IL-4, IL-10 and IL-13 down-regulate monocyte-chemoattracting protein-1 (MCP-1) production in activated intestinal epithelial cells

Several studies have demonstrated that intestinal epithelial cells play a major role in the initiation and perpetuation of intestinal inflammation by secreting proinflammatory cytokines and chemokines. MCP-1 is suggested to be a chemokine that plays a major part during intestinal inflammation in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL-4, IL-10 and IL-13 have been described to exert anti-inflammatory properties on various cell types. The aim of our study was to determine the effect of Th2 cytokines on the production of MCP-1 by activated intestinal epithelial cells. We examined Caco-2 cells as well as intestinal epithelial cells which were isolated from surgical specimens. Production of the chemokine MCP-1 was determined under stimulated and non-stimulated conditions. IL-4, IL-10 and IL-13 were added to stimulated epithelial cells under various culture conditions. Supernatants were analysed for cytokine concentrations using ELISAs. Under stimulation with physiological agents like IL-1β or tumour necrosis factor-alpha (TNF-α), we observed markedly increased concentrations of MCP-1 in supernatants of Caco-2 cells and intestinal epithelial cells. IL-4, IL-10 and IL-13 all had the capacity to down-regulate the production of MCP-1 in Caco-2 cells as well as in freshly isolated epithelial cells. Caco-2 cells which were primed with Th2 cytokines 24 h before stimulation were subsequently decreased in their ability to be stimulated by IL-1β or TNF-α for MCP-1 production. As MCP-1 has been shown to play a major role during intestinal inflammation, the in vitro suppression of MCP-1 in enterocytes suggests the in vivo use of regulatory cytokines in patients with active IBD.     

Enhanced liver regeneration in IL-10-deficient mice after partial hepatectomy via stimulating inflammatory response and activating hepatocyte STAT3

Emerging evidence suggests that proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a critical role in the initiation and progression of liver regeneration; however, relatively little is known about the role of anti-inflammatory cytokine IL-10 in liver regeneration after partial hepatectomy (PHx). Here, we examined the role of IL-10 in liver regeneration using a model of PHx in several strains of genetically modified mice. After PHx, expression of IL-10 mRNA in the liver and spleen was significantly elevated. Such elevation was diminished in TLR4 mutant mice. Compared with wild-type mice, IL-10(-/-) mice had higher levels of expression of proinflammatory cytokines (IL-6, TNF-α, and IFN-γ) and inflammatory markers (CCR2 and F4/80) in the liver, as well as higher serum levels of proinflammatory cytokines after PHx. The number of neutrophils and macrophages was also higher in the livers of IL-10(-/-) mice than in wild-type mice after PHx. Liver regeneration as determined by BrdU incorporation after PHx was higher in IL-10(-/-) mice than in wild-type mice, which was associated with higher levels of activation of IL-6 downstream signal STAT3 in the liver. An additional deletion of STAT3 in hepatocytes significantly reduced liver regeneration in IL-10(-/-) mice after PHx. Collectively, IL-10 plays an important role in negatively regulating liver regeneration via limiting inflammatory response and subsequently tempering hepatic STAT3 activation.     

Interleukin-4 (IL-4) and IL-13 suppress excessive neutrophil infiltration and hepatocyte damage during acute murine schistosomiasis japonica

Due to the importance of neutrophils and proinflammatory cytokines in schistosomal liver damage, we analyzed the mechanisms underlying neutrophil and proinflammatory responses in murine schistosomiasis japonica. We found that granulomatous inflammation around parasite eggs in the liver was greater in Schistosoma japonicum-infected IL-4-/- IL-13-/- (double-knockout [DKO]) mice than in infected wild-type (WT) mice at 6 weeks, but not at 8 weeks, postinfection, suggesting the importance of Th2 responses in these typical hepatic lesions. Infected DKO mice also showed increased neutrophil infiltration accompanying more severe pathology, as shown by the enhanced necrosis of hepatocytes. This was not likely due to a Th1/Th2 imbalance, because there was no detectable increase in gamma interferon (IFN-γ) production in these DKO mice. mRNA expression of interleukin-17A (IL-17A), proinflammatory cytokines, and the neutrophil chemoattractant CXCL2 in liver was higher in infected DKO mice than in WT mice. However, in IL-4-/- IL-13-/- IL-17A-/- (triple-knockout [TKO]) mice, the absence of IL-17A was associated with only marginal differences in schistosomal liver damage, suggesting that IL-17A is only partially responsible for neutrophil-driven hepatic damage. Furthermore, the expression of mRNAs encoding proinflammatory cytokines was not under the control of IL-17A in TKO mice. These findings indicate that IL-4 and IL-13 suppress excessive neutrophil recruitment, proinflammatory cytokine production, and hepatic damage during the acute stage of S. japonicum infection, suggesting that neutrophils and proinflammatory cytokines are mainly responsible for hepatocyte damage during acute murine schistosomiasis japonica. However, neutrophil induction and the production of proinflammatory cytokines were not due solely to IL-17A.     

