Differential regulation of TSG-14 expression in murine fibroblasts and peritoneal macrophages

Tumor necrosis factor (TNF)-stimulated gene 14 (TSG-14, also termed PTX3) encodes a secreted glycoprotein whose carboxy-terminal half shares sequence similarity with the pentraxin family of acute phase proteins (C-reactive protein and serum amyloid P component). We compared TSG-14 mRNA expression in cultures of murine BALB/c 3T3 fibroblasts and thioglycollate-elicited peritoneal macrophages. TNF and interleukin-1 (IL-1) potently induced TSG-14 expression in 3T3 fibroblasts but not in peritoneal macrophages. Lipopolysaccharide (LPS) elicited TSG-14 expression in both cell types, but induction in 3T3 cells and macrophages showed several distinct characteristics. Whereas in 3T3 fibroblasts TSG-14 mRNA was rapidly up-regulated by LPS, expression in macrophages was substantially delayed. Furthermore, cycloheximide greatly reduced LPS-induced TSG-14 mRNA up-regulation in macrophages but not in 3T3 cells. Finally, interferon-gamma (IFN-gamma; but not IFN-alpha/beta) inhibited LPS-induced TSG-14 expression in macrophages and not in 3T3 fibroblasts. The antioxidant pyrrolidine dithiocarbamate inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation and TSG-14 expression in macrophages. In contrast, IFN-gamma did not inhibit NF-kappaB function as measured by IkappaB-alpha and IkappaB-beta degradation, IkappaB-alpha resynthesis, or electrophoretic mobility shift analysis. Inhibition of LPS-induced TSG-14 mRNA expression by IFN-gamma in macrophages was also observed in the presence of cycloheximide and in cells from STAT1 null mice, suggesting that IFN-gamma inhibits TSG-14 expression through an unconventional mechanism.     

CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml).     

IL-10对小鼠肺泡巨噬细胞清道夫受体及CD14表达的影响

本研究旨在观察IL-10对肺泡巨噬细胞（AM），清道夫受体（SR），CD14表达的影响。探讨IL-10在内毒素血症时防止AM由免疫防御向炎症效应转变中的作用。分离培养小鼠AM，以不同剂量（0,0.01,0.1,1,10,100μg/L）IL-10刺激细胞16h或以100μg/LIL-10刺激细胞不同时间（0,2,4,6,8,12,16h)，采用免疫细胞化学及RT-PCR方法观察SR，CD14表达变化。结果显示低至10μg/LIL-10刺激16h或100μg/LIL-10刺激12-24h能显著增强SRmRNA并抑制CD14mRNA表达，SR蛋白表达也显著增强，但CD14蛋白表达无明显变化，IL-10刺激下对SR蛋白表达的增强能降低AM对LPS的反应性，在内毒素血症时防止AM由免疫防御型向炎症效应型转变中发挥重要作用。     

TNF-α对小鼠肺泡巨噬细胞CD14和清道夫受体表达的影响

本研究旨在通过观察TNF-α对肺泡巨噬细胞(AM)清道夫受体(SR)、CD14表达的影响，探讨TNF-α在内毒素血症时AM由早期防御性(清除、灭活内毒素)向后期效应性(释放大量的致炎与抗炎因子)转变中的作用。分离培养小鼠AM，以不同剂量TNF-α(0，0．001，0．01，0．1，1，10，100μg／L)刺激细胞16h或以100μg／L TNF-α刺激细胞不同时间(0，2，4，6，8，12，16，24h)，采用免疫细胞化学及RT-PCR方法观察SR、CD14表达变化。结果显示，以低至0．1μg／L TNF-α刺激16h或100μg／L TNF-α刺激6h以上就能显著增强CD14并抑制SR蛋白的表达，实验同时显示，CD14mRNA、SRmRNA表达也显著增强，SRmRNA表达变化与SR蛋白表达趋势不一致。研究结果提示，TNF-α能通过增强CD14蛋白表达并抑制SR蛋白表达而在内毒素血症时小鼠AM由免疫防御型向炎症效应型转变中发挥重要作用。     

