (n = 6, aged 9-13.5 years). Intracellular levels of interferon (IFN)-gamma, interleukin (IL)-2, IL-8 and IL-10 were determined by flow cytometry after whole blood culture. The data identified T lymphocyte subsets producing either low levels (M1) or high levels (M2) of cytokine under steady-state conditions. We found that the production of IFN-gamma and IL-10 by T lymphocytes was similar between young CF patients and healthy subjects. In contrast, after 4 h of activation with PMA and ionomycin, the percentage of T cells producing high levels of IL-2 (M2) was greater in CF patients (P = 0.02). Moreover, T cells from CF patients produced lower levels of IL-8, before and after activation (P = 0.007). We conclude that a systemic immune imbalance is present in young CF patients, even when clinically stable. This disorder is characterized by the capability of circulating T lymphocytes to produce low levels of IL-8 and by the emergence of more numerous T cells producing high levels of IL-2. This imbalance may contribute to immune dysregulation in CF.     

Distal regulatory elements play an important role in regulation of the human IL-5 gene

Eosinophil infiltration of the lung is a feature of both allergic and nonallergic asthma, and IL-5 is the key cytokine regulating the production and activation of these cells. Despite many studies focusing on the IL-5 promoter in both humans and mice there is as yet no clear picture of how the IL-5 gene is regulated. The aim of this study was to determine if distal regulatory elements contribute to appropriate regulation of the human IL-5 (hIL-5) gene. Activity of the -507/+44 hIL-5 promoter was compared to expression of the endogenous IL-5 gene in PER-117 T cells. The IL-5 promoter was not sufficient to reproduce a physiological pattern of IL-5 expression. Further, functional analysis of the 5' and 3' intergenic regions revealed a number of novel regulatory elements. We have identified a conserved enhancer located approximately 6.2 kb upstream of the hIL-5 gene. This region contains two potential GATA-3-binding sites and increases expression from the hIL-5 promoter by up to ninefold.