Soluble Receptors Neutralizing TNF-α and IL-1 Block Stress-Triggered Murine Abortion

PROBLEM: In several models of abortion in rodents, the success or failure of the implanted embryos is determined by a balance between pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-2, interleukin-1 (IL-1), and γ-interferon, and cytokines that counteract the former, such as interleukin-10 and transforming growth factor-β2 (TGF-β2)-related suppressor factor. Stress can trigger abortions in susceptible strains of mice and is thought to reflect the pathogenesis of some types of miscarriage in human pregnancy. In mice, stress increases levels of the abortogenic cytokine TNF-α and decreases the suppressive activity of TGF-β2-related factor via a neurotransmitter substance P (SP)-dependent pathway. Evidence for a role of pro-inflammatory cytokines in SP-mediated abortions in vivo is indirect. METHODS: Direct evidence for a role of IL-1 and TNF-α in stress-triggered abortions was sought by injecting pregnant female mice with soluble receptors neutralizing TNF-α (rhuTNFR: Fc) or IL-1 (rmIL-1R) beginning 1 day after implantation and prior to stress. RESULTS: The stress-triggered abortion rate was reduced by 68% when either TNF-α or IL-1 antagonists were injected. The stress-triggered decreased TGF-β2-like suppressive activity in the maternal uterine decidua was not restored by injection of either antagonist; indeed the soluble IL-1 receptor significantly reduced suppressive activity in unstressed control mice, and soluble TNF-α receptor had a lesser effect. CONCLUSIONS: Both IL-1 and TNF-α play a role in the pathogenesis of stress-triggered abortions, and may induce a compensatory physiological increase in suppressive activity in normal pregnancy counteracting pro-inflammatory cytokines.     

Human Recombinant IL-2 Augments Immunoglobulin and Induces Rheumatoid Factor Production by Rheumatoid Arthritis Lymphocytes Engrafted into Severe Combined Immunodeficient Mice

Recombinant (r) human IL-2 was administered in vivo to improve homing and engraftment of rheumatoid arthritis (RA) patients' peripheral blood mononuclear cells (PBMC) into severe combined immunodeficient (SCID) mice. Human rIL-2 treatment resulted in augmented human Ig production and induced IgM rheumatoid factor (RF) of human origin in SCID-RA chimeras. The increment of human serum IgG in SCID-RA chimeras after IL-2 treatment ranged between 15 and 43% and for IgM between 50 and 98% during 2-8 weeks postengraftment. Human IgM-RF was detectable after 1 to 2 weeks after engraftment and persisted over a period of 10-13 weeks. No RF was produced in SCID mice engrafted with PBMC from healthy individuals with or without exogenous rIL-2 administration. Thus, human rIL-2 expanded autoreactive clones involved in the production of RF in the SCIDRA chimeras. The present study provides a novel approach to establish an in vivo SCID-RA model to study the cellular and molecular mechanisms involved in the production of RF and development of a RA-like lesion.     

Interleukin-12 is required for interferon-γ production and lethality in lipopolysaccharide-induced shock in mice

Several cytokines, in particular tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 γg/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-γ production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-γ production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1–3 ng/ml at 3–6 h after LPS injection, whereas IFN-γ production was observed only in BCG-primed mice. The priming effect of BCG on IFN-γ production appears to be mostly due to its ability to increase TNF-α production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-γ production, as shown by the ability of injection of TNF-α and LPS (1 γg/mouse), but not LPS alone, to induce IFN-γ production. However, in addition to TNF-α, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-γ production, because co-injection of TNF-α and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-γ production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-γ production, also completely protect BCG-primed mice injected with up to 10 μg of LPS from shock-induced death. Thus, IL-12 is required for IFN-γ production and lethality in an endotoxic shock model in mice.     

Reduced secretion of the inflammatory cytokine IL-1β by stimulated peritoneal macrophages of radiosensitive Balb/c mice after exposure to 0.5 or 0.7Gy of ionizing radiation

Since the beginning of the 20th century, low dose radiotherapy (LD-RT) has been practiced and established as therapy of inflammatory diseases. Several clinical studies already have proven the anti-inflammatory effect of low doses of ionizing irradiation (LDR). However, further research is inevitable to reveal the underlying immune-biological mechanisms. Focus has been set on the modulation of activated macrophages by LDR, since they participate in both, initiation and resolution of inflammation. Here we examined with an ex vivo peritoneal mouse macrophage model how LDR modulates the secretion of the inflammatory cytokines IL-1β and TNF-α by activated macrophages and whether the basal radiosensitivity of the immune cells has influence on it. Peritoneal macrophages of Balb/c mice responded to exposure of 0.5 or 0.7 Gy of ionizing irradiation (X-ray) with significant decreased release of IL-1β and slightly, but not significantly, reduced release of TNF-α. Macrophages of the less radiosensitive C57BL/6 mice did not show this anti-inflammatory reaction. This was observed in both wild type and human TNF-α transgenic animals with C57BL/6 background. We conclude that only the inflammatory phenotype of more radiosensitive macrophages is reduced by LDR and that ex vivo and in vivo models with primary cells should be applied to examine how the immune system is modulated by LDR.     

The Adenosine-Dependent Angiogenic Switch of Macrophages to an M2-Like Phenotype is Independent of Interleukin-4 Receptor Alpha (IL-4Rα) Signaling

Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as “alternatively activated” (M2a) macrophages. We have shown previously that adenosine A_2A receptor (A_2AR) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an “M2-like” phenotype that we have termed “M2d.” Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα^?/? mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify “M2” macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.     

Encapsulation in hollow fibres of xenogeneic cells engineered to secrete IL-4 or IL-13 ameliorates murine collagen-induced arthritis (CIA).

A strategy of gene therapy using IL-4 or IL-13 xenogeneic transfected cells encapsulated into permeable hollow fibres (HF) was used to treat CIA. Hydrogel-based hollow fibres were obtained from AN-69 copolymer, already known for its biocompatibility and tolerance in rodents. Permeability to IL-4 and lack of cell leakage from the fibres were ascertained in vitro and in vivo. Chinese hamster ovary (CHO) fibroblasts transfected with mouse IL-4 gene were encapsulated in HF (6.25 x 105 cells/HF). IL-4 was detected in vitro in the culture supernatant of filled fibres for at least 19 days. IL-4 or IL-13 transfected CHO cells encapsulated in HF were implanted in the peritoneum of mice on days 11-13 after immunization with type II collagen. Control mice were treated with fibre containing CHO cells transfected with beta-galactosidase (betagal) gene; a positive control group consisted of mice treated by subcutaneous injection of 106 cells on days 10 and 25. Mice were monitored for signs of arthritis by observers unaware of the status of animals. Results of these experiments indicate that severity of the articular disease was significantly reduced in the groups of mice treated with CHO/IL-4 or CHO/IL-13 cells encapsulated in HF, compared with control groups receiving CHO/betagal cells encapsulated in HF. Histological analysis confirmed these data and extended them to a better inhibitory effect of encapsulated cells compared with free cells on inflammatory and destructive joint disease. Moreover, such long-term treatment with HF was well tolerated; macroscopic and histological aspects of peritoneal cavity were moderately inflammatory. Thus, our results may have important implications for clinical use of gene transfected cells as therapeutic agents in the treatment of autoimmune diseases.     

The contribution of interleukin (IL)-4 and IL-13 to the epithelial-mesenchymal trophic unit in asthma

Interleukin (IL)-4 and IL-13 are key proinflammatory cytokines in asthma. Studies in transgenic mice show that both cytokines cause inflammation, but only IL-13 causes subepithelial fibrosis, a characteristic feature of asthma. We compared the in vitro profibrogenic effects of IL-4 and IL-13 using bronchial fibroblasts from asthmatic subjects. In the presence of transforming growth factor (TGF)-beta the cells transformed into contractile myofibroblasts and expressed alpha-smooth muscle actin and procollagen I. IL-4 and IL-13 also stimulated proliferation, but were relatively ineffective in promoting myofibroblast transformation. TGF-beta was more potent than the cytokines in stimulating release of endothelin-1 and vascular endothelial growth factor, whereas IL-4 and IL-13 were more potent stimuli for eotaxin release. Although neither IL-4 nor IL-13 induced profibrotic responses, both cytokines caused a corticosteroid-insensitive stimulation of TGF-beta2 release from primary bronchial epithelial cells. These data indicate that epithelial activation by IL-13 or IL-4 plays a critical role in initiating remodeling through release of TGF-beta2. TGF-beta2 then activates the underlying myofibroblasts to secrete matrix proteins and smooth muscle and vascular mitogens to propagate remodeling changes into the submucosa. In contrast, direct activation of submucosal fibroblasts by IL-4 and IL-13 has a proinflammatory effect via eotaxin release and recruitment of eosinophils into the airways.     

Osteoclastogenesis and arthritis

There is emerging interest for osteoclasts as key players in the erosive and inflammatory events leading to joint destruction in chronic arthritis. In fact, chronic inflammatory joint diseases such as psoriatic arthritis and rheumatoid arthritis are often characterized by destruction of juxta-articular bone and erosions due to the elevated activity of osteoclasts, which are involved in bone resorption. The main step in inflammatory bone erosion is an imbalance between bone resorption and bone formation: osteoclast formation is enhanced by proinflammatory cytokines such as TNF-α, IL-1β, and IL-17 and is not balanced by increased activity of bone-forming osteoblasts. T-cells, stromal cells, and synoviocytes enhance osteoclast formation via expression of RANKL and, under pathologic conditions, of proinflammatory cytokines. In rheumatoid arthritis, accumulation of osteoclasts in synovial tissues and their activation associated with osteoclastogenic cytokines and chemokines at cartilage erosion sites suggest that they could be usefully selected as therapeutic target. In particular, in consideration of the primary role of RANKL and TNF-α in osteoclastogenesis, the control of the production of RANKL and the inhibition of TNF-α represent important strategies for reducing bone damage in this disease.     

Immunoregulatory properties of IL-13 in patients with inflammatory bowel disease; comparison with IL-4 and IL-10

Activated monocytes with increased expression of proinflammatory cytokines play a major role in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL-4 and IL-10 can effectively suppress the proinflammatory response of activated monocytes. IL-13 is a recently described antiinflammatory agent in vitro. The aim of our study was to determine the in vitro immunosuppressive capacity of IL-13, IL-4 and IL-10 in patients with IBD. Peripheral blood monocytes were isolated from 27 patients with ulcerative colitis (UC), 27 patients with Crohn's disease (CD) and 16 healthy controls. Cells were stimulated with pokeweed mitogen (PWM) after treatment with IL-13, IL-4 and IL-10, and secretion of IL-1β, tumour necrosis factor-alpha (TNF-α) and IL-6 was assessed using sandwich ELISA systems. Peripheral blood monocytes secreted significantly increased amounts of TNF-α and IL-6 under stimulation with PWM in patients with CD, while UC patients showed significantly elevated levels of IL-1β. The antiinflammatory cytokines IL-13, IL-4 and IL-10 were all capable of inhibiting monocyte secretion of IL-1β in a dose-dependent manner. With regard to IL-13 and IL-4, there was no significant suppression of TNF-α and IL-6 in patients with active IBD. By contrast, IL-10 was able to down-regulate all proinflammatory cytokines in active IBD as well as in controls. Proinflammatory cytokines from patients with inactive IBD could be significantly down-regulated by all three immunoregulatory cytokines. The inhibitory effect of IL-13 on TNF-α and IL-6 production in differentiated macrophages was diminished in IBD patients, as well as in controls. In disease controls we also observed a reduced inhibition of TNF-α and IL-6 after treatment with IL-13. In conclusion, the antiinflammatory activity of IL-13 is partially reduced in patients with active IBD. The hyporesponsiveness of activated and differentiated monocytes to IL-13 and IL-4 does not seem to be a disease-specific phenomenon.     

CCK-8对LPS攻击小鼠IL-1β、IL-6、IL-4、IL-10表达的影响

目的： 观察LPS攻击小鼠IL-1β、IL-6、IL-4、IL-10的动态变化规律及八肽胆囊收缩素（CCK-8）对其表达的影响。方法: 将小鼠分为4组：对照组、LPS组（腹腔注射LPS）、LPS+CCK-8组（注射LPS前 30 min 腹腔注射CCK-8）及CCK-8组（单独注射CCK-8）。用ELISA及PT-PCR方法检测各组小鼠血清、肺组织中IL-1β、IL-6、IL-4、IL-10的含量及mRNA的表达情况。结果: LPS攻击可使小鼠血清及肺组织中 IL-1β、IL-6、IL-4、IL-10蛋白及mRNA的表达增加，预先注入CCK-8可显著抑制IL-1β、IL-6的表达，并使 IL-4、IL-10的表达进一步增加。结论: CCK-8可能通过抑制LPS攻击小鼠IL-1β、IL-6的表达和进一步增加 IL-4、IL-10的表达参与抗炎反应过程，从而减轻LPS引起的肺组织炎症反应。