Th17细胞及白细胞介素-17在肝移植急性排斥反应中的意义

目的探讨Th17细胞及相关因子白细胞介素-17（IL-17）在肝移植急性排斥反应中的变化及意义。方法收集2011年1月至2012年12月大连医科大学附属第二医院肝移植手术患者28例,根据移植肝组织穿刺活检病理诊断结果将肝移植的28例患者分为急性排异反应组6例和无排斥反应稳定组22例,15名健康体检者作为对照组。急性排斥组及稳定组在移植术后3 d和7 d,行肝穿刺活检病理检查;同时检测受检者外周血Th17细胞,受检者血清中IL-17水平。结果移植肝穿刺活检病理诊断显示急性排斥组随着移植时间延长,排斥反应逐渐增强。肝组织出现典型的细胞免疫性病理损伤,术后7 d肝脏汇管区、肝实质、小静脉壁、胆管上皮内及小叶间胆管被大量的淋巴细胞及嗜中性粒细胞包绕及浸润,胆管上皮细胞内空泡形成、上皮细胞凋亡。病理改变明显比术后3 d严重;急性排斥组患者术后3 d和7 d外周血Th17细胞比例及血清中IL-17含量较稳定组和对照组均明显增多（P〈0.05）,且Th17细胞及IL-17在术后急性排斥期7 d值均明显高于3 d（P〈0.05）。结论 Th17细胞及IL-17在肝移植急性排斥反应的发生、发展中可能起着促进作用,外周血Th17细胞及IL-17的检测有可能成为肝移植急性排斥反应的早期诊断指标。     

PDIA3 mRNA expression and IL-2, IL-4, IL-6, and CRP levels of acute kidney allograft rejection in rat

Kidney transplantation to treat end-stage renal disease has evolved rapidly from the first successful transplantations to the current widespread use of grafts from both cadaveric and living donors. But acute rejection is still a strong risk factor for chronic rejection in recipients of renal grafts. To investigate possible mechanisms, we describe a comparison between differentially proteins expression and immune markers profile (IL-2, IL-4, IL-6, and CRP) of acute rejection and the controls. Through quantitative real-time RT-PCR confirmation, PDIA3 mRNA and protein expression levels in serum and transplanted kidney in experiment group was significantly (P < 0.05) higher than that in control group. Immunity analysis showed that plasma IL-2, IL-4, IL-6, and CRP levels were higher in experimental rats than those in control rats. Our data thus indicate that PDIA3 might be potentially involve into the occurence and development of acute rejection response in renal transplantation and increased plasma IL-2, IL-4, IL-6, and CRP levels play an important role to prevent acute kidney allograft rejection in rats.     

Total body irradiation of donors can alter the course of tolerance and induce acute rejection in a spontaneous tolerance rat liver transplantation model

Liver transplantation is an established therapy for end-stage liver diseases. Graft rejection occurs unless the recipient receives immunosuppression after transplantation. This study aimed to explore the mechanism of acute rejection of liver allografts in rats pre-treated with total body irradiation to eliminate passenger lymphocytes and to define the role of CD4⁺CD25⁺ regulatory T cells in the induction of immunotolerance in the recipient. Male Lewis rats were used as donors and male DA rats were recipients. Rats were randomly assigned to the following four groups: control group, homogeneity liver transplantation group, idio-immunotolerance group and acute rejection group. After transplantation, the survival time of each group, serum alanine aminotransferase, total bilirubin levels, number of Foxp3⁺CD4⁺CD25⁺ regulatory T cells, expression of glucocorticoid-induced tumor necrosis factor receptor on T cell subgroups, histopathology of the hepatic graft and spleen cytotoxic T lymphocyte lytic activity were measured. In the acute rejection group, where donors were preconditioned with total body irradiation before liver transplantation, all recipients died between day 17 and day 21. On day 14, serum alanine aminotransferase increased significantly to (459.2±76.9) U L^?1, total bilirubin increased to (124.1±33.7) ??mol L^?1 (P<0.05) and the ratio of Foxp3⁺CD4⁺CD25⁺ regulatory T cells decreased significantly to 1.50%±0.50% (P<0.05) compared with the other groups. Analysis of the T cell subpopulations in the acute rejection group varied from the other groups. Histological analysis showed typical changes of acute rejection in the acute rejection group only. Preconditioning of the donors with total body irradiation eliminated passenger lymphocytes of the liver graft, and thus affected the course of tolerance and induced acute rejection after liver transplantation.     

