| BackgroundIntestinal ischemia-reperfusion (IIR) is an important pathophysiological factor, contributory to systemic inflammatory response syndrome (SIRS), resulting in multiple organ dysfunction syndrome (MODS). Early intervention on the inflammatory response during IIR is an effective way to reduing MODS-associated mortality of critically ill patients.Development of SIRS depends on body's immune response that consists of adaptive immunity and innate immunity. Our prior study revealed activation of TLRs-NF-[kappa]B-proinflammatory gene expression, but decreased IgA, SC, SIgA in intestine during IIR macaque, reflecting excessive innate immunity and reduced adaptive immunity in intestinal mucosal immunity. IFN-γis an important cytokine responsible for regulation of adaptive and innate immunity. However, whether and how IFN-γ, is involved in intestinal mucosal immunity and SIRS post IIR remains to be determined. Cytokines always works closely with other hormones, neuropeptides and neurotransmitters, constituting a particular intercellular network. Role of somatostatin (SST), as a kind of important physiological neuropeptide in the production and regulation of IFN-γis also needed to characterize. Our research may help with understanding intestinal mucosal immunity after IIR and providing a new insight into treatment of SIRS and MODS.Intestinal ischemia reperfusion plays an important role in pathophysiological process of sever acute pancreatitis (SAP) to multiple organ disfunction. It is unclear if IIR aggravate inversely the inflammatory and histopathologic changes of pancreas. With the use of SST in treating SAP patients, it remarkedly reduced the incidence of MODS after SAP. Is it the result of suppressing the production of serum amylase levels? Does SST have a pivotal role in preventing second hit of pancreas during SIRS secondary to IIR? By answering this question, it will change our thoughts on prevention and treatment of SIRS and MODS, and will help to understand pharmacological mechanism of anti-inflammatory of SST.Traditionally, adaptive immunity mediated by B lymphocyte is regarded as an important factor in intestinal mucosal immunity. Our prior study indicates that increased homing of intestinal mucosal lymphocytes is involved in intestinal ischemia-reperfusion of rats; B lymphocytes in macaques peyer's patches are obviously increased after IIR. But sIgA, as a feature of intestinal acquired immunity, is found to decrease. Recent studies have proved that that B lymphocytes is involved in innate immunity and in acquired immunity. Do B lymphocytes aggragating in peyer's patches participate in excessive intestinal innate immunity? If yes, which type or which stage B lymphocytes have such function? Do immunological memory B lymphoblasts participate in innate immunity? By answering these questions, we should recognize new checkpoints and are able to understand the mechanism of SIRS and MODS better.Material and Methods1. 15 macaques with weighing 6.9±1.7Kg obtained from ChengDu safari park (ChengDu, china) were divided into three groups: normal control, IIR group and IIR+SST group.2. The animals were anesthetized with intramuscular xylazine compound (The military veterinary institute, quartermaster university of PLA, Changchun, China) at a dosage of 0.2±0.1ml/kg, followed by intravenous administration of diazepam at a dose of 0.16±0.09mg/kg/h. In IIR and IIR+SST group, A 5-cm vertical median incision at the epigastrium was made in animals after asepsis and antisepsis were completed. About 20% of initial blood volume removed [removed blood volume(ml)=weight(g)×5%] from inferior caval vein with a syringe and partial blood samples were used for organ functional laboratory tests. The superior mesenteric artery (SMA) was isolated near its aortic origin and was looped with a 7 silk suture. The SMA was occluded for 1h and reopened with this silk suture loop and a vascular clamp. During the experiment, the abdominal wall was kept approximated to prevent fluid and heat loss. One hour later, normal saline solution containing 20g glucose was infused into animal via