Whole-Blood Assay to Measure Oxidative Burst and Degranulation of Neutrophils for Monitoring Trauma Patients

AbstractBackground and Purpose: Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms, but excessive PMN activation after trauma causes tissue injury. Rapid monitoring of PMN function is critical for the assessment of the inflammatory state of trauma patients. Here, the authors adapted two simple and rapid methods to measure oxidative burst and degranulation of human PMNs in whole blood to avoid potential interference of cell isolation procedures with the assessment of PMN function.Material and Methods: Heparinized blood was drawn from healthy volunteers or trauma patients, preincubated at 37 °C for 5 min, and stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Four assays for oxidative burst were tested: (1) cytochrome C; (2) homovanillic acid (HVA); (3) Amplex^® Red; and (4) flow cytometry with dihydrorhodamine 123 (DHR). PMN degranulation was assessed with flow cytometry using antibodies to: (1) CD11b/Mac-1 (CD18); (2) CD63; and (3) CD66b (CD67).Results: With the exception of the DHR method, all methods to measure oxidative burst were found to be unsuitable in whole blood due to interference of plasma proteins and hemoglobin with the fluorimetric or photometric readouts. By contrast, all degranulation methods were suitable for whole-blood studies. However, for the assessment of formyl peptide-induced degranulation, anti-antibodies to CD11b/Mac-1 and CD66b were up to five times more sensitive than antibodies to CD63. Thus, the degranulation and DHR methods were optimized for increased sensitivity, speed, and specificity and their usefulness to measure PMN function in trauma patients was tested.Conclusion: The whole-blood methods based on flow cytometry with DHR, anti-CD11b/Mac-1, and anti- CD66b are rapid, simple, and reliable techniques to assess PMN function for trauma research.     

Allergen extracts directly mobilize and activate human eosinophils

Allergic diseases are characterized by the presence of eosinophils, which are recruited to the affected tissues by chemoattractants produced by T cells, mast cells and epithelium. Our objective was to evaluate if allergens can directly activate human eosinophils. The capacity of purified allergen extracts to elicit eosinophil chemotaxis, respiratory burst, degranulation and up-regulation of the adhesion molecule complement receptor 3 (CR3) was determined in eosinophils isolated from healthy blood donors. Eosinophils stimulated with an extract from house dust mite (HDM) released the granule protein major basic protein (MBP) and up-regulated the surface expression of CR3. Cat allergen extracts also induced the up-regulation of CR3, but not the release of MBP; instead cat, as well as birch and grass allergens, elicited the release of eosinophil peroxidase (EPO). In addition, grass pollen extract caused the secretion of MBP. None of the allergens stimulated eosinophilic cationic protein release, nor production of free oxygen radicals. Both HDM and birch extracts were chemotactic for eosinophils. These findings establish that common aeroallergens can directly activate eosinophils in vitro. We propose that eosinophil activation in vivo is not exclusively mediated by cytokines and chemokines of the allergic inflammatory reaction, but could partly be the result of direct interaction between allergens and eosinophils.     

Rapid non-genomic inhibitory effects of glucocorticoids on human neutrophil degranulation

Background: Glucocorticoids acting as anti-inflammatory or immunosuppressive drugs have been shown to exert most of their effects genomically. Recent findings suggest that non-genomic activity might be relatively more important in mediating the therapeutic effects of high-dose pulsed glucocorticoid. However, few non-genomic anti-inflammatory effects were reported, much less non-genomic mechanisms.Objective: This study was performed to investigate the nongenomic effects of glucocorticoids on human neutrophil degranulation.Methods: Purified human neutrophils were pretreated with 6 -methylprednisolone or hydrocortisone for 5 min, and then primed with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10^–6 M) or phorbol myristate acetate (PMA) (50 ng/ml) in the presence of cytochalasin B. The release of two markers of neutrophil granules, lactoferrin and myeloperoxidase, was measured by ELISA and enzymology methods respectively.Results: Both 6 -methylprednisolone (10^–5–10^–4 M) and hydrocortisone (10^–4 M) showed significant inhibitory effects on neutrophil degranulation within 5 min after fMLP administration. For PMA stimulated degranulation, 6 -methylprednisolone (10^–4 M) showed significant inhibitory effects (p < 0.01), while hydrocortisone (10^–4 M) only showed an inhibitory tendency (P > 0.05). Neither RU486 (10^–5 M) nor cycloheximide (10^–4 M) could alter the inhibitory effects of glucocorticoids.Conclusion: Our results demonstrate that megadoses of glucocorticoids exert rapid inhibitory effects on human neutrophil degranulation at the cellular level via a new mechanism that is independent of corticosteroid type II receptor occupation or protein synthesis. We infer that these effects may be very important when glucocorticoids act as anti-inflammatory drugs during pulse therapy.Received 20 May 2004; returned for revision 21 July 2004; accepted by M.J. Parnham 23 September 2004L. Liu and Y. X. Wang contributed equally to this work.     