An atypical population of NK cells that spontaneously secrete IFN-gamma and IL-4 is present in the intraepithelial lymphoid compartment of the rat

The intestinal lymphoid compartment of the rat is large and diverse, but the phenotype and functions of its constituent cell populations are not fully characterized. Using new methodology for the isolation and purification of rat intestinal intraepithelial lymphocytes (IELs), we previously identified a population of alphabeta- and gammadelta-TCR- NKR-P1A+ NK cells. These cells were almost completely restricted to the CD4-CD8- IEL population, and unlike peripheral NK cells in the rat, they were CD2-. We now report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, however, intraepithelial and splenic NK cells differ markedly in two respects. First, IENK cells express high levels of ADP-ribosyltransferase 2 (a marker of regulatory T cells in the rat) and CD25, whereas peripheral NK cells do not. Second, unlike splenic NK cells, a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-gamma. We conclude that the rat IEL compartment harbors a large population of NKR-P1A+CD3- cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive IENK cells may participate in the regulation of mucosal immunity.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis

Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43). To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P. brasiliensis-sensitized subjects. gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10. Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM. gp43-pulsed adherent cells, but not non-adherent cells, were the main source of IL-10. Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects. Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients.     

Restricted T cell expression of IL-2/IFN-gamma mRNA in human inflammatory disease

It has been difficult to demonstrate functionally distinct T cell populations in humans on the basis of cytokine secretion. As previous investigators have examined the T cell cytokine profile from immunized animals, we examined whether Th1 or Th2 type T cells could be identified in the peripheral blood or cerebrospinal fluid (CSF) immune compartments from subjects with or without inflammatory diseases. Using limiting dilution analysis and growth with PHA and IL-2/IL-4, we directly cloned a total of 177 T cells from the peripheral blood and CSF of seven subjects, four with inflammatory disease and three control subjects, and examined the cytokine message profile after stimulation with ionomycin and PMA. We found that most clones from both the peripheral blood and CSF express IL-1, IL-2, IL-4, IFN-gamma, or TNF-alpha cytokine mRNA after activation with ionomycin and PMA. All T cell clones tested produced TNF-alpha mRNA, and all but 14 produced IFN-gamma mRNA. As reported previously, Th0 cells, which produced IFN-gamma, IL-2, IL-4, and IL-5 mRNA, were found in most subjects. In striking contrast, Th1 cells, which expressed IL-2 and IFN-gamma but not IL-4 or IL-5 mRNA, were present in both peripheral blood and CSF of subjects with inflammatory disease but not found in peripheral blood or CSF of subjects without systemic inflammation. Th2 cells, expressing IL-4 and IL-5 but not IFN-gamma or IL-2 mRNA, were not found in any subject. These data present the first evidence for Th1 T cell clones in humans that may be associated with systemic inflammation.     

Postoperative peak serum C-reactive protein is a predictor of outcome following liver transplantation for hepatocellular carcinoma

Context: C-reactive protein (CRP), a biomarker of inflammation, may correlate with prognosis in several malignancies.

Objective: To investigate the prognostic impact of early postoperative peak serum levels of CRP on tumor-specific outcome in 106 liver transplant patients with hepatocellular carcinoma (HCC).

