LPS stimulation of purified human platelets is partly dependent on plasma soluble CD14 to secrete their main secreted product,soluble-CD40-Ligand

Background

Platelets are instrumental to primary haemostasis; in addition, as they are central to endothelium vascular repair, they play a role in physiological inflammation. Platelets have also been demonstrated to be key players in innate immunity and inflammation, expressing Toll-like receptors (TLRs) to sense microbial infection and initiate inflammatory responses. They are equipped to decipher distinct signals, to use alternate pathways of signalling through a complete signalosome, despite their lack of a nucleus, and to adjust the innate immune response appropriately for pathogens exhibiting different types of ‘danger’ signals. Previous work has described the two main LPS isoforms-TLR4 activation pathways in purified platelets. However, the precise mechanism of TLR4 signalling in platelets is not completely unravelled, especially how this signalling may occur since platelets do not express CD14, the TLR4 pathophysiological companion for LPS sensing. Thus, we investigated from what source the CD14 molecules required for TLR4 signalling in platelets could come.

Results

Here we show that CD14, required for optimal response to LPS stimulation, is obtained from plasma, but used with restrictive regulation. These data add to the body of evidence that platelets are closer to regulatory cells than to first line defenders. The readout of our experiments is the canonical secreted cytokine-like protein, soluble (s)CD40L, a molecule that is central in physiology and pathology and that is abundantly secreted by platelets from the alpha-granules upon stimulation.

Conclusions

We show that sCD14 from plasma contributes to LPS/TLR4 signalling in platelets to allow significant release of soluble CD40L, thereby elucidating the mechanism of LPS-induced platelet responses and providing new insights for reducing LPS toxicity in the circulation.     

Release from a human monocyte-like cell line of two different soluble forms of the lipopolysaccharide receptor,CD14

Lipopolysaccharide (LPS) stimulates mononuclear phagocytes to synthesize and secrete immunoregulatory and inflammatory molecules such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). LPS forms complexes with either the serum protein termed LPS-binding protein or a serum factor, septin. These complexes are more stimulatory than LPS alone. The myeloid differentiation antigen CD14 is known to be the receptor for such complexes. In the present study, by using a monocytic cell line, we demonstrate the release of two different soluble forms of CD14 (sCD14) which are secreted by different mechanisms. We show that the two sCD14 forms differ in their electrophoretic mobility, two-dimensional gel electrophoretic patterns, sensitivity to endoglycosidases and peptide maps. One of the sCD14 molecules, apparent molecular mass 48 kDa, was found in supernatants of both surface iodinated and [³⁵S] methionine biosynthetically labeled cells. The other sCD14 molecule (56 kDa) was found labeled only in supernatants of [³⁵S] methionine-labeled cells. Furthermore, purified 48 kDa sCD14 enhanced the LPS-induced TNF-a and IL-6 release by the monocytic cells suggesting that a cell-surface signal transducer molecule may be involved in signaling. The data suggest a possible novel role for sCD14 in the monocyte response to LPS.     

Both membrane-bound and soluble forms of CD14 bind to Gram-negative bacteria

Tissue macrophages and their precursors – the blood monocytes – respond rapidly to a bacterial infection with the release of inflammatory mediators. These mediators are involved in the recruitment of phagocytic cells, principally neutrophils, from the blood to the site of infection. To initiate this process macrophages and monocytes must be able to detect the presence of bacteria in a reliable, but nevertheless nonspecific, fashion. It is thought that this is achieved by means of receptors on the cell surface which recognize structures common to many different bacteria. One candidate for such a “pattern recognition element” is the cell surface glycoprotein CD 14. CD 14 has been shown to bind components of the Gram-positive cell wall and it also binds soluble lipopolysaccharide released from Gram-negative bacteria. In both cases the interaction with CD 14 leads to an activation of the cell. Here we show that human peripheral blood monocytes can, in addition, bind intact Gram-negative bacteria in the presence of serum and that this process involves CD14. When CD14 expression is induced on the myelomonocytic cell line U937 by treatment with vitamin D3 the cells concomittently acquire the capacity to bind bacteria. Furthermore, a non-monocytic cell line which does not bind bacteria acquires the capacity to do so when transfected with either the human or mouse CD14 gene. This binding can be inhibited by blocking the CD14 receptor with anti-CD14 antibody or by blocking the ligand on the bacteria with soluble CD14. Finally we demonstrate binding of sCD14 to Escherichia coli. We conclude that in the presence of serum both membrane-bound and soluble forms of CD14 can bind to Gram-negative bacteria. This suggests that CD14 may play a role in the detection and elimination of intact bacteria in vivo.     

