mRNA expression and RNA editing (2451 C-to-U) of IL-12 receptor beta2 in adult atopic patients

Interleukin (IL)-12 activates T helper (Th) 1 cells to produce interferon (IFN)-gamma which inhibits atopic inflammation. IL-12 acts through interaction with its receptor, especially beta(2) subunit. In several studies, the low production of IFN-gamma in peripheral mononuclear cells of atopic patients on response to IL-12 stimulation has been reported. Therefore we investigated the IL-12 receptor beta(2) (IL-12R beta(2)) mRNA expression and RNA editing, nucleotide 2451 C-to-U conversion, to find the cause of low responsiveness to IL-12 in atopy. Quantitative real time PCR for mRNA expression and sequence analysis for RNA editing were performed in 80 atopic patients and 54 healthy controls. The expression of IL-12R beta(2) mRNA was significantly lower in atopic patients than healthy controls (p<0.05). In sequence analysis, RNA editing on nucleotide 2451 was not found from either atopic patients or healthy controls. In additional evaluation, there was no relationship between expression of IL-12R beta(2) mRNA and serum total IgE or blood eosinophil count. Reduced IL-12R beta(2) mRNA expression in atopic patients indicate the reduced capacity to respond to IL-12 which induce IFN-gamma production and this may contribute to Th2-skewed immune response in atopy.     

IL-1 beta and IL-3-like activity in preterm infants.

The capacity of peripheral blood mononuclear cells (PBMC) of preterm neonates to release IL-1 beta and IL-3-like activity (IL-3-LA) has been investigated. In the present study it was found that this capacity is significantly lower than that of their mothers and of control adults. In addition, the results showed that preterm serum has a lower stimulatory effect on IL-1 beta production and an inhibitory effect on IL-3-LA secretion by PBMC of adult controls, in comparison with maternal and adult sera. These findings suggest an additional feedback mechanism for control of haematopoiesis in premature neonates. It is possible that the lower production of IL-1 beta and IL-3-LA may be involved in the increased susceptibility to infections of preterm newborns.     

Early induction of IL-1 receptor antagonist (IL-1Ra) in infants and children undergoing surgery.

The cytokine response to injury or trauma is of interest in terms of both its mediation of the acute phase response and its possible relation to the immunological depression observed after major surgery. In this study, the production of cytokines IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6 and the naturally occurring inhibitor of IL-1, IL-1Ra, have been investigated in infants and children undergoing Swenson's pull-through operation for Hirschsprung's disease. Samples of peripheral blood were taken before, during and after surgery for the measurement of cytokines. IL-1Ra levels increased significantly (P < 0.01) at 2 h after commencement of surgery, with maximal levels for individual patients being attained between 3 h and 5 h (range 7.6-67.9 ng/ml). The mean level of IL-1Ra was maximal (26.2 ng/ml) at 5 h and returned to baseline levels between 24 h and 72 h. There were no changes observed in the circulating levels of IL-1 beta in nine out of 11 patients following commencement of surgery. TNF-alpha levels did not increase in any of the patients studied. IL-6 levels increased significantly (P < 0.02) 3 h after commencement of surgery, reaching maximum concentrations at 24 h (range 20-670 pg/ml), with levels falling between 48 h and 72 h. This study demonstrates, in vivo, the independent induction of IL-1Ra without a concomitant increase of IL-1 beta levels after major surgery. It also shows that IL-1Ra is the earliest cytokine produced in response to surgical stress.     

Allelic polymorphism in IL-1 beta and IL-1 receptor antagonist (IL-1Ra) genes in inflammatory bowel disease.

Recent reports have shown that allele 2 of the IL-1 receptor antagonist (IL-1Ra) gene is over-represented in ulcerative colitis (UC). Healthy individuals carrying allele 2 of this gene have increased production of IL-1Ra protein. Since the final outcome of the biological effects of IL-1 beta may depend on the relative proportion of these two cytokines, we have studied if a TaqI polymorphism in the IL-1 beta gene, which is relevant to IL-1 beta protein production, may be involved in the genetic susceptibility to UC and Crohn's disease (CD), in association with the established IL-1Ra gene polymorphism. Polymorphisms in the closely linked genes for IL-1 beta and IL-1Ra were typed in 100 unrelated Dutch patients with UC, 79 with CD, and 71 healthy controls. The polymorphic regions in exon 5 of the IL-1 beta gene and in intron 2 of the IL-1Ra gene, were studied by polymerase chain reaction (PCR)-based methods. The IL-1 beta allele frequencies in UC and CD patients did not differ from those in healthy controls. In order to study if the IL-1 beta gene polymorphism might participate synergistically with the IL-1Ra gene polymorphism in susceptibility to UC and CD, individuals were distributed into carriers and non-carriers of allele 2 of the genes encoding IL-1 beta and IL-1Ra, in each of the patient groups and controls. Results indicated a significant association of this pair of genes, estimated by the odds ratio (OR) after performing Fisher's exact test, in the UC group (P = 0.023, OR = 2.81), as well as in the CD group (P = 0.01, OR = 3.79). Thus, non-carriers of IL-1 beta allele 2 were more often present in the subgroup of patients carrying the IL-1Ra allele 2. By contrast, no association of these alleles was detected in the group of healthy controls (P = 1.00, OR = 0.92). These results suggest that the IL-1 beta/IL-1Ra allelic cluster may participate in defining the biological basis of predisposition to chronic inflammatory bowel diseases.     

IL-2 receptor gene expression and IL-2 production by human preterm newborns' cells.

IL-2 receptor (IL-2R) gene expression in human umbilical cord blood mononuclear cells (CBMC) of preterm and term newborns was examined following stimulation for 18 h with phytohaemagglutinin (PHA) and compared with that of adult peripheral blood mononuclear cells (PBMC; mothers and control group). mRNA for IL-2R could not be detected in CBMC of preterm infants, whereas the mRNA levels for IL-2R found in full term neonates were similar to those observed in PBMC of adults. IL-2 activity in conditioned medium (CM) of mononuclear cells stimulated with either optimal or suboptimal PHA concentrations for 24 h and 48 h was also determined. At 24 h of stimulation, IL-2 activity found in CM obtained from CBMC of preterm and term newborns was significantly higher than that found in CM of adults' PBMC. A further enhancement of IL-2 activity (six to eight times) was observed in CM of preterm and term cells stimulated for 48 h, whereas no significant difference was found in IL-2 activity in CM from adult cells tested at the two incubation periods. The present findings may provide an additional explanation for the impaired function of the immune system, and the high susceptibility to infections observed in preterm newborns.     

IL-1 alpha gene expression and protein production by fibroblasts from patients with systemic sclerosis.

We examined IL-1 alpha and IL-1 beta gene expression and protein production in human dermal fibroblasts from patients with systemic sclerosis (SSc) to investigate the abnormal function of SSc fibroblasts. Human dermal fibroblasts were biopsied from 13 patients with SSc, three patients with rheumatoid arthritis (RA) and five healthy normal controls (NC). Cells were cultured in serum-free media and total RNA was collected from second or third passage fibroblasts. In cultured SSc fibroblasts, IL-1 alpha and IL-1 beta mRNAs were constitutively expressed and intracellular pro-IL-1 alpha was present. These observations suggest that an autocrine effect of IL-1 alpha contributes to the fibrosis in SSc.     

IL-12 receptor deficiency revisited: IL-23-mediated signaling is also impaired in human genetic IL-12 receptor beta1 deficiency

IFN-gamma and IL-12 are crucial cytokines for cell-mediated immunity against intracellular pathogens. We have previously shown that human IL-12Rbeta1-deficiency leads to impaired IL-12 responsiveness and unusual susceptibility to infections due to mycobacteria and salmonellae. IL-23 is a cytokine with functions that partially overlap with those of IL-12. IL-23 consists of IL-12p40 and a novel p19 protein, and binds to a receptor complex comprising IL-12Rbeta1 and IL-23R. Thus, IL-12Rbeta1-deficiency may impair both IL-12- and IL-23 signaling, and both may contribute to the immunological phenotypes. To examine whether IL-12Rbeta1 is essential for IL-23 signaling in human T cells, we have studied IL-23 responsiveness of four IL-12Rbeta1-deficient individuals. Whereas IL-23 promoted IFN-gamma production by CD4(+) and CD8(+) T cells in controls, IL-12Rbeta1-deficient T cells lacked IL-23-induced IFN-gamma secretion, but responded normally to IL-2, IL-4, IL-15 and IL-18. We also show that induction of IFN-gamma production by IL-23 depends upon TCR-ligation and is enhanced by CD28-costimulation. Furthermore, IL-23 cooperates with IL-12 and IL-18 in promoting IFN-gamma production in controls, but not in patients. We conclude that IL-12Rbeta1-deficiency impairs IL-12- and IL-23-dependent signaling in human T cells. The syndrome caused by IL-12Rbeta1-deficiency thus needs to be reinterpreted as resulting from defective IL-12-as well as IL-23-mediated immunity.     

Different lymphoid cell populations produce varied levels of neopterin, beta 2-microglobulin and soluble IL-2 receptor when stimulated with IL-2, interferon-gamma or tumour necrosis factor-alpha.

Immune activation is central to many immune disorders. Clinical investigations have shown that immune activation can be quantified by measurements of soluble immune activation products in serum. Most in vitro studies of these immune activation products have focused on single products. In this study the specific cell sources and the major lymphokines inducing multiple activation products were investigated. In vitro addition of interferon-gamma (IFN-gamma) or IL-2 stimulated peripheral blood mononuclear cells to produce neopterin, beta 2-microglobulin (beta 2-M) and soluble IL-2 receptor (sIL-2R). These two lymphokines can act independently, because neutralizing antibodies to one of the lymphokines did not block the inducing activity of the other. Tumour necrosis factor-alpha (TNF-alpha) was also investigated and shown to be a less powerful inducer than IL-2 or INF-gamma. Separated lymphoid subpopulations responded differently to specific lymphokines. Monocytes produced only neopterin and only in response to INF-gamma. T cells released beta 2-M and sIL-2R in response to IL-2. B cells, however, were capable of producing all three immune activation products. Neopterin production in B cells was induced by either INF-gamma of IL-2, indicating that B cells have additional mechanisms for responding to lymphokines. To investigate whether these in vitro findings also occur in vivo, sera from patients who had received either rIL-2 or INF-gamma treatment were tested. INF-gamma administration led to substantial increases in serum neopterin but only a moderate beta 2-M increase and no increase in the serum sIL-2R levels. rIL-2 administration caused a substantial increase of all three serum immune activation products, consistent with our in vitro findings. The results confirm that increased serum levels of soluble immune activation products are indicators of increased cytokine production by lymphocytes and monocytes and also that B cells can be a prominent source of immune activation products.     

Significance of IL-1β and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12·9 ± 9·5 months (mean ±s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL-1β and IL-8 were significantly higher in DPB compared with healthy volunteers (P < 0·05, P < 0·05, respectively). IL-1 Ra in patients is considered to have a weak inhibitory activity for IL-1β, with approximately five-fold concentration of IL-1β compared with that in healthy volunteers (approx. nine-fold concentration of IL-1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL-1Ra in BALF. The level of IL-1β correlated significantly with the concentration of neutrophils in BALF (r = 0·72, P < 0·01), as well as with the level of IL-1Ra (r = 0·688, P < 0·05) and IL-8 (r = 0·653, P < 0·05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL-1Ra or IL-8 in BALF (r = 0·526, P = 0·053 or r = 0·776, P < 0·01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r = 0·524, P < 0·05). Our results suggest that the relative amounts of IL-1β and IL-1Ra or IL-8 may contribute, at least in part, to the neutrophil-mediated chronic airway inflammation in patients with chronic airway disease, and long-term erythromycin therapy may down-regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil-mediated inflammatory response.     

Role of IL-18 in the secretion of Il-1beta, sIL-1RII, and IL-1Ra by human neutrophils

In the present study we investigated the effect of IL-18 on the production of IL-1β, IL-1Ra and sIL-1RII by human neutrophils. Our observations indicate that rhIL-18 induces IL-1β and, to a lesser extend, IL-1Ra and sIL-1RII production by human neutrophils isolated form peripheral blood. However, this effect was less important in comparison with LPS-stimulation. Moreover, the results obtained suggest that IL-18 can induce priming of neutrophils for IL-1β and, to a lesser extend, IL-1Ra and sIL-1RII production by LPS-stimulated cells. The capacity of IL-18 to serve as an effective modulator for IL-1β and its regulatory proteins may have significance in the inflammatory and immune reactions mediated by IL-1β.     

Detection of cytoplasmic IL-1 beta in peripheral blood mononuclear cells from intensive care unit (ICU) patients.

Cytokines including IL-1 beta have been implicated in the pathophysiology of sepsis and the systemic inflammatory response. It is believed that certain critically ill patients may be 'primed' with respect to cytokine production, and that subsequent 'triggers' may cause exaggerated cytokine production in these patients with exacerbation of their clinical condition; however, no means of identifying 'primed' patients has been described. The presence of cytoplasmic IL-1 beta within peripheral blood mononuclear cells (PBMC) from patients in the ICU was investigated as a means of identifying 'primed' patients, using fluorescent antibody labelling and flow cytometry. The study revealed that PBMC from ICU patients had a different staining pattern for IL-1 beta than those from healthy subjects, and that PBMC from certain ICU patients did indeed stain strongly for IL-1 beta; however, the presence of these strongly staining cells was not associated with clinical condition or outcome. It is concluded that whilst it might be possible to identify 'primed' patients in the ICU using this technique, this is of no clinical value as a predictor of clinical course.     

人IL-2mRNA的特性研究

通过测定人IL-2和IL-2mRNA的诱生动力学,在高峰时相从PHA+TPA刺激的人脾脏单个核细胞内用冷酚法提取了大量富含IL-2mRNA的胞浆RNA。用制备型柱状7M尿素—SDS—PAGE分离胞浆RNA,并结合麦胚无细胞转译体系和IL-2生物活性检测,测定出人IL-2mRNA全长片段约为14S。将转译IL-2(tIL-2)与基因工程IL-2(rIL-2)和部分纯化的天然IL-2(nIL-2)进行分子筛层析,证实三种IL-2洗脱峰基本相同,分子量约为14000道尔顿。tIL-2与IL-2R具良好的结合特性。     

Evidence for cell-specific changes with age in expression of oestrogen receptor (ER) alpha and beta in bone fractures from men and women

Oestrogen is recognized as important for maintaining bone mass in men and women. Oestrogen receptor (ER) alpha and the recently described ER-beta are both expressed in bone cells, but have different affinities for oestrogen agonists and plant oestrogens, which could be important in developing treatments for bone loss in both men and women. It is unclear, however, which isoform predominates in bone; cell type and age may influence their relative expression. The present study has compared ER-alpha and ER-beta expression in serial sections of human fracture callus from males (n = 19, age range 5-72 years) and females (n = 15, age range 3-86 years) by indirect immunoperoxidase. Fracture callus was used as it can be readily obtained from individuals over a wide age range and contains a variety of bone cells. Antibody specificity was confirmed by western blotting and comparison of immunoreactivity in sections of breast tumour and benign prostate hyperplasia. No gender difference in ER expression was found in bone from individuals less than 40 years old. Proliferative chondrocytes were positive for both isoforms, but few larger hypertrophic cells were immunoreactive. ER-alpha and ER-beta were co-expressed in osteoclasts, suggesting that oestrogen may act directly on these cells. Osteoblasts, osteocytes, and mesenchymal cells also expressed both isoforms. In women over 40 years of age, however, relatively fewer biopsies contained osteocytes positive for ER-alpha and ER-beta. Likewise, the proportions of osteoblasts and mesenchymal cells expressing ER-beta were reduced but ER-alpha remained unaffected. In contrast, in men over 40 years, only the proportion of biopsies containing ER-beta-positive mesenchymal cells was lower. In these older men and women, ER-alpha and ER-beta expression was retained by the small proliferative chondrocytes. These results demonstrate that gender, age, and cell type are important determinants of ER isoform expression in skeletal cells.     

Perioperative serum levels of tumour-necrosis-factor alpha (TNF-alpha), IL-1 beta, IL-6, IL-10 and soluble IL-2 receptor in patients undergoing cardiac surgery with cardiopulmonary bypass without and with correction for haemodilution.

Cardiac surgery with cardiopulmonary bypass (CPB) leads to a systemic inflammatory response with secretion of cytokines. Alterations in the serum concentrations of cytokines have important prognostic significance. Reports on cytokine release during cardiac surgery with CPB have yielded conflicting results. Haemodilution occurs with the onset of CPB resulting in large fluid shifts during the perioperative course of cardiac procedures. In the present study we compare the perioperative course of serum concentrations of TNF-alpha, IL-1beta, IL-6, IL-10 and sIL-2R with and without correction for haemodilution in patients undergoing coronary artery bypass grafting (CABG) surgery. Twenty male patients undergoing elective CABG surgery with CPB and general anaesthesia using a balanced technique with sufentanil, isoflurane and midazolam were enrolled into the study. Serum levels of TNF-alpha, IL-1beta, IL-6, IL-10 and sIL-2R were measured using commercially available ELISA kits. Simultaneous haematocrit values were obtained at all sample times. Statistical analysis was performed by non-parametric analysis of variance and t-tests for data corrected for haemodilution and data that were not corrected for haemodilution. Adjusted significance level was P < 0.01. Intra-operatively, up to the second post-operative day PCV values were significantly decreased compared with preoperative values. Cytokine measurements not corrected for haemodilution were significantly lower than the corrected values. The perioperative haemodilution and decrease in PCV may lead to an underestimation of the cytokine secretion in post-operative patients. We conclude that cytokine measurements were significantly influenced by the perioperative haemodilution and the subsequent decrease in PCV and may in part account for the varying results reported in the literature regarding cytokine release in patients undergoing CABG surgery.     

The Th1 and Th2 cytokines IFN-γ and IL-4 antagonize the inhibition of monocyte IL-1 receptor antagonist by glucocorticoids: involvement of IL-1

Monocytes express IL-1 and IL-1 receptor antagonist (IL-1Ra) in response to lipopolysaccharide (LPS). IL-1 self-induction contributes to the increase in IL-1 following LPS stimulation. LPS-stimulated IL-1 and IL-1Ra production are inhibited by glucocorticoids. In the present work we examined the regulation of IL-1Ra by Th1 cytokine IFN-γ, Th2 cytokine IL-4, glucocorticoids and IL-1 in human monocytes. We demonstrate that IL-1 contributes to LPS-induced IL-1Ra expression as shown by IL-1 blockade in LPS-stimulated monocytes using a specific anti-IL-1β antibody or recombinant IL-1Ra. Glucocorticoids inhibited IL-1β-stimulated IL-1Ra mRNA expression and protein production. Glucocorticoids inhibited both IL-1-mediated and non-mediated LPS stimulation of IL-1Ra expression. Both IFN-γ and IL-4 reversed the inhibitory effect of glucocorticoids on IL-1Ra expression and secretion. The effect of IFN-γ was blocked by pretreatment of monocytes with an anti-IL-1β blocking antibody, whereas the effect of IL-4 could not be blocked, demonstrating that IFN-γ acts through a mechanism dependent on endogenous IL-1 production, whereas IL-4 acts through an IL-1-independent one. Consistent with this finding, IFN-γ (but not IL-4) failed to reverse the inhibitory effect of glucocorticoids when stimulated by IL-1, and only IL-4 combined with IL-1 showed synergism resulting in an increase in IL-1Ra production. The differential regulation and involvement of IL-1 in the expression of IL-1Ra by IFN-γ, IL-4 and glucocorticoids sets the level of monocyte responsiveness during the Th1 or Th2 responses.     

Differences in the interleukin-2 (IL-2) receptor system in human and mouse: α chain is required for formation of the functional mouse IL-2 receptor

Reconstitution with mouse interleukin-2 (IL-2) receptor subunits demonstrated that the mouse IL-2 receptor complex was different from the human complex in the α chain requirement for the functional mouse receptor complex. The heterotrimeric complex of the mouse exogenous α and β chains and the endogenous γ chain on mouse lymphoid BW5147 cells showed the ability to bind IL-2 with high affinity, resulting in IL-2-induced tyrosine phosphorylation of a cytosolic tyrosine kinase, JAK3, which is involved in IL-2-dependent signals. Exogenous introduction of the β chain with the endogenous γ chain, however, could neither confer appreciable IL-2 binding nor IL-2-induced signal transduction on BW5147 cells, unlike the human βγ heterodimer. Mouse spleen CD8⁺ cells, not having the α chain initially, showed IL-2-dependent cell proliferation only when expression of the α chain was induced. Collectively, these results illustrate that the functional mouse IL-2 receptor complex necessarily includes the α chain, and that the regulation of CD8⁺ T cell growth during immune reaction depends upon α chain expression.