MRP8/MRP14, CD11b and HLA-DR expression of alveolar macrophages in pneumonia

Activation of alveolar macrophages is characterised by specific alterations to the expression pattern of surface markers under certain pathological conditions. MRP8/MRP14 and CD11b are involved in the regulation of macrophage migration and adhesion. HLA-DR regulates the antigen presentation by alveolar macrophages. The aim of this study was to investigate the phenotype of alveolar macrophages in pneumonia particularly in relationship to the changes in concentrations of TGF-beta1 and IL-8. Using cytofluorimetry, we analysed the surface expression of MRP8/MRP14, CD11b, and HLA-DR on alveolar macrophages of 42 pneumonia (PN) patients, 14 patients with interstitial lung diseases (ILD), five patients with chronic obstructive lung disease (COPD), and 58 patients without lung disease. Phenotypic characteristics were correlated to the concentration of TGF-beta1 and IL-8 in the bronchoalveolar lavage fluid (BALF) of the same patients. The direct influence of TGF-beta1 and IL-8 on expression of MRP8/MRP14, CD11b and HLA-DR of cultured monocytes and MonoMac cells was analysed. Significantly more MRP8/MRP14 and CD11b positive macrophages and less HLA-DR-positive macrophages were found in PN but not in ILD or COPD. The percentage of CD11b-positive macrophages correlated with the TGF-beta1 as well as the IL-8 concentrations. The amount of HLA-DR-positive macrophages correlated negatively to the concentration of TGF-beta1 and IL-8. These findings document a significant activation of alveolar macrophages during pneumonia. TGF-beta1 led to a modulation of HLA-DR and MRP8/MRP14-antigen expression in vitro. In conclusion, it was shown that in pneumonia but not in ILD or COPD alveolar macrophages were characterised by an increased MRP8/MRP14 and CD11b expression and a diminished HLA-DR expression. The characterisation of subpopulations within the alveolar macrophages may be a useful tool for the monitoring of disease progression.     

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。     

Differential role of CD80 and CD86 on alveolar macrophages in the presentation of allergen to T lymphocytes in asthma

In the asthmatic lung the altered expression of costimulatory molecules (CD80, CD86) by alveolar macrophages contributes to T lymphocyte activation and expansion. We hypothesized that CD80 and CD86 on alveolar macrophages could differentially support allergic inflammation in adult asthma. Here we studied 11 subjects with mild allergic asthma and 11 atopic non-asthmatics as controls. Dermatophagoides-specific T cell lines were derived from peripheral blood from each subject. Bronchoalveolar lavage with evaluation of lung inflammatory cells was performed in all individuals at baseline and 24 h after allergen challenge. The expression of CD80 and CD86 costimulatory molecules by alveolar macrophages was studied and, in parallel, the efficiency of antigen presentation was measured in terms of IL-4 and IL-5 production by allergen-stimulated autologous T cells. We found that in asthmatic subjects (i) the percent of CD80+, but not CD86+ alveolar macrophages was increased at baseline and did not change following allergen challenge; (ii) CD86, but not CD80, membrane expression was up-regulated following allergen challenge; (iii) both CD80 and CD86 were required to support Th2 cytokine production by allergen-specific T cells, with a prevalent role of CD86 after allergen challenge. Our data indicate that alveolar macrophages deliver costimulatory signals via CD80 and CD86, which support the production of Th2 cytokines by allergen-specific T cells. They also indicate that CD86 in vivo is up-regulated in the 24 h following allergen exposure and that this modulation is functionally relevant.     

Differential regulation of tumor necrosis factor-alpha in human alveolar macrophages and peripheral blood monocytes: a cellular and molecular analysis

Human tumor necrosis factor-alpha (TNF), a mononuclear phagocyte (MO)-derived peptide, is increasingly being recognized for its pleomorphic immunologic effects. A number of studies have demonstrated that LPS can induce TNF synthesis, but data examining the production and regulation of TNF in human MO populations are lacking. In this study, we present data demonstrating that alveolar macrophages (AMO) and peripheral blood monocytes (PBM) obtained from 10 normal volunteers display a significant difference in both the production of TNF and their susceptibility to TNF regulation by prostaglandin E2 (PGE2) and dexamethasone (Dex). Adherent populations of PBM and AMO were incubated for 18 h in the presence of either LPS (10 micrograms/ml) alone, PGE2 for 1 h prior to LPS challenge, Dex for 1 h prior to LPS challenge, or control media alone. Cell-free supernatants were examined for TNF bioactivity and cellular TNF mRNA was assessed via in situ hybridization and Northern blot analysis. PGE2 and Dex treatment of PBM suppressed LPS-induced TNF production by 78% and 72%, respectively, while AMO-TNF production was suppressed by only 22% and 33%. The accumulation of TNF mRNA in PBM was reduced 63% by PGE2 and 45% by Dex, as assessed by laser densitometry. Similar studies demonstrated that TNF mRNA accumulation in AMO was reduced 12% and 13% by PGE2 and Dex, respectively. A 1,000-fold increase in PGE2 levels was necessary to induce 50% suppression of the maximal response to AMO as compared to PBM. These data support the notion that human MO derived from different compartments or stages of differentiation exhibit differential responsiveness to immunomodulators.     

