英文版 重症医学会

Associations of plasma neutrophil-derived microparticles with the prognosis of patients with sepsis or septic shock(Department of Critical Care Medicine, Affiliated Hospital of Guangdong Medical College, 524001)Lijun Ye1 *Liang Zhang1 *Jinle lin2Huaguo Yao1Liehua Deng1 && Corresponding authorWenwu Zhang2 && Corresponding author1Department of Critical Care Medicine, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong.2Department of Critical Care Medicine, People’s hospital ofinBao District, Shenzhen, Guangdong.大 Equal contributorsAbstractObjective : Neutrophil-derived microparticles are small plasma membrane vesicles releasing from stilling, activation or apoptosis neutrophils, and play an important role in regulating inflammatory response. In this study, we investigated the associations of plasma neutrophil-derived microparticles with the prognosis of patients with sepsis or septic shock.Methods: We enrolled 111 ICU patients: 48 with sepsis, 63 with septic shock. Plasma NDMPs, TNF-a, IL-6, and sTREM-1 levels were measured on ICU day 1, 3, 5, 7. At the same time points, we recorded APACHE II score , MODS score , PCT. Mechanical ventilation time, ICU hospitalization time and total hospitalization time were counted. 19 healthy individuals were enrolled acting as control group. A P value < 0.05 was considered significant.Results: NDMPs were presented in the plasma of both healthy individuals and patients with sepsis or septic shock, and plasma AnxA1 and CD62L in sepsis and septic shock significantly exceeded those of healthy individuals (P < 0.05). Basically, compared with sepsis group, plasma NDMPs levels were significantly higher in septic shock group (P < 0.05). In the comparison between survival group and no-survival group, the analysis results showed in no-survival group, plasma AnxA1 was significantly higher at any time point (P < 0.05), plasma CD11b were significantly higher at 7d (P < 0.05), plasma CD62L were markedly higher at 3d and 7d (P < 0.05).Conclusions: The study suggested that plasma NDMPs, especially AnxA1 and CD62L were significantly associated with prognosis by promoting the releasing of inflammatory mediators in patients with sepsis or septic shock.1. IntroductionNeutrophil-derived microparticles (NDMPs) are small plasma membrane vesicles releasing from stilling, activation or apoptosis neutrophils, which contain cell surface proteins and cytoplasmic matter, with biological activities. There are different protein markers on the surface, including the specific protein markers cell surface Annexin1 (AnxA1), L-selectin (CD62L), CD11b and CD66b, and intracellular proteins myeloperoxidase, elastase, and other proteolytic enzymes.In recent years , microparticles (MPs) become a focus of attention in researches. As to NDMPs, the studies in vitro are still constantly development. Researchers obtained NDMPs by setting up animal models, separating health and the patient's body neutrophils, and analyzed them to show the biological activities of these vesicles: NDMPs contributing to anti-/pro-inflammatory responses [1-4], promoting thrombosis, maintaining vascular barrier function and preventing vascular leakage [3, 5, 6], damaging vascular endothelial cells and tissues [7], antibacterial effect [8].A rare clinical research by collecting abdominal washings and bronchoalveolar lavage fluid from sites of inflammation of sepsis patients suggested NDMPs may play an important role in regulating the inflammatory response to sepsis in patients with critical illness [9], which may be suggested that NDMPs were not only involved in the development of sepsis, but also associated with the prognosis of it.This study firstly collected the plasma of patients with sepsis and septic shock, and detected plasma NDMPs, for the sake of assessing whether the NDMPs can act as biomarkers or even successful markers in the prognosis assessment of patients with sepsis or septic shock. Owing to CD62L, CD11b and AnxA1 are special protein markers of NDMPs, we only analyze the three proteins.2. Materials and Methods2.1 MaterialsAnti-Annexin antibody (clone ab33061), secondary antibody (goat anti-rabbit lgG H&L, clone ab150077) and isotype control (clone ab172730) are purchased from abcom trading company ltd. Anti-Human CD11b (clone CBRM1/5), anti-Human CD62L (clone DREG-56) and isotype control (clone 3.6.2.8.1) are purchased from eBioscience Affymetrix company. Human Tumor necrosis factor alpha (TNF- a) Enzyme Linked Immunosorbent Assay (ELISA) Kit and human Interleukin-6 (IL-6) ELISA Kit are purchased from NeoBioscience. Human Souble serum triggering rceptor expressed on myeloid cells-1 (sTREM-1) ELISA Kit is purchased from RayBiotech, Inc. All these reagents are stored in the 4 °C refrigerator for the experiment using.2.2.1 Patients of studyThe study was conducted in 111 patients with sepsis in department of Intensive Care Unit (ICU), affiliated hospital of Guangdong Medical College, Zhanjiang Guangdong and the People’s hospital of Bao’an District, Shenzhen Guangdong, between January and November 2014. The patients were divided into 2 groups : 48 patients diagnosed with sepsis setting as sepsis group , 63 patients diagnosed with severe sepsis and septic shock setting as septic shock group. Taking 19 healthy subjects, from