Methods and results: In multivariate Cox regression analysis, a posttransplant elevated peak CRP level (>versus?≤?3.5?mg/dl) was identified as an independent predictor of poor recurrence-free survival (p?=?0.01; HR?=?4.04; CI?=?1.399–11.640).

Conclusion: Early postoperative serum CRP may serve as a useful inflammation-based biomarker of outcome in liver transplant patients with HCC.     

Establishment of animal model of dual liver transplantation in rat

The animal model of the whole-size and reduced-size liver transplantation in both rat and mouse has been successfully established. Because of the difficulties and complexities in microsurgical technology, the animal model of dual liver transplantation was still not established for twelve years since the first human dual liver transplantation has been made a success. There is an essential need to establish this animal model to lay a basic foundation for clinical practice. To study the physiological and histopathological changes of dual liver transplantation, "Y" type vein from the cross part between vena cava and two iliac of donor and "Y' type prosthesis were employed to recanalize portal vein and the bile duct between dual liver grafts and recipient. The dual right upper lobes about 45-50% of the recipient liver volume were taken as donor, one was orthotopically implanted at its original position, the other was rotated 180° sagitally and heterotopically positioned in the left upper quadrant. Microcirculation parameters, liver function, immunohistochemistry and survival were analyzed to evaluate the function of dual liver grafts. No significant difference in the hepatic microcirculatory flow was found between two grafts in the first 90 minutes after reperfusion. Light and electronic microscope showed the liver architecture was maintained without obvious features of cellular destruction and the continuity of the endothelium was preserved. Only 3 heterotopically positioned graft appeared patchy desquamation of endothelial cell, mitochondrial swelling and hepatocytes cytoplasmic vacuolization. Immunohistochemistry revealed there is no difference in hepatocyte activity and the ability of endothelia to contract and relax after reperfusion between dual grafts. Dual grafts made a rapid amelioration of liver function after reperfusion. 7 rats survived more than 7 days with survival rate of 58.3.%. Using "Y" type vein and bile duct prosthesis, we successfully established a novel rat model of dual right upper liver lobe transplantation.     

Identification of rat IL-1beta, IL-2, IFN-gamma and TNF-alpha in activated splenocytes by intracellular immunostaining.

We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1beta, IL-2, IFN-gamma and TNF-alpha. Cultured rat splenocytes were polyclonally activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-acetate) and a calcium ionophore (ionomycin). Careful selection of either antigen affinity-purified polyclonal or monoclonal cytokine-detecting antibodies combined with gentle fixation and permeabilization of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-gamma and TNF-alpha could be identified by a characteristic, intracellular, rounded, juxtanuclear immunofluorescence signal. This staining pattern reflected the accumulation of the intracellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1beta, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1beta producing macrophages expressed intense nuclear immunofluorescence with additional reticular, cytoplasmic signals. Furthermore, the use of biologically neutralizing detecting antibodies against the cytokines under study prevented recognition of surface-stained target cells that had bound secreted cytokines by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing cells in cultures of rat splenocytes after various modes of polyclonal activation.     

Gene and protein expression profiles in the foetal liver of the pregnant rat fed a low protein diet

Foetal growth is particularly sensitive to the protein content of the mother’s diet. Microarray data from the foetal liver of pregnant rats fed normal (HP) or reduced protein diets (LP) were compared by gene set enrichment analysis. Soluble proteins from a second portion of the liver were analysed by two-dimensional gel electrophoresis. Genes associated with progesterone, insulin-like growth factor-1 and vascular endothelial growth factor were upregulated in HP compared to LP, in addition to genes associated with cell differentiation and signalling from the extracellular matrix. In contrast, cytokine signalling was downregulated. Proteomics showed that proteins associated with amino acid metabolism, mitochondrial function and cell motility were differentially abundant in the HP compared to the LP groups. These growth factor and extracellular matrix signalling pathways linked to cell motility may be important mediators of the changes in liver structure that occur in utero and persist into adult life.     