Critical investigation of the CD14 promoter polymorphism: lack of a role for in vitro cytokine response and membrane CD14 expression

Blood of volunteers, genotyped for the CD14 C(-159)-->T polymorphism, showed no difference in cytokine release when stimulated with nine CD14-dependent immune stimuli. An analysis of the published data on the proposed association of CD14 genotype with membrane CD14 density revealed no significant correlation, questioning a functional impact of the CD14 polymorphism.     

Differential regulation of soluble interleukin 1 release and membrane expression by pharmacologic agents

Membrane IL-1 (mIL-1) expression was compared with release of soluble IL-1 (sIL-1) by C3H/HeNCrl mouse peritoneal macrophages. Selective antagonists of protein kinase C (PKC) (H-7) and calmodulin (W-7) in combination inhibited LPS-induced mIL-1 and sIL-1 production, suggesting a role for these activation pathways in IL-1 induction. Low levels of A23187, when combined with OAG (a direct activator of PKC), stimulated mIL-1 expression in the absence of sIL-1 release. Induction of mIL-1 by LPS was inhibited by PGE₂ and dibutyryl cAMP, but higher concentrations were required to inhibit mIL-1 expression compared with sIL-1 release. LTB₄ alone did not induce mIL-1 or sIL-1 production. LTB₄ did enhance LPS-induced mIL-1 expression but not sIL-1 release. These results indicate that mIL-1 expression and sIL-1 release are differentially regulated. Membrane IL-1 is induced by lower levels of certain stimuli and is less effectively inhibited than is sIL-1 release. This differential regulation is further evidence to support the existance of membrane IL-1.     

Differential B-lymphocyte regulation by CD40 and its viral mimic,latent membrane protein 1

Summary: CD40 plays a vital role in humoral immunity, via its potent and multifaceted function as an activating receptor of various immune cells, most notably B lymphocytes. The Epstein-Barr virus-encoded transforming protein latent membrane protein 1 (LMP1) serves as a functional mimic of CD40 signals to B cells but lacks key regulatory controls that restrain CD40 signaling. This allows LMP1 to activate B cells in an abnormal manner that can contribute to the pathogenesis of human B-cell lymphoma and autoimmune disease. This review focuses upon a comparative analysis of CD40 versus LMP1 functions and mechanisms of action in B lymphocytes, discussing how this comparison can provide valuable information on both how CD40 signaling is normally regulated and how LMP1 disrupts the normal CD40 pathways, which can provide information of value to therapeutic design.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

Differential prostaglandin production by unfractionated and density-fractionated human monocytes and alveolar macrophages

Mononuclear phagocyte elaboration of E series prostaglandins (PGE) may be important in the regulation of inflammatory and fibrotic reactions. Mononuclear phagocytes are morphologically and functionally heterogeneous cells. To further understand the processes controlling inflammation and fibrosis, in particular that in the human lung, we characterized the ability of unfractionated and density-fractionated human alveolar macrophages and blood monocytes to elaborate PGE. Alveolar macrophages and blood monocytes constitutively elaborated small amounts of PGE, and their elaboration of PGE was increased with lipopolysaccharide (LPS) stimulation. Monocytes elaborated more PGE than autologous alveolar macrophages. In addition, denser monocytes (specific gravity greater than 1.055) and denser alveolar macrophages (specific gravity greater than 1.044) elaborated more PGE than less dense monocytes and alveolar macrophages, respectively. When monocytes were incubated in vitro, their constitutive PGE elaboration decreased with time. However, in vitro incubation did not cause monocytes to lose their capacity to elaborate PGE in response to LPS. Thus, mononuclear phagocyte populations differ in their ability to elaborate PGE. These differences can be only partially attributed to differences in cell maturation.