Serum protects against azurophil granule dependent down-regulation of complement receptor type 1 (CR1) on human neutrophils

We have investigated the effect of gradual degranulation on the expression of functional receptors (CR1 and CR3) on human neutrophils. Incubation with increasing concentrations of fMLP (10^–10–10^–7M) translocated CR1 and CR3 to the cell surface in a similar kinetic pattern. When reaching maximal expression of receptors (10^–7 M fMLP), 78 ± 10% and 87 ± 9% of the total pool of CR1 and CR3, respectively, were translocated to the cell surface. To drive the mobilization process further, cytochalasin B was introduced to increase the stimulatory effect of fMLP. No further increase in CR1 surface expression was obtained. However, we found a characteristic time course of surface appearance of CR1 and CR3 with a maximal surface expression within 1 minute, followed by a time-related down-regulation of CR1 but not CR3. In addition, the total pool of CR1 in cytochalasin B treated neutrophils was reduced after 15 minutes stimulation with fMLP measured by flow cytometry and immunoblotting, indicating degradation of CR1. The down-regulation of CR1 was concomitant with a translocation of azurophil granules, in terms of upregulation of CD63. Azurophil, but not specific nor secretory, granule fractions caused a down-regulation of CR1 on fMLP activated neutrophils. The presence of human sera and serine protease inhibitor protected CR1 from down-regulation. Together, these findings indicate that intracellular stored proteases, released in the late part of the sequential mobilization process, alters the expression of functional receptors mobilized in the early part of the mobilization process. The findings also focus on the importance of the microenvironment for the net outcome of neutrophil activation in terms of functional receptor expression.     

肥大细胞脱颗粒在牙周炎病理中的作用

目的:探讨肥大细胞脱颗粒在牙周炎病理中的意义。方法:将自愿接受研究的受试者分为3组:轻度慢性牙周炎组(轻度组,17例)、重度慢性牙周炎组(重度组,18例)、正常对照组(15例)。取牙龈活检组织制作连续切片,HE染色在光镜下观察各组牙周组织的组织学改变;甲苯胺蓝染色法观察各组肥大细胞的数量及肥大细胞脱颗粒状况;天狼猩红染色法观察各组牙周组织纤维化的程度。结果:与正常对照组相比,各慢性牙周炎组的牙周组织中肥大细胞显著增多(P<0.01);重度组与轻度组相比较肥大细胞数量和脱颗粒率均明显升高(P<0.01),牙周组织纤维化程度更加严重(P<0.05)。结论:肥大细胞的募集、脱颗粒和纤维化程度与牙周炎的严重程度相关,提示肥大细胞可能在牙周炎的发病和疾病进程中起着重要的作用。     

Up-regulation of DNAM-1 and NKp30, associated with improvement of NK cells activation after long-term culture of mononuclear cells from patients with ovarian neoplasia

This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I–IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56⁺ lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p < 0.05), however, the long-term procedure supported an even higher increase (p < 0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells.     

Novel and atypical splicing mutation in a compound heterozygous UNC13D defect presenting in Familial Hemophagocytic Lymphohistiocytosis triggered by EBV infection

Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule priming factor Munc13-4 that intervenes in NK and T cell cytotoxic function. Here we report the case of a 17-month-old girl with prolonged symptomatic EBV infectious mononucleosis and clinical symptoms of hemophagocytic syndrome. In vitro functional analysis pointed to a degranulation defect. The genetic analysis of UNC13D gene identified initially a heterozygous mutation (c.753 + 1G > T) in the donor splice-site that resulted in exon 9 skipping (maternal allele). Mutations in other genes were considered, but additional analysis of UNC13D cDNA revealed in the paternal allele a heterozygous transition from G to A (c.2448 − 13G > A) at the 3′ acceptor splice-site in intron 25, generating a new acceptor splice-site that leads to a frameshift and a premature STOP codon. Allele specific amplification of the cDNA confirmed the absence of a functional mRNA from the paternal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype.     

伴放线放线杆菌超声粉碎滤液诱导外周血中多形核白细胞脱粒的体外研究

目的:研究伴放线放线杆菌(actinobac illus actinomycetem com itans,Aa)可溶性产物对体外培养的多形核白细胞(polymorphonuc lear leukocytes,PMNLs)的影响,探讨该菌在牙周炎发病过程中的作用。方法:采集健康青少年外周血,Percoll梯度离心法分离PMNLs,将Aa超声粉碎滤液分别加入PMNLs(实验组)与含抗毒血清的PMNLs(对照组)中,在1m in、5m in时电镜观察摄片,20m in、40m in和60m in时细胞学检测并记数。结果:实验组PMNLs在透射电子显微镜下可见明显的形态学变化,其颗粒伴部分胞液脱出细胞外,对照组细胞无明显变化;实验组脱粒的PMNLs百分数较对照组显著增多(P<0.01)。结论:Aa可溶性产物可诱导外周血PMNL中颗粒向细胞周边移动并脱出,降低宿主抵抗力,参与牙周炎的发生。     

茶属植物及茶多酚类成分对抗原诱导RBL-2H3细胞过敏反应的抑制作用

目的:研究茶属植物及茶多酚类和嘌呤碱类成分对抗原刺激RBL-2H3细胞脱颗粒及肿瘤坏死因子α(TNF-α)和白细胞介素4(IL-4)释放的抑制作用。方法:测定β-hexosaminidase的释放以评价脱颗粒反应的指标,酶联免疫吸附法测定TNF-α和IL-4的释放,评价茶中主要成分的抗过敏活性。结果:茶属植物均有抑制RBL-2H3细胞脱颗粒作用,其中花类表现出较叶类更强的抗过敏活性;EGCG对抗原刺激的RBL-2H3细胞即刻相(IC50:234μmol·L-1)和延缓相反应(IL4,IC50:158μmol·L-1),及钙离子载体A23187诱导的脱颗粒反应均有抑制作用(IC50:126μmol·L-1)。结论:茶属植物和EGCG具有明显的抗过敏作用。     

射干甙抑制嗜酸性粒细胞脱颗粒实验研究

目的:研究射干甙是否具有抑制嗜酸性粒细胞(EOS)脱颗粒作用。方法:哮喘病人外周血采用Percoll液密度梯度分离,收集EOS培养后检测射干甙作用前后主碱蛋白(MBP)及EOS阳离子蛋白(ECP)含量的变化。结果:射干甙作用后MBP和ECP含量降低。结论:射干甙具抑制嗜酸性粒细胞脱颗粒作用。