Role of IFN-gamma and IL-2 in rat lung epithelial cell migration and apoptosis after oxidant injury

In the present study, IFN-gamma exposure to primary cultures of rat type II epithelial cells (TIIP) upregulated membrane expression of the common gamma-chain of the IL-2 receptor (approximately 2.5- to 4-fold increase) and redistributed receptor affinity in TIIP, as assessed by Western blot, cell, and tissue histochemistry and Scatchard analysis. As for restitution processes of the lung epithelium, functionality of IL-2R on TIIP was conditional to IFN-gamma exposure: 1) IFN-gamma priming promoted a fivefold increase of IL-2-driven TIIP locomotion (P < 0.05 vs. control at 100 U/ml) and 2) IFN-gamma coincubation with IL-2 reduced bleomycin-induced TIIP apoptosis in vitro by 25% (caspase-3 activity) and by approximately 70% (TdT-mediated dUTP nick end labeling/4',6'-diamidino-2-phenylindole assay) as well as in vivo by approximately 90% (caspase-3 activity; P < 0.05 vs. control). Sustained p42/44 extracellular signal-regulated kinase activity played a protective role in this process, whereas specific inhibition by PD-98059 (50 microM) significantly reversed bleomycin-induced TIIP apoptosis (P < 0.05 vs. control). From these in vitro and in vivo data, it is proposed that combinations of IFN-gamma and IL-2 can drive repair activity of TIIP by stimulating migration and preventing programmed cell death, both of which are speculated to be very fast restitution events after oxidant-induced acute lung injury.     

Polymorphisms in IL-4R alpha correlate with airways hyperreactivity, eosinophilia, and Ym protein expression in allergic IL-13-/- mice

The development of airways hyperreactivity in allergic IL-13(-/-) mice is controversial and appears to correlate with the number of times that the original 129 x C57BL/6 founder strain has been crossed to the BALB/c background. In this investigation, we compared allergic responses in founder IL-13(-/-) mice crossed for either 5 (N5) or 10 (N10) generations to BALB/c mice. Whereas allergic N5 IL-13(-/-) mice developed airways hyperreactivity, tissue eosinophilia, elevated IgE, and pulmonary expression of Ym proteins, these processes were attenuated in N5 IL-13(-/-) mice treated with an IL-4-neutralizing Ab, and in N10 IL-13(-/-) mice. These data showed that IL-4 was more effective in regulating allergic responses in N5 IL-13(-/-) mice than in N10 IL-13(-/-) mice. To elucidate the mechanism associated with these observations, we show by restriction and sequence analysis that N5 IL-13(-/-) mice express the C57BL/6 form of IL-4Ralpha and N10 IL-13(-/-) mice express the BALB/c form. Despite the near identical predicted molecular mass of these isoforms, IL-4Ralpha from N5 IL-13(-/-) mice migrates with a slower electrophoretic mobility than IL-4Ralpha from N10 IL-13(-/-) mice, suggesting more extensive posttranslational modification of the N5 form. The Thre(49)Ile polymorphism in the extracellular domain of BALB/c IL-4Ralpha has been demonstrated to disrupt N-linked glycosylation of Asn(47) and increase the dissociation rate of the IL-4Ralpha/IL-4 interaction. Collectively, these data show that polymorphisms in IL-4Ralpha, which have been shown to affect the interaction with IL-4, correlate with the ability of IL-4 to regulate allergic responses in IL-13(-/-) mice.     