posterior limb venous at a rate of 0.1～0.2ml/kg/min throughout the study.3. All 5 macaques were treated with somatostatin-14(Serono, Genevese, Switzerland) added to the normal saline solution at a dose of 5ug/kg/h iv prior to the onset of occluding superior mesenteric and through out 24 hours. Otherwise in control group, all animals received the same treatment except IIR operation.4. Tissues of ileum and pancrea were fixed in 4% buffered-paraformaldehyde solution and embedded in paraffin. Paraffin sections (5μn) were prepared and stained with haematoxylin and eosin. Injury was observed under macro- and microscopic examination while sections were scored with a semiquantitative scale designed to evaluate the degree of organ' injury.5. SST concentration in peripheral intravenous blood was measured using a radioimmunoassay method.6. Ileum tissues and specimens of ileac epithelial cells, ileac lymphocytes, supernatant fluids ofileac lymphocytes incubation, peripheral blood, portal vein blood were obtained for detection the level of IFN-γusing ELISA.7. The concentration of tumor necrosis factor-alpha (TNF-alpha), interleukin-1belta(IL-1β) and interleukin-6 (IL-6) in peripheral intravenous blood and Ileum tissues were measured using ELISA kit.8. The serum levels of amylase (AMY) and lipase were determined by chromometry.9. Immunohistochemistry were performed to identify the distribution of IFN-γ, CD4, CD8, CD57, CD20, CD5, PAX-5, plasma cell, TLR4, TLR2, NF-κB, integrinα4β7 and semi-quantitative immunohistochemical analyse was used to score integrated optical density(IOD) and ratio of area of object to total area of image(per area).10. Blood culture ofpotal vein. 11. Human B lymphoblast cell line was cultured and divided into two groups. One is interferenced in medium supplemented with LPS (10μg/ml) and the other is control group. Stock cultures and the experimental cultures were maintained at 37℃in a moist atmosphere of 95% air and 5% CO2.12. The concentration of IL-6 and IgM in culture cells supernatant fluid was determined by ELISA.13. The expression of TLR2 and TLR4 ofhmy2. cir was detected by immunofluorescence.14. MTT assay was used to measure the proliferation.Results1. IFN-γlevel in the ileac epithelial cells and potal vein blood after IIR were both remarkably increased ( 30.7±20.1 pg vs 9.4±2.4pg/mg total protein; 18.1±1.1 pg/ml vs 8.74±1.3 pg/ml), p＜0.05; Whereas IFN-γlevel in the ileac lymphocytes and supematant fluid of that of IIR were reduced (1.7±0.02pg/mg vs 11.1±4.0pg/mg total protein; 2.2±0.3pg/mg vs 5.2±1.5pg/mg total protein).2. After the injection of SST, IFN-γlevel decreased to 13.6±4.9pg/mg total protein, p=0.055 in the ileac epithelial cells, while in the potal vein blood to 8.9±2.5, p＜0.05. However, SST had no effect on IFN-γin the ileac lymphocytes and supernatant fluid.3. There is no change of IFN-γlevel in tissue homogenates and peripheral blood among three groups. Distribution of CD4+, CD57+ cells has no relationship with IFN-γ, while that of CD8+ cells demonstrated a conflicting tendency to IFN-γ.4. Numbers and areas of peyer's patches were significantly increased postoperatively in IIR groups as compared to NC group.5. In peyer's patches, the phenotype of B lymphocyte is PAX-5+CD20+CD5- in NC groups deemed as B2 lymphocyte. In contrast, PAX-5+ B lymphocytes are increased (p＜0.05) and PAX-5+CD20-D5- B lymphocytes(pro-B cells) are observed in peyer's patches of IIR groups. In NC group, plasm cells of mucosa locate mainly in lamina propria but decrease significantly in IIR group(p＜0.05).6. In NC group, there is weak expression of TLR2, TLR4, NF-κB and IFN-γin peyer's patches. IIR significantly increases the expression of the four molecules.7. Regarding toα4β7, there is sporadic expression in normal while IIR group has no expression in peyer's patches.8. Compared to IL-1β(peripheral intravenous blood: 27.3±7.17ng/ml; ileac tissues: 82.8±20.5pg/g),IL-6(peripheral intravenous blood: 13.9±10.50ng/ml; ileac tissues: 709.6±211.2pg/g),TNF-α(peripheral intravenous blood: 3.04±1.01ng/ml; ileac tissues: 56.0±10.04pg/g)in normal group, these are