Physical examination center of affiliated hospital of Guangdong Medical College, Zhanjiang Guangdong, set as control group.The diagnosis and classification of sepsis referred to international guidelines for severe sepsis and septic shock 2012 [17]. All entrants were signed informed consent before experiment, and this study was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical College (Zhanjing, China) and People’s hospital of Bao’an District (Shenzhen, China).2.2.2 Cases of exclusion criteriaPatient histories with malignant tumor, or patients in the immunosuppression state or poisoning, were count out.2.3 Methods 2.3.1 Neutrophil-derived microparticles preparation and Flow cytometry for surface stainingIn total, 5ml of heparinized blood were obtained from each patient and healthy donor. The blood samples were initially centrifuged at 1000g for 10 minutes, the plasma supernatants were collected and centrifuged at 9900 g, 4 °C for 10 minutes [9] prior to further ultracentrifugation at 15000g, 4Cfor 1 hour [3]. The microparticles pellets were then suspended in the last supernatant and stored at -80 C until use. After thaw, the MPs were initially stained with the following mAb at 10pg/mL: anti-AnxA1 (clone ab33061), anti-CD62L (clone DREG-56), anti-CD11b (clone CBRM1/5), or isotype control (clone ab172730) and clone P3.6.2.8.1) for 30 minutes at 37 C with gentle shaking, a final staining with secondary antibody (goat anti-rabbit lgG H&L) for 30 minutes at 37 C was conducted. The immune labeled MPs were finally resuspended in 500p! sheath fluid. Fluorescence-activated cell sorting (FACS) analysis was conducted using FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson) [1].2.3.2 ELISA AssaysThe levels of sTREM-1, TNF-a and IL-6 in the plasma from sepsis group and septic shock group at 1, 3, 5 ,7d and control group were determined using enzyme-linked immunosorbent assays according to the manufacturer's protocol (RayBiotech, Inc, NeoBioscience, China and NeoBioscience, China, respectively).2.4 Data collectionUpon admission into the ICUs, the following items were recorded for each patient: source of patient, age, gender, primary disease, medical history, and diagnosis. Within (first day of study) after ICU admission and in the morning of days 3, 5 and 7, recorded Acute Physiology and Chronic Health Evaluation (APACHE) II score, Multiple Organ Dysfunction Syndrome (MODS) score and assay results of Procalcitonin (PCT). Counted up the mechanical ventilation time, ICU hospitalization time and total hospitalization time and recorded the final outcome (survival or no-survival) after the patient was discharge.2.5 Statistical analysisSPSS15.0 software was used for statistical analysis. All values were presented as means 土 standard deviation. Significant differences among three groups were determined by one-way analysis of variance with subsequent Student-Neman-Keuls test. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, California, USA). A P value of less than 0.05 was considered statistically significant.3. Results3.1.1 NDMPs AnxAl, CD11b and CD62L were detected in the plasma of sepsis, septic shock and control groups by FACSOn FACS analysis of the plasma NDMPs, we used a size gating (Figure 1) varying approximately between 0.1 and 2 pm to identify MPs. The results showed that all NDMPs were detected in the plasma of sepsis, septic shock and control groups (Figure 2).3.1.2 The distribution characteristic of AnxA1, CD11b and CD62L in sepsis, septic shock and control groupCompared with control group, AnxA1 and CD62L in sepsis or septic shock group were significantly higher (P < 0.05). As to CD11b, there was no markedly difference between sepsis group and control group (P > 0.05), but the CD11b in septic shock group was significantly higher than that in sepsis group and control group ( P < 0.05). (shown in Table 1)3.2 Comparison of various experimental variables between sepsis and septic shock patientsThe levels of CD11b and CD62L in septic shock group were significantly higher than those in sepsis group at 1, 3, 5, 7d and at 5, 7d, respectively (all P < 0.05). Compared with sepsis group, plasma TNF- a and sTREM-1 were markedly higher at 3, 5, 7d in septic shock group ( P < 0.05). The levels of PCT and APACHE II score in septic shock group were significantly higher than those in sepsis group at any time points (P < 0.05). The mechanical ventilation time of septic shock group was significantly longer than that of sepsis group ( P < 0.05). However, there were no difference of MODS score, ICU hospitalization time andtotal hospitalization time between the two groups ( P <0.05). (shown inTable 2)3.3 Dynamic changes of Annxin1, CD11b and CD62L within sepsis or septic shock patientsIn sepsis group, according to analyzing the dynamic change of the plasma concentration of AnxA1, CD11b and CD62L at different time points, the results were showed as follows : i) the discrepant levels of AnxA1 between the 1d and 3d, the 5d and 7d were statistically significant (Figure 3.1 A, P < 0.05). ii) The levels of CD11b at 3d and 7d were significantly higher than that of 1d, compared with that of 3d, the levels of CD11b at 7d were also significantly higher (Figure 3.1 B, P < 0.05). iii) The levels of CD62L at 3, 5 and 7d were