IFN-gamma up-regulates IL-18 gene expression via IFN consensus sequence-binding protein and activator protein-1 elements in macrophages

Constitutive IL-18 expression is detected from many different cells, including macrophages, keratinocytes, and osteoblasts. It has been known that IL-18 gene expression is regulated by two different promoters (p1 promoter and p2 promoter). When RAW 264.7 macrophages were treated with IFN-gamma, IL-18 gene expression was increased in a dose- and time-dependent manner. IFN-gamma activated the inducible promoter 1, but not the constitutive promoter 2. Mutagenesis studies indicated that an IFN consensus sequence-binding protein (ICSBP) binding site between -39 and -22 was critical for the IFN-gamma inducibility. EMSA using an ICSBP oligonucleotide probe showed that IFN-gamma treatment increased the formation of DNA-binding complex, which was supershifted with anti-IFN regulatory factor-1 Ab and anti-ICSBP Ab. Another element, an AP-1 site between -1120 and -1083, was important. EMSA using an AP-1-specific oligonucleotide demonstrated that IFN-gamma or LPS treatment increased the AP-1-binding activity. The addition of anti-c-Jun Ab or anti-c-Fos Ab to IFN-gamma- or LPS-treated nuclear extracts resulted in the reduction of AP-1 complex or the formation of a supershifted complex. Taken together, these results indicate that IFN-gamma increased IL-18 gene expression via ICSBP and AP-1 elements.     

Regional differences in sexually dimorphic protein expression in the spontaneously hypertensive rat (SHR)

Hypertension is sexually dimorphic and modified by removal of endogenous sex steroids. This study tested the hypothesis that endogenous gonadal hormones exert differential effects on protein expression in the kidney and mesentery of SHR. At ~5 weeks of age male and female SHR underwent sham operation, orchidectomy, or ovariectomy (OVX). At 20-23 weeks of age, mean arterial pressure (MAP) was measured in conscious rats. The mesenteric arterial tree and kidneys were collected, processed for Western blots, and probed for Cu Zn superoxide dismutase (SOD1), soluble epoxide hydrolase (sEH), and Alpha 2A adrenergic receptor (A2AR) expression. MAP was unaffected by ovariectomy (Sham 164 ± 4: Ovariecttomy 159 ± 3 mm Hg). MAP was reduced by orchidectomy (Sham 189 ± 5:Orchidectomy 167 ± 2 mm Hg). In mesenteric artery, SOD1 expression was greater in male versus female SHR. Orchidectomy increased while ovariectomy decreased SOD1 expression. The kidney exhibited a different pattern of response. SOD1 expression was reduced in male compared to female SHR but gonadectomy had no effect. sEH expression was not significantly different among the groups in mesenteric artery. In kidney, sEH expression was greater in males compared to females. Ovariectomy but not orchidectomy increased sEH expression. A2AR expression was greater in female than male SHR in mesentery artery and kidney. Gonadectomy had no effect in either tissue. We conclude that sexually dimorphic hypertension is associated with regionally specific changes in expression of three key proteins involved in blood pressure control. These data suggest that broad spectrum inhibition or stimulation of these systems may not be the best approach for hypertension treatment. Instead regionally targeted manipulation of these systems should be investigated.     

IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.     

Reduced expression of GSTM2 and increased oxidative stress in spontaneously hypertensive rat

Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. The spontaneously hypertensive rat (SHR) is a well-characterized experimental model for essential hypertension. By comparative proteomics, we previously identified glutathione S-transferase, mu 2 (GSTM2), a protein involved in detoxification of reactive oxygen species, which had a significant reduction in left ventricles of 16-week-old SHR compared with WKY rats. In parallel, Western blotting and RT-PCR showed a similar reduction of GSTM2 in left ventricles and aortas of 4-, 8-, and 16-week-old SHR, which is before the onset of hypertension. This suggests that differential expression is not attributable to long-term changes in blood pressure. Meanwhile, the activities of GSTM2 were significantly decreased in different ages old SHR. Conversely, there was an enhanced generation of superoxide anion and activation of NADPH oxidase in SHR, which was accompanied by an increase in the protein expression of p47phox, a subunit of NADPH oxidase. These data suggest that it maybe a reduction in antioxidant defenses, evident by a reduced expression and activity of GSTM2, in the left ventricles and aortas of SHR that leads to increased levels of superoxide anion and activation of NADPH oxidase.