MBL对LPS刺激的不同分化和活化状态THP1/CD14细胞产生TNF-α和IL-12的影响

目的探讨甘露聚糖结合凝集素（MBL）对脂多糖（LPS）刺激的不同分化和活化状态THP1/CD14细胞产生TNF-α和IL-12的影响.方法用PMA和/或IFN-γ诱导不同分化和活化状态的THP1/CD14细胞,以不同浓度人MBL预处理2 h后再用光滑型LPS刺激24h.收集培养上清,以ELISA从蛋白水平分析其TNF-α和IL-12 p40＋p70的产生.收集细胞提取总RNA,以RT-PCR从转录水平评估TNF-α和IL-12的表达.结果ELISA检测发现,LPS可刺激各组细胞分泌TNF-α和IL-12 p40＋p70;IFN-γ活化的THP1/CD14细胞产生IL-12 p40＋p70最高,PMA分化的THP1/CD14细胞最低;PMA分化＋IFN-γ活化的THP1/CD14细胞分泌TNF-α最高,未用PMA或IFN-γ预刺激的THP1/CD14细胞最低.高浓度MBL（50～100 mg/L）可抑制LPS诱导细胞分泌TNF-α和IL-12 p40＋ p70的作用,低浓度MBL（1～10 mg/L）则几无影响.RT-PCR分析亦显示,与相应只用LPS刺激的实验组相比,高浓度MBL（50 mg/L）对不同实验组LPS诱导的TNF-α、IL-12 p35和p40的mRNA表达均有不同程度的抑制作用.结论MBL可抑制LPS诱导的不同分化和活化状态THP1/CD14细胞产生TNF-α和IL-12,THP1/CD14细胞可用做进一步剖析MBL在免疫应答与细胞因子网络调控中的作用及其机理的模型.     

CD14lowCD16high: A cytokine-producing monocyte subset which expands during human immunodeficiency virus infection

Infection with the human immunodeficiency virus HIV-1 is associated with the expansion of a CD14^lowCD16^high monocyte subset in peripheral blood. This subset, which represents a minor subpopulation of monocytes in healthy individuals, increases during HIV infection and, in patients with AIDS, may represent up to 40% of the total circulating monocyte cell population. The CD14^lowCD16^high circulating monocytes co-express MAX.1, p150,95 and HLADR which are typical of tissue macrophage markers. These cells also express higher levels of intracellular interleukin (IL)-1α and tumor necrosis factor (TNF)-α than the CD14^highCD16^low monocyte population from the same patients. The CD14^lowCD16^high cells also express low levels of CD35, CD11a and CD4 in common with normal monocytes. When cultured in vitro, monocytes from HIV-seropositive individuals differentiated within a few hours into an elongated fibroblastoid shape characteristic of migratory cells. Our results suggest that the expansion of the CD14^lowCD16^high monocyte subset, which produce high amounts of TNF-α and IL-1α, may participate in the immune dysfunction observed during HIV infection.     

CD14-dependent and -independent cytokine and chemokine production by human THP-1 monocytes stimulated by Streptococcus suis capsular type 2

Streptococcus suis capsular type 2 is an important aetiologic agent of swine meningitis, and it has been highlighted as a cause of occupational disease leading to meningitis and fulminant sepsis in humans. The objective of the present work was to study the ability of S. suis type 2 to induce the release of tumour necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, IL-8 and monocyte chemotactic protein one (MCP-1) by human monocytic THP-1 cells. The induction of these five cytokines was dose- and incubation time-dependent, and it was significantly enhanced by pre-treatment of cells with interferon gamma. IL-8 levels were markedly higher compared with those obtained with the other cytokines. However, elevated levels of MCP-1 and IL-6 were also observed. Levels of cytokine induced by heat-killed or live bacteria were similar. Pre-treatment of cells with anti-CD14 monoclonal antibodies suggested that this important host receptor is partially implicated in TNF, IL-1, IL-6 and MCP-1 production, while CD14-independent pathways seem to be responsible for IL-8 production after S. suis stimulation. In addition, blocking studies with anti-TNF and anti-IL-1 antibodies revealed that these cytokines are involved in amplification of the S. suis-induced cytokine cascade. When several different S. suis strains of human or porcine origin were compared, a very heterogeneous pattern of cytokine production was observed. Human strains did not exhibit a clear tendency to induce higher cytokine release by human THP-1 monocytes. The synergistic effect of the up-regulation of cytokines during S. suis meningitis may mediate many of the inflammatory reactions, including the sequestration of leucocytes at the site of infection.     

Enhanced frequencies of CD14++CD16+, but not CD14+CD16+, peripheral blood monocytes in severe asthmatic patients

CD16+ monocytes are expanded in various inflammatory conditions. Recently it was reported that CD16+ monocytes can be divided into two subsets with contrasting potential of modulating inflammatory responses, namely CD14++CD16+ and CD14+CD16+ monocytes. Here, we characterized and quantified CD14++CD16+ and CD14+CD16+ monocyte subsets in asthmatic patients in the context of severity of disease and different treatment options. Subjects included seventeen severe asthmatics and eighteen moderate asthmatics treated with moderate-to-high doses of inhaled glucocorticosteroids (GCS), twenty nine steroid-naive mild asthmatics and fifteen healthy controls.First, we demonstrated that CD14++CD16+ monocytes, in contrast to CD14+CD16+ monocytes, present significantly higher expression of anti-inflammatory molecule CD163. The frequency of CD14++CD16+, but not CD14+CD16+ monocytes, was significantly higher in patients with severe asthma as compared to mild and moderate asthmatics. However, the frequency of both CD16+ monocyte subsets did not correlate directly with exhaled nitric oxide levels. Short-term administration of oral GCS in patients with exacerbations resulted in a preferential decrease of CD14+CD16+ monocytes. Our study indicates that CD14++CD16+ and CD14+CD16+ monocyte subsets in asthmatics are differentially modulated by both the inflammatory process and GCS treatment.     

Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen

To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.     

Reduced levels of soluble CD14 in atopic children

BACKGROUND: A reduced microbial stimulation has been reported as a reason for the increasing prevalence of atopic diseases in industrialized countries. Antigen-presenting cells (APC), responding to microbial signals by pattern recognition receptors such as CD14, have an important role in the development of the Th1/Th2 balance. OBJECTIVE: We hypothesized that atopic children have a lower expression of CD14 on monocytes and lower soluble CD14 levels (sCD14). METHODS: Seventy-six children were followed prospectively from birth and signs of atopic disease were evaluated. The expression of CD14 on monocytes was analysed with flow cytometry at 0, 3, 6, 12 and 18 months. Circulating levels of sCD14 were analysed by ELISA and total IgE was analysed by fluoroenzymo immunoassay at these ages, and in a subgroup, followed up at 7 years. RESULTS: Levels of sCD14 were reduced at 7 years both in children with a current or a cumulative history of atopy compared to non-atopic children with P=0.002 and 0.001, respectively. Sensitized children with atopic symptoms had lower sCD14 at 3 and 18 months and at 7 years of age than non-atopic non-sensitized children with P=0.023, 0.039 and 0.008, respectively. CONCLUSION: The lower levels of sCD14 observed in atopic children may be a consequence of an atopic family heredity and/or atopic disease, but it may also reflect a reduced capacity to respond to microbial signals.     

CD38 ligation induces discrete cytokine mRNA expression in human cultured lymphocytes

Human CD38 is a surface glycoprotein expressed by different immuno-competent cells such as immature and activated lymphocytes, plasma cells and natural killer cells. It has recently been reported that the CD38 molecule exerts adenosine diphosphate ribosyl cyclase activity and is associated with distinct transmembrane signaling molecules. This study reports that ligation of CD38 by specific monoclonal antibodies (mAb) induces multiple cytokine mRNA expression in cultured peripheral blood mononuclear cells (PBMC). The mRNA for tumor necrosis factor-α, interleukin (IL)-1β, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-12 were always detected, whereas interferon-γ and IL-10 mRNA expression were seen in most, but not all PBMC cultures. Low levels of IL-2, IL-4 and IL-5 mRNA were also found. The key observation of this work is that CD38 ligation in PBMC induces a large spectrum of cytokines, many of which overlap with those induced via CD3 activation. The main differences between CD38 and CD3 activation are the low to undetectable levels of IL-2 mRNA, and the sustained IL-1β and IL-6 mRNA accumulation found in PBMC cultures following treatment with anti-CD38 mAb. Furthermore, PBMC proliferation was not found to be a prerequisite for CD38-mediated cytokine induction. Together, these results suggest that human CD38 activates a signaling pathway which leads to the induction of a discrete array of cytokines, and that this pathway only partially overlaps with that controlled by T cell receptor CD3.     

CD244抑制活动性肺结核患者CD8+T细胞细胞因子分泌的实验研究

目的 探讨表面受体CD244在活动性肺结核患者CD8+T细胞中的功能.方法 密度梯度离心法提取活动性肺结核患者和健康对照者的外周血单个核细胞,通过流式细胞术检测CD244在CD3+ CD8+细胞中的表达;通过细胞内染色方法检测CD244与细胞因子IFN-γ和TNF-α表达的关系.结果 CD244在活动性肺结核患者CD8+T细胞表达强度显著高于健康对照者(P=0.0003);复治肺结核患者的CD244表达强度显著高于新发肺结核患者(P=0.0011);CD244-细胞表达IFN-γ比例显著高于CD244+细胞(P=0.0013);CD244-细胞表达TNF-α比例显著高于CD244+细胞(P =0.0016).结论 CD244抑制活动性肺结核患者CD8+T细胞的细胞因子分泌表达.     

Association of CD14 promoter polymorphisms and soluble CD14 levels in mite allergen sensitization of children in Taiwan

CD14 is responsible for environmental lipopolysaccharide recognition and is a positional candidate gene for allergy. We hypothesized that genetic polymorphisms in the promoter region of the CD14 gene may be associated with Dermatophagoides pteronysinnus (Der p) allergen sensitization in children. Three single nucleotide polymorphisms (SNPs) of the CD14 promoter region, C(–159)T, A(–1,145)G, and G(–1,359)T were genotyped, and analyzed in 240 randomized case–control school-age children in Taiwan. Serum concentrations of IgE and soluble CD14 (sCD14) were also assayed. We found a significant inverse correlation of sCD14 and total serum IgE levels in our study population. Moreover, sCD14 binds Der p allergen in vitro in a dose-dependent manner. The distribution of three SNPs genotypes was similar in asthmatic children and the control group. However, there was a significant difference in the distribution of genotype CD14 G(–1,359)T, but not C(–159)T, between mite-sensitive and non-sensitive children. Haplotype analysis showed strong linkage disequilibrium among these three SNPs in the CD14 promoter region. Carriers of the CD14–159C/–1,145A/–1,359T haplotype had the highest IgE and lowest sCD14 levels as compared to other haplotypes. Our results support the hypothesis that CD14 gene variants may play an important role in influencing allergen sensitization of children in Taiwan.     