all markedly higher than that of 1d, the difference between the 3d and 7d was also significant (Figure 3.1 C, P < 0.05).As to septic shock group, seemly whether the levels of CD11b or CD62L presented a trend of increasing from 1d to 7d, but not all of the increasing were statistically significant, concrete analysis was as follows: i) plasma AnxA1 in septic group presented the same significant change tendency with that in sepsis group. ii) At 3d and 5d, the levels of CD11b were markedly higher than that of 1d, and from 3d to 7d the increased levels were also significant (Figure 3.2 E, P < 0.05). iii) The levels of CD62L at 3d were markedly higher than that of 1d, and the increased trend from 3d to 7d were also statistically significant (Figure 3.2 F, P < 0.05).3.4 Comparison of various experimental variables in survival and no-survival patientsAccording to the final outcome of patients, we divided patients into two groups: survival and no-survival group. Compared with survival group, plasma AnxA1 in no-survival group were all significantly higher at 1, 3, 5, 7d (P = 0.002, P = 0.000, P= 0.010 and P = 0.000, respectively), plasma CD11b were significantly higher at 7d (P = 0.000), plasma CD62L were markedly higher at 3d and 7d ( P = 0.028 and P = 0.000, respectively). The plasma TNF- a IL-6 in no-survival group were markedly higher at 1, 3, 5, 7d, however plasma sTREM-1 in no-survival group was significantly higher at 1d and 3d ( P = 0.006 and P = 0.033, respectively). In no-survival group, the mechanical ventilation time and total hospitalization time were significantly longer than those in survival group (P = 0.000 and P = 0.000, respectively). (shown in Table 3)4. DiscussionPrevious studies have showed that the NDMPs are released by neutrophils in vitro [1-2, 5-8, 10-15, 18-19] and in vivo [3, 9, 20], in local and systemic inflammation, and they are involved in inflammation and damage. Prakash et al. have found NDMPs by collecting bronchoalveolar lavage fluid and abdominal drainage fluid in the inflamed foci of patients with sepsis or infection, and concluded NDMPs with the role of modulating the immune response to sepsis [9]. In present study, we have observed that the three NDMPs AnxA1, CD11b and CD62L not only generate in the plasma of sepsis and septic shock patients, but also do in healthy individuals , what’ more, the levels of AnxA1 and CD62L were significantly increased in sepsis and septic patients. Thus, it is suggested that the high-level of AnxA1 and CD62L symbolize the presence of inflammation, even maybe an ideal biomarker for early sepsis diagnosis. Further, our data have demonstrated that in patients with septic shock, the plasma NDMPs are especially higher than those of sepsis patients, which suggest that the more severe of the disease , the plasma leves of NDMPs are higher. In terms of plasma CD11b, its level showed significantly higher at all time points, suggesting that it may play an important role in the development of sepsis and septic shock. There is evidence that AnxA1 carries anti-inflammatory and pro-inflammatory properties [1], while the levels of AnxA1 had no markedly difference between sepsis group and septic shock group, which may demonstrate that plasma AnxA1 is not increasing along with aggravating of inflammation. During the early period of the disease, plasma CD62L in septic shock patients was not significantly higher than that in sepsis patients, but gradually presented markedly higher along with the progress of the disease.By observing the dynamic changes of AnxAl, CD11b and CD62L within sepsis or septic shock patients, we have discovered that plasma AnxA1 has no obvious change trend in the two groups, while both plasma CD11b and CD62L presented increasing trend, which indicating that AnxA1 may act as relatively stationary NDMPs, however, CD11b and CD62L function importantly through the process of sepsis or septic shock acting as relatively active NDMPs, as to the function of the two NDMPs are anti- or pro- properties need to further study.At the last of the study, by comparing survival group and no-survival group, we have observed that there are significantly difference in plasma levels of AnxA1 and CD62L, suggesting that they are closely associated with prognosis. Furthermore, in no-survival group, mechanical ventilation time and total hospitalization time are also longer than that in survival group, which is accord with the previous studies. Therefore, plasma NDMPs, especially AnxA1 and CD62L, may provide new reference index for prognosis assessment of sepsis.5. ConclusionIn summary, our present study demonstrated for the first time that the association between the plasma of NDMPs and the prognosis of sepsis in vivo study, as described above, the plasma concentration of NDMPs indeed involved in sepsis, closely related with the prognosis of sepsis, aggravating state of illness the severity of illness and organ damage.Competing interestsThe authors have no competing interests.AcknowledgementsThis study was supported in part by the Clinical Laboratory Center of Guangdong Medical College Affiliated Hospital and the Department Critical Care Medicine People’s hospital of Bao'an District, Shenzhen Guangdong.References1. 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