increased in IIR group: IL-1β(peripheral intravenous blood: 79.2±14.4ng/ml; ileac tissues: 294.0±46.4pg/g), IL-6(peripheral intravenous blood: 1261±297.5ng/ml; ileac tissues: 1527±160.8pg/g), TNF-α(peripheral intravenous blood: 64.8±18.7ng/ml; ileac tissues: 213.2±29.2pg/g)significantly(p＜0.05). Pretreatment of the macaques with somatostatin significantly inhibited the increased secretion of IL-1β(40.0±9.9 ng/ml), IL-6(244.4±70.0 ng/ml),TNF-α(19.2±10.1 ng/ml) compared with those of IIR group (p＜0.05).9. In NC group, all blood cultures of potal vein were negative, whereas all animals' speciments in IIR group were positively. Colon bacillus is the only bacterial translocation ingredient.10. Supernatant IgM levels(0.528±0.002 ng/ug total protein) were decreased 72 h after LPS treatment, with significant difference compared to control((0.617±0.057ng/ug total protein) (p＜0.05).11. Hmy2. cir cells showed a higher IOD of TLR4 (1201.3±913.4)and TLR2(174.8±80.2) 72 h after LPS stimulation compared with those of control group (TLR4: 5078.2±2483.2; TLR2: 1463.5±51.2) (p＜0.05).12. LPS interference significantly decreases IL-6(0.528±0.002 pg/ug total protein) secretion from hmy2. cir cells compared to that of control group(0.406±0.218pg/ug total protein(p＜0.05).13. Hmy2. cir cells displayed declined proliferation(0.389±0.061) after 24 h in response to LPS than that of control group(0.727±0.245). 72 h after LPS treatment enhanced proliferation(0.562±0.219) was found in comparation to control group(0.173±0.079) (p＜0.05).14. Activities of AMY (2 492.0±2 102.8 IU/L vs 385.4±136.1 IU/L) and lipase (1 278.0±1,446. 3 IU/L vs 25.8±13.0 IU/L) were increased (p＜0.05). Pretreatment of the macaques with somatostatin significantly inhibited secretion of AMY(567.0±338.0 IU/L)and lipase(77.2±77.9 IU/L) in peripherial blood (p＜0.05)。15. After IIR injury, somatostatin leveI detected by RIA significantly decreased (62.17±13.56 pg/ml) in comparation to NC group(160.61±33.84 pg/ml) (p＜0.05) and got correct after SST treatment(156.32±30.25 pg/ml)(p＜0.05).16. I1R group exhibited histopathologic injury of pancreas which was reduced from +++ to ++ after SST pretreatment.Conclusion1. After IIR, intestinal epithelial cells and pro-B lymphocytes produced a large number of IFN-γ, interacting with FcεR on mast cells in intestine, mucosa and tung where resulting in excessive innate immunity. In other words, IFN-γ, connected TLR- NF-κB pathway of innate immunity with mast cells. Thus it produced a sophisicated and highly active network of innate immunity.2. After IIR, pro-B lymphocytes in intestine participate in intestinal mucosal innate immunity. In addition, we hypothesize that serum or even marrow pro-B lymphocytes may be activated by LPS through TLR. Then is also involved in innate immunity and SIRS states of circulation by producing numerous IL-1β, IL-6, TNF-α. This is one of prominent causes of SIRS.3. After IIR, lack of rapid and vigorous response, PAX-5+CD20+ CD5- memory B lymphoblast does not exhibit function of innate immunity.4. B lymphocytes are functional both in innate immunity and in acquired immunity but exert one immunomodulatory function at each stages of development. Pro-B cells may be activated by LPS through expression of TLRs. TLRs signal through NF-κB results in a rapid and vigorous innate immune response characterized by the production of pro-inflammatory cytokines; mature B cells has weak innate immune function but are central components of the adaptive immunity; the lymphoblast though expressing TLRs, does not participate in innate immunity because it is lack of activation function of LPS to TLRs.5. From IIR to MODS, pancreas is observed to damage in early stage. The inflammatory changes of pancreas after IIR are not induced by oxidative stress from oxygen deficiency, but are associated with NF-kB activation by inflammatory mediators in the circulation. Intravenous supplement of somatostatin shoud be benefitial in preventing the second hit to pancreas. It thus can avoid exacerbation of acute pancreatitis by suppressing systemic inflammatory reaction.
|
PDF Full Text Request