孕酮对小鼠子宫巨噬细胞数量及膜受体CD14、CD204表达的影响

目的 探讨孕酮对子宫巨噬细胞数量及其膜受体CD14、CD204表达的影响,以及孕酮和巨噬细胞在子宫局部免疫中的作用。 方法 1. 25只正常非孕鼠随机分为空白对照组、阴性对照组和低、中、高3个不同浓度孕酮处理实验组,每组5只。低、中、高浓度实验组每天分别皮下注射0.4mg、2.0mg、4.0mg孕酮,阴性对照组注射等体积芝麻油,连续注射5d;空白对照组不做任何处理。2.孕4d和5d小鼠各15只,分别随机分为空白对照组、阴性对照组和实验组,每组5只。实验组分别于当天早晨皮下注射4mg孕酮,阴性对照组注射等体积芝麻油,空白对照组不做任何处理。以上各组均于末次注射后6h取子宫,通过非特异性酯酶（α-NAE）染色和免疫组织化学检测子宫α-NAE⁺ 、CD14⁺ 、CD204⁺巨噬细胞的变化。 结果 与对照组相比,注射孕酮后,非孕鼠子宫内膜、肌层和外膜的αNAE⁺及CD14⁺巨噬细胞极显著减少（P<0.01）,并与剂量呈正相关,但未检测到CD204⁺巨噬细胞;孕4d、5d小鼠子宫α-NAE⁺ 、CD14⁺ 、CD204⁺巨噬细胞均显著增多（P<0.01）。 结论 孕酮可通过调节巨噬细胞数量、分布及膜受体的表达影响子宫局部免疫状态,替代途径活化的巨噬细胞可能介导了妊娠期母胎免疫耐受的维持。     

Effect of CD14 promoter polymorphism and H. pylori infection and its clinical outcomes on circulating CD14

CD14 is a pattern recognition receptor on the membranes of monocytes and macrophages for several microbial products, of which lipopolysaccharide (LPS) is the best known. A shed form of CD14 is present in serum. As the CD14 gene promoter polymorphism -159C/T and some bacterial infections may affect the sCD14 levels, we compared the impact of both the CD14 promoter polymorphism and Helicobacter pylori infection on serum sCD14 levels in 201 dyspeptic patients (group 1) who had undergone gastroscopy, and 127 staff members (group 2) with no endoscopy. sCD14 was measured from the sera by a commercial enzyme immunoassay (EIA), and CD14 genotyping was carried out with PCR. Helicobacter pylori infection was detected by serology and/or culture or PCR. sCD14 levels were elevated in the subjects carrying the T allele (CT or TT genotype) in both groups when compared with subjects with the CC genotype. Overall, H. pylori-positive subjects tended to have higher sCD14 levels compared with H. pylori-negative subjects. In group 1 consisting of dyspeptic patients, those with gastric ulcer, gastric erosion or duodenal ulcer had significantly elevated levels of sCD14 compared with the patients with normal endoscopic findings or macroscopic gastritis. The recent use of NSAIDs was also associated with enhanced sCD14. Thus, we were able to show several factors, one genetic and the other environmental (H. pylori infection and mucosal lesion), to have an impact on sCD14.     

用标记磁微粒技术克隆牛CD14基因

为获得牛ＣＤ１４ 的全长编码基因，以探讨牛ＣＤ１４ 基因结构与功能的关系，进一步阐明其作用机理，我们用抗牛ＣＤ１４ 的单克隆抗体（ｍＡｂ）ＣＣＧ３３ 标记的磁微粒作为探针，从转染有牛肺泡巨噬细胞ｃＤＮＡ文库的ＣＯＳ７细胞中进行筛选。并对获得的牛ＣＤ１４ ｃＤＮＡ进行了克隆和序列分析。结果表明，牛ＣＤ１４ 基因全长为１１２２ ｂｐ，共编码３７３ 个氨基酸，其中含有信号肽序列和３ 个可识别的Ｎ连接糖基化位点，但缺少穿膜结构域及细胞内区。证实牛ＣＤ１４ 以糖基磷脂酰肌醇（ＧＰＩ） 锚定在靶细胞膜上；其氨基酸序列与人和小鼠ＣＤ１４ 的同源性分别为７３－５ ％ 和６１－４ ％ ，三者共有的保守序列约为５５％ 。