单核细胞趋化蛋白－1在大肠杆菌中的表达

将人单核细胞趋化蛋白－１（ＭＣＰ－１）的ｃＤＮＡ插入融合蛋白表达载体ｐＧＥＸ－４Ｔ－１中，构建成质粒ｐＧＥＸ／ＭＣＰ转化大肠杆菌ＪＭ１０９，经ＩＰＴＧ诱导后合成ＧＳＴ－ＭＣＰ－１融合蛋白。用１２％ＳＤＳ－ＰＡＧＥ检测在３０ｋＤ左右有新生蛋白条带出现，表达量约占菌体总蛋白的３１．７％。趋化实验证明，该产物具备明确的单核细胞趋化活性。     

Human monocyte chemoattractant protein-1 (MCP-1)

During the past three years great advances have been made in the chemistry and biology of chemoattractants for human leukocytes. Two chemoattractant cytokines have been isolated, sequenced and cloned, each with distinctive leukocyte attractant specificity. Monocyte chemoattractant protein 1 (MCP-1), the subject of this review by Edward Leonard and Teizo Yoshimura, is secreted by PHA-stimulated mononuclear cells and can be identified by northern blotting in response to LPS or PHA. It attracts monocytes but not neutrophils. In contrast, neutrophil attractant/activation protein (NAP-1) (also known as interleukin 8 (IL-8)) attracts and activates human neutrophils but it is not a chemoattractant for human monocytes. Based on amino acid sequence analysis, each of these attractants has been assigned to one of two distinct families of cytokines that are thought to participate in host defense and inflammatory responses.     

Acute Alcohol Consumption Attenuates Interleukin-8 (IL-8) and Monocyte Chemoattractant Peptide-1 (MCP-1) Induction in Response to Ex Vivo Stimulation

Chronic alcohol use is associated with impaired immunity and host defense. Even acute ethanol treatment both in vitro and in vivo has been shown to result in decreased inflammatory cytokine production. However, the potential immunoregulatory effects of acute, moderate alcohol use are yet to be fully explored. Here we show that in vitro acute alcohol treatment of normal blood monocytes resulted in a significant, dose-dependent (25–100 mM) attenuation of staphylococcus enterotoxin B (SEB), phytohemagglutinin (PHA), or IFN-induced monocyte IL-8 and MCP-1 production (P < 0.01). Likewise, ethanol (100 mM) in vitro reduced MCP-1 levels in response to SEB, PHA, or IFN stimulation in mononuclear cells (31–62% reduction). Furthermore, acute alcohol consumption (0.85 g/kg body weight) significantly attenuated SEB- or LPS-induced IL-8 and MCP-1 levels in whole-blood samples obtained 4 hr after alcohol consumption from normal nonalcoholic individuals (P < 0.01). RANTES and MIP-1 were only minimally inhibited (16–25% inhibition) by in vitro ethanol (100 mM) in mononuclear cells, suggesting that ethanol may have a selective effect on the regulation of various chemokines. These results demonstrate that acute alcohol, in vivo as well as in vitro, attenuates monocyte-derived chemokine production in response to a subsequent immune challenge. Our data show for the first time that activation of nuclear regulatory factor B (NF-B), a common regulator binding in the promoter region of IL-8 and MCP-1 genes, is inhibited by acute ethanol (25 mM) treatment in SEB-stimulated human monocytes. These results imply that inhibition of NF-B activation may be one of the intracellular mechanisms for the ethanol-induced inhibition of IL-8 and MCP-1 production in monocytes. Thus, impaired chemokine (particularly MCP-1 and IL-8) induction upon an immune challenge is likely to contribute to compromised host defense after acute alcohol consumption and may also affect progression of diseases such as atherosclerosis or HIV infection where chemokines contribute to progression of the disease.     

Tetracyclines and Chemically Modified Tetracycline-3 (CMT-3) Modulate Cytokine Secretion by Lipopolysaccharide-Stimulated Whole Blood

In addition to their bacteriostatic effect, tetracyclines, which are often used in the treatment of periodontitis, also present anti-inflammatory properties. In the present study, we investigated the effects of tetracycline (TC), doxycycline (doxy), and chemically modified tetracycline-3 (CMT-3) on the production of pro-inflammatory mediators and matrix metalloproteinases (MMPs) in an ex vivo human whole blood (WB) model stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). WB samples obtained from three periodontitis patients and six healthy subjects were stimulated with P. gingivalis LPS in the absence and presence of TC, doxy, or CMT-3. The secretion of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), MMP-8, and MMP-9 by the WB samples was determined using enzyme-linked immunosorbent assays. P. gingivalis LPS significantly increased the secretion of all cytokines and MMPs tested. While we observed inter-patient variations, TC, doxy, and CMT-3 caused reductions of LPS-induced cytokine secretion to various degrees. TC, doxy, and CMT-3 had no significant effect on MMP-8 and MMP-9 secretion by LPS-stimulated WB samples. In conclusion, we used a human WB model that takes into consideration relevant in vivo immune cell interactions in the presence of plasma proteins to show that TC, doxy, and CMT-3 can reduce the production of pro-inflammatory mediators. This property may contribute to the clinically proven benefits of these molecules in the treatment of periodontitis and other chronic inflammatory diseases.     

Reassessment of Immune Correlates in Human Visceral Leishmaniasis as Defined by Cytokine Release in Whole Blood

Depressed cell-mediated immunity in human visceral leishmaniasis (VL) (also known as kala-azar), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond to Leishmania antigen, remains a hallmark of and is thought to underlie the progressive nature of this disease. We recently reported the ability of a whole-blood, gamma interferon (IFN-γ) release assay to detect subclinical infections among healthy individuals living in an area where kala-azar is endemic (Bihar, India) and the surprising result that patients with active VL also secreted significant levels of antigen-specific IFN-γ in this assay. We were interested in ascertaining whether these findings would be true for a larger cohort of subjects and in employing the whole-blood assay to detect additional cytokines that might better correlate with the disease status of infected individuals. We evaluated IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) release in 35 patients with active VL, 54 patients with VL who were cured, 27 patients with other diseases, 52 healthy controls who lived in regions where VL or kala-azar is not endemic (NEHCs [for nonendemic healthy controls]), and 147 healthy controls who lived in regions where kala-azar is endemic (EHCs [for endemic healthy controls]). The cellular responses of the EHCs were correlated with their serological antibody titers against Leishmania donovani and Phlebotomus argentipes saliva. The whole-blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed subclinical infections, produced significantly elevated levels of IFN-γ. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the patients with active VL also produced IL-10, which in conjunction with IFN-γ better reflects the immune responses that distinguish individuals with active disease from cured or subclinically infected, immune individuals.     

Delayed Addition of Glucocorticoids Selectively Suppresses Cytokine Production in Stimulated Human Whole Blood

Glucocorticoids (GC) are potent drugs proven to effectively treat inflammatory diseases, although patients typically begin therapy after the onset of symptoms. Clinical studies with cytokine inhibitors prove that these mediators drive inflammatory responses in diseases such as rheumatoid arthritis and Crohn''s disease. Despite the clear sequence of cytokine-induced inflammation followed by effective GC treatment, most basic science investigations have examined the ability of GC to prevent an inflammatory response rather than halt its progression. The current studies used the Toll-like receptor 2 (TLR2) agonist palmitoyl₃-cysteine-serine-lysine₄ (PAM) or the TLR4 agonist lipopolysaccharide (LPS) to stimulate human whole blood and determine whether postponing the addition of the GC dexamethasone (DEX) limits its ability to decrease cytokine production. Twenty-four hours after stimulation, tumor necrosis factor (TNF), interleukin-1β (IL-1β), IL-6, and IL-8 levels were measured, in addition to the cytokine inhibitors IL-1 soluble receptor II (SRII), IL-1 receptor antagonist, and TNF SRII. LPS rapidly induced all of the proinflammatory mediators over 24 h while failing to induce any of the cytokine inhibitors. PAM stimulation also induced IL-1β, IL-6, and IL-8. Concomitant addition of DEX plus LPS or PAM significantly suppressed all cytokine levels. Delaying the addition of DEX until 6 h after LPS stimulation failed to decrease TNF or IL-6. In contrast, delayed DEX addition significantly suppressed PAM-induced IL-1β, IL-6, or IL-8 and also suppressed LPS-induced IL-1β and IL-8. Our results show that cytokines which typically increase in concentration between 6 and 24 h after stimulation were significantly suppressed by the addition of DEX 6 h after stimulation.Glucocorticoids (GC) are members of the corticosteroid family whose anti-inflammatory properties have been widely exploited both as a clinical therapy and as a tool for understanding the mechanisms of inflammation (20). GC are widely used to treat a number of inflammatory conditions such as rheumatoid arthritis (30), bacterial meningitis (11), and allergic asthma (29). GC have been shown to have a beneficial effect when administered after the onset of some inflammatory disorders. Inhaled corticosteroids, for example, are commonly used to treat acute allergic asthma. Early systemic administration of corticosteroids has significantly reduced the incidence of hospitalization and relapse and expedited recovery for severe asthma patients presenting in the clinic with an acute exacerbation (28). A small study showed that GC rescue in patients with established acute respiratory distress syndrome significantly improved sequential organ failure assessment scores in trauma patients (17). Cytokines have been implicated in the pathogenesis of several of these diseases (16), and cytokine inhibitors have revolutionized the treatment of diseases such as rheumatoid arthritis.Multiple stimuli such as infections, trauma, autoimmune disorders, and allergies activate immune responses and initiate inflammation by stimulating the secretion of cytokines. These mediators drive both the innate and adaptive immune responses by perpetuating inflammatory responses via paracrine and autocrine mechanisms (15). Invading pathogens shed their outer membrane components, which, upon binding to cell surface receptors, initiate cytokine and chemokine secretion by inflammatory cells over several hours to days, and in some instances, chemokines are produced over several weeks (26). Published data report that in response to various stimuli, cytokines and chemokines often display distinct kinetic profiles (4, 9, 10). Cytokines such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6 are known to be rapidly induced and cleared, while chemokines such as IL-8 have been shown to be steadily, continuously produced over time (10).Despite proven clinical efficacy when GC are given after the onset of inflammation, virtually no studies have examined the ability of GC to inhibit the production of cytokines if added several hours after the initial stimulus. The experiments described here were designed to determine if dexamethasone (DEX) could decrease cytokine production after inflammation had been initiated in the whole-blood model. While the whole-blood model does not provide the same complexity as in vivo studies, the model has been used extensively by numerous investigators to study the regulation of cytokine production (6) and has become a standardized method (18). These studies determined the mechanism by which delayed treatment with GC controls inflammation by determining which mediators could potentially be decreased.     

Cytokine Gene Expression by Peripheral Blood Leukocytes in Horses Experimentally Infected with Anaplasma phagocytophila

Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A. phagocytophila or by allowing feeding by infected ticks. The p44 genes encoding the major outer membrane protein P44s of A. phagocytophila were detected by PCR in PBLs of all four horses from 4 to 20 days postexposure. During the 20-day infection period, interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA expression was upregulated in PBLs of all four horses, and IL-8 mRNA expression was upregulated in three horses. Gamma interferon, IL-10, and IL-12 p35 mRNAs were weakly expressed in only one horse each. IL-2, IL-4, IL-6, and IL-12 p40 mRNA expression , however, could not be detected in the PBLs of any of the four horses. These results suggest that IL-1β, TNF-α, and IL-8 generation during A. phagocytophila infection has a primary role in HGE pathogenesis and immunomodulation.     

Cytokine Gene Expression Occurs More Rapