IL-1ra Transcripts Research Articles

Altered Treg and IL-1A Expression in the Immune Microenvironment of Lung Squamous-cell Cancer after EGFR Blockade

背景与目的精确靶向表皮生长因子受体（epidermal growth factor receptor, EGFR）的治疗在肺鳞癌、口腔和肠胃癌中取得了一定疗效，但会引发系统性炎症。本实验旨在探究EGFR抑制剂治疗癌引发的肿瘤内部免疫变化。方法我们通过含H-ras基因逆转录病毒转染EGFR基因缺失或野生型小鼠角质细胞，将改造的细胞同源移植至小鼠以成瘤，吉非替尼治疗荷瘤小鼠，流式细胞仪检测T细胞比例与程序性死亡受体1（programmed death 1, PD-1）表达，RT-PCR检测细胞因子与趋化因子的表达。结果敲除EGFR基因形成的肿瘤较野生型小，并且肿瘤微环境中浸润FoxP3+调节性T细胞（regulatory cells, Treg）细胞较少，FoxP3 RNA较少，程序性死亡受体1（programmed death 1, PD-1）阳性CD4+细胞比例降低。表明肿瘤细胞可以自主调节肿瘤微环境。野生型成瘤模型使用吉非替尼治疗1周显示，相对于对照组肿瘤较小；在这短期的药理模型中，同样观察到FoxP3+细胞、FoxP3 RNA减少的趋势，同时IL-1A/IL-1RA比例明显升高，表明相对短暂的系统性抑制EGFR信号通路可改变靶向肿瘤的免疫微环境。结论肿瘤细胞自发（基因）或系统性（药理作用）的抑制EGFR信号通路可减少肿瘤的生长和肿瘤微环境中Treg的渗透。EGFR依赖性Treg细胞增强肺鳞癌的生长，是EGFR抑制剂治疗的靶标。

Cell autonomous or systemic EGFR blockade alters the immune-environment in squamous cell carcinomas.

Targeting mutations and amplifications in the EGFR has been successful precision therapy for cancers of the lung, oral cavity and gastrointestinal track. However, a systemic immune reaction manifested by dose-limiting inflammation in the skin and gut has been a consistent adverse effect. To address the possibility that intra-tumoral immune changes contribute to the anti-cancer activity of EGFR inhibition, squamous cancers were produced by syngeneic orthografts of either EGFR null or wildtype mouse primary keratinocytes transduced with an oncogenic H-ras retrovirus. Flow cytometric, RNA and Bioplex immunoassay analyses of the tumor immune milieu were performed. Cancers forming from keratinocytes genetically depleted of EGFR were smaller than wildtype cancers and had fewer infiltrating FoxP3 Treg cells, lower Foxp3 RNA and a lower percentage of CD4 PD1 positive cells indicating a tumor cell autonomous regulation of its microenvironment. Hosts bearing wildtype cancers treated with gefitinib for 1 week showed a trend for smaller tumors. In this short term pharmacological model, there was also a trend to reduced FoxP3 cells and FoxP3 RNA in the tumors of treated mice as well as a substantial increase in the ratio of IL-1A/IL-1RA transcripts. These results suggest that relatively brief systemic inhibition of EGFR signaling alters the immune environment of the targeted cancer. Together these data imply that an EGFR dependent Treg function supports the growth of squamous cancers and is a target for the therapeutic activity of EGFR inhibition.

A cytokine network involving brain-borne IL-1β, IL-1ra, IL-18, IL-6, and TNFα operates during long-term potentiation and learning

We have previously shown that long-term potentiation (LTP) induces hippocampal IL-1β and IL-6 over-expression, and interfering their signalling either inhibits or supports, respectively, LTP maintenance. Consistently, blockade of endogenous IL-1 or IL-6 restricts or favours hippocampal-dependent memory, effects that were confirmed in genetically manipulated mice. Since cytokines are known for their high degree of mutual crosstalk, here we studied whether a network of cytokines with known neuromodulatory actions is activated during LTP and learning. We found that, besides IL-1β and IL-6, also IL-1 receptor antagonist (IL-1ra) and IL-18, but not TNFα are over-expressed during LTP maintenance in freely moving rats. The increased expression of these cytokines is causally related to an increase in synaptic strength since it was abrogated when LTP was interfered by blockade of NMDA-glutamate receptors. Likewise, IL-1 and IL-6 were found to be over-expressed in defined regions of the hippocampus during learning a hippocampus-dependent task. However, during learning, changes in IL-18 were restricted to the dorsal hippocampus, and no differences in TNFα and IL1-ra expression were noticed in the hippocampus. Noticeably, IL-1ra transcripts were significantly reduced in the prefrontal cortex. The relation between cytokine expression and learning was causal because such changes were not observed in animals from a pseudo-trained group that was subject to the same manipulation but could not learn the task. Taken together with previous studies, we conclude that activation of a cytokine network in the brain is a physiologic relevant phenomenon not only for LTP maintenance but also for certain types of learning.

A cytokine network in the brain during synaptic plasticity and learning

Our previous studies have shown, in vivo and in vitro , that long-term potentiation (LTP) induces an NMDA receptor-dependent expression of IL-1β and IL-6 in the hippocampus. Blockade of the receptors for these cytokines either decreases (IL-1) or supports (IL-6) LTP maintenance, indicating the physiological relevance of their effects. Consistently, interference with IL-1 or IL-6 signalling also restricts or favours, respectively, hippocampus-dependent learning. The question that arises is whether brain-borne cytokines that interact with each other affect learning and memory consolidation just because they have a tonic, permissive effect or because their production increases during learning and are therefore part of the neuro-chemical changes involved in this process, as it occurs during LTP. Here we provide evidence that, besides IL-1β and IL-6, the endogenous IL-1 receptor antagonist (IL-1ra) and IL-18, but not TNF α , are produced during LTP in freely moving rats. Furthermore, we found that IL-1β and IL-6 genes are over-expressed in different hippocampal regions soon after learning a hippocampus-dependent alternation task. Restricted changes in IL-18 and no differences in TNF α and IL1-ra expression were noticed in the hippocampus during this learning process, but IL-1ra transcripts were significantly reduced in the prefrontal cortex. These effects were not noticed in pseudo-trained rats that could not learn the task. Taken together with previous studies, we conclude that activation of a cytokine network in the brain is a physiologic relevant condition not only for LTP maintenance but also for the consolidation of the learning process.

Molecular cloning and expression of a functional anti-inflammatory protein, Sj16, of Schistosoma japonicum

Schistosomes are the causative agent of schistosomiasis. In the infected host, significant inflammatory response to the parasite is not observed. Previous studies of Schistosoma mansoni showed that this subdued inflammatory response was due to a 16-kDa protein, Sm16, which is present in high levels in the secretions of schistosomula. Here we report the cloning and characterization of a gene (named Sj16) from Schistosoma japonicum. Sequence analysis showed that Sj16 shares 99% identity with Sm16 in its nucleotide sequence, and 100% identity in its protein sequence. While previous studies reportedly failed to obtain the soluble recombinant protein of Sm16, we expressed and purified recombinant Sj16 (rSj16) from Escherichia coli. Western blot and ELISA analyses showed that S. japonicum-infected rabbit sera could not recognize rSj16, indicating that native Sj16 may fail to induce circulating antibodies during S. japonicum infection. In vivo, rSj16 dramatically suppressed the recruitment of thioglycollate-mediated leukocytes to the peritoneal cavity of BALB/c mice, accompanied by marked up-regulation of IL-10 and IL-1RA transcripts, and down-regulation of IL-12p35, IL-1β and MIP-2 transcripts in peritoneal cells. Further analysis revealed that rSj16 also suppressed thioglycollate-induced peritoneal macrophage maturation. These results demonstrate that rSj16 has an anti-inflammatory function.

Leptin induces IL-1 receptor antagonist expression in the brain

The ob gene product, leptin, is known to be an important circulating signal for regulation of food intake and body weight. We report here that peripherally applied leptin increased interleukin-1 receptor antagonist (IL-1ra) transcripts in the hypothalamus. Interestingly, we observed leptin-induced increase in IL-1ra transcripts not only in the hypothalamus but also in other brain regions such as the hippocampus, the cortex, the cerebellum, and the brain stem. Moreover, leptin applied to the db/db mice, which lack functional Ob-Rb receptor, increased IL-1ra mRNA levels in the hyothalamus to a similar extent as in normal mice. These results indicate that the leptin-induced IL-1ra expression in the brain may be mediated through STAT3 independent mechanisms.

A POSSIBLE ROLE OF IL-1RA 3′-UNTRANSLATED REGION IN MODULATION OF PROTEIN PRODUCTION

Human interleukin 1 (IL-1) receptor antagonist (hIL-1ra), an anti-inflammatory cytokine and naturally occurring antagonist of IL-1, may be regulated at both the transcriptional and post-transcriptional levels. The aim of this study was to evaluate post-transcriptional regulation of hIL-1ra with specific focus on the 3′-untranslated region (3′-UTR) of hIL-1ra mRNA using the luciferase reporter gene system. Constructs were created containing the luciferase reporter gene followed by the hIL-1ra 3′-UTR or its modified variants. In monocyte/macrophage cell lines RAW264.7 and U937, the presence of the hIL-1ra 3′-UTR resulted in a 5.7-fold (n=6, P<0.001) and a 3.9-fold (n=7,P <0.001) decrease in transient reporter gene expression, respectively, with only a less than 2-fold mean difference in steady-state mRNA levels in the former case. In a cell-free translation system, the presence of the middle segment of the 3′-UTR caused a 5.2-fold (n=5, P<0.001) decrease in the amount of luciferase synthesized. In contrast to synthetic 3′-UTR and that derived from bovine growth hormone RNA, the presence of hIL-1ra 3′-UTR resulted in accumulation of unprocessed transcripts in transfected cells. We conclude that hIL-1ra synthesis may be regulated at the post-transcriptional level through mechanisms involving the 3′-UTR of IL-1ra transcripts.

Expression and localization of IL-1beta mRNA is interrelated with cytoskeletal rearrangement in monocytes stimulated by adherence: a light microscopy in situ hybridization study.

Differences in IL-1beta mRNA expression, stability and translation between non-adherent monocytes and those stimulated by adherence suggest that cytokine regulation is coupled to the function and assembly of cytoskeletal structures. In situ hybridization studies were performed to visualize expression and positioning of IL-1beta mRNA in adherently cultivated monocytes. IL-1beta mRNA expression was heterogeneous with high transcript levels found in spread or polarized cells. Transcripts were compartmentalized to the perinuclear region in spread cells, and partially redistributed with polarization. In contrast to mRNA distribution in other motile cell populations, IL-1beta mRNA did not localize to the distal or proximal actin cytoskeleton. Perinuclear confinement of transcripts required intact actin microfilaments. Treatment with cytoskeleton disruption and detergent extraction suggested that most non-translated IL-1beta mRNA was associated with intermediate filaments. In monocytes stimulated by LPS, IL-1beta, but not IL-1Ra transcripts were redistributed and partially associated, yet not bound to actin microfilaments. The present study demonstrates that IL-1beta mRNA expression and localization in adherent monocytes is interrelated with the cytoskeletal rearrangement upon adherence, spreading and polarization.

SCF, IL-1β, IL-1ra AND GM-CSF IN THE BONE MARROW AND SERUM OF NORMAL INDIVIDUALS AND OF AML AND CML PATIENTS

This study compared cytokine transcript and protein levels in BM cells of normal individuals and leukemic patients. AML differed from normal in that: (1) AML marrow cells contain more IL-1β protein than normal cells, (2) IL-1ra transcripts are absent from AML marrow cells, (3) AML marrow serum contains less IL-1ra protein than normal, (4) peripheral blood and marrow serum of AML patireents contains more SCF protein than normal serum, and (5) SCF transcripts have been detected in AML marrow biopsies and not in aspirate cells. These data suggest that unbalanced cytokine production may make a significant contribution to the abnormal behaviour of AML cells.

Expression of interleukin-1 genes and interleukin-1 receptors in the mouse brain after hippocampal injury.

In order to evaluate the role of IL-1 production in post-traumatic brain, transcripts for IL-1 (alpha, beta, RA) have been quantified following RT-PCR, in hippocampus and cortex after injury of either hippocampus (Hip) or striatum (Stri). Moreover, 125I IL-1alpha binding sites have been directly quantified using binding experiments on brain sections and quantitative autoradiography. Under basal conditions, levels of PCR products were very low. On day 1, IL-1RA transcripts only were strongly increased in the hippocampus after Hip-lesions and in cortex after Stri lesion. Transcripts were back to control values on day 7 post-lesion. IL-1 receptor densities in the hippocampus (dentate gyrus) were decreased at day 1 around the site of the lesion (but not on the contralateral side) and were back to controls on day 7 indicating a transient and local IL-1 production in the surroundings of the lesion. No changes were found following Stri lesion. This study provides further evidence of the role of the IL-1 molecules family, notably IL-1RA, in the brain reaction to trauma.

INTERLEUKIN 1 RECEPTOR ANTAGONIST LOCALIZATION EXCLUSIVELY IN NON-PARENCHYMAL CELLS OF THE MOUSE LIVER

The natural interleukin 1 receptor antagonist (IL-1ra) is induced in mouse liver after lipopolysaccharide (LPS) or recombinant interleukin 1 (IL-1) administration. Changes in hepatic IL-1ra mRNA concentration were measured following LPS injection to CBA mice. The results showed, that in vivo, IL-1ra transcripts were stimulated early at about 2 h with maximal elevation at 4–6 h. Northern blot analysis of RNA extracted from primary cultures of mouse hepatocytes did not detect IL-1ra mRNA, although constitutive and stimulated contrapsin mRNA expression were easily demonstrated. Therefore, induction of IL-1ra mRNA in sinusoid lining cells of the mouse liver at 4 h after LPS injection was evidenced by in situ hybridization using the same specific cDNA probe as in the Northern blotting technique. Additionally, immunohistochemical studies revealed that IL-1 receptor antagonist protein was induced with LPS after 19 hours in sinusoid lining cells. The data deriving from Northern blotting, in situ hybridization and immunohistochemical studies, suggest that IL-1ra is formed exclusively in non-parenchymal liver cells.

Induction by transforming growth factor‐β1 of the interleukin‐1 receptor antagonist and of its intracellular form in human polymorphonuclear cells

The aim of this study was to examine the expression of interleukin-1 receptor antagonist (IL-1ra) in human polymorphonuclear cells (PMN) treated with transforming growth factor-beta 1 (TGF beta 1). TGF beta 1 induced IL-1ra transcripts in human circulating PMN and the induction was not blocked by protein synthesis inhibitors. Actinomycin D blocked induction by TGF beta 1 of IL-1ra transcripts, suggesting the involvement of gene transcription. The half life of IL-1ra transcripts was prolonged by TGF beta 1. By reverse transcriptase-polymerase chain reaction, TGF beta 1 was found to augment the transcripts coding for both the intracellular (keratinocyte type) and the secreted form of IL-1ra. TGF beta 1 induced the production of IL-1ra in PMN. Induction of IL-1ra by TGF beta 1 in PMN may represent a further mechanism by which this molecule can counteract the potent pro-inflammatory properties of IL-1.

Interleukin-13 Induces the Production of Interleukin-1 Receptor Antagonist (IL-lra) and the Expression of the mRNA for the Intracellular (Keratinocyte) Form of IL-lra in Human Myelomonocytic Cells

The aim of this study was to examine the expression of interleukin-1 receptor antagonist (IL-1ra) in human myelomonocytic cells treated with IL-13. IL-13 induced IL-1ra transcripts in human circulating monocytes and polymorphonuclear cells (PMN). Induction by IL-13 was not blocked, but rather superinduced, in the presence of the protein synthesis inhibitor cycloheximide. Actinomycin D blocked induction, suggesting involvement of gene transcription. The half-life of IL-1ra transcripts was prolonged by IL-13 from 1.3 hours to 4.5 hours in monocytes and to 12 hours in PMN. By reverse transcriptase-polymerase chain reaction, IL-13 was found to augment the transcripts coding for the soluble form of IL-1ra, but also to induce the expression of the intracellular (keratinocyte) form of IL-1ra, the latter being extremely low or undetectable in myelomonocytic cells. IL-13 induced production of IL-1ra in myelomonocytic cells, augmenting both cell-associated and released protein. Induction of IL-1ra by IL-13 may represent a further mechanism by which this molecule can counteract the potent proinflammatory properties of IL-1.

Interleukin-13 induces the production of interleukin-1 receptor antagonist (IL-1ra) and the expression of the mRNA for the intracellular (keratinocyte) form of IL-1ra in human myelomonocytic cells

The aim of this study was to examine the expression of interleukin-1 receptor antagonist (IL-1ra) in human myelomonocytic cells treated with IL-13. IL-13 induced IL-1ra transcripts in human circulating monocytes and polymorphonuclear cells (PMN). Induction by IL-13 was not blocked, but rather superinduced, in the presence of the protein synthesis inhibitor cycloheximide. Actinomycin D blocked induction, suggesting involvement of gene transcription. The half-life of IL-1ra transcripts was prolonged by IL-13 from 1.3 hours to 4.5 hours in monocytes and to 12 hours in PMN. By reverse transcriptase-polymerase chain reaction, IL- 13 was found to augment the transcripts coding for the soluble form of IL-1ra, but also to induce the expression of the intracellular (keratinocyte) form of IL-1ra, the latter being extremely low or undetectable in myelomonocytic cells. IL-13 induced production of IL- 1ra in myelomonocytic cells, augmenting both cell-associated and released protein. Induction of IL-1ra by IL-13 may represent a further mechanism by which this molecule can counteract the potent proinflammatory properties of IL-1.

Expression of interleukin-1 receptor antagonist (IL-1ra) by human circulating polymorphonuclear cells.

After appropriate stimulation, mononuclear phagocytes express a specific inhibitor of interleukin (IL)-1, now re-named IL-1 receptor antagonist (IL-1ra). In this study we have examined the production of IL-1ra by polymorphonuclear cells (PMN). Human PMN isolated from peripheral blood expressed low but detectable levels of IL-1ra transcripts, which were considerably augmented after treatment with lipopolysaccharides (LPS) and cytokines [IL-4, granulocyte (G)- and granulocyte macrophage (GM)-Colony Stimulating factor (CSF), and tumor necrosis factor (TNF)]. The levels of induced IL-1 ra transcripts were comparable to those observed in endotoxin-stimulated human monocytes. By contrast IL-1 beta, interferon (IFN)-gamma and chemotactic factors (fMLP, C5a and IL-8) failed to promote IL-1ra expression in PMN. IL-1ra induction by LPS reached peak levels at 10 ng/ml after 3-6 h and remained sustained 24 h after stimulation. Induction by LPS and GM-CSF appears to be at the transcriptional level, as assessed by inhibiting mRNA synthesis with actinomycin D. Inhibition of protein synthesis by cycloheximide superinduced both basal and inducible IL-1ra mRNA. In addition to expressing mRNA, PMN also produce IL-1ra protein. Secretion of IL-1ra was induced in PMN treated with LPS, IL-4 and GM-CSF, but not by IL-1 beta, IFN-gamma and fMLP, thus yielding results that paralleled those seen in Northern blot experiments. These data indicate that, among myelomonocytic cells, PMN, in addition to mononuclear phagocytes, can express IL-1ra, suggesting that PMN, while exerting a series of pro-inflammatory activities, may also modulate the inflammatory potential of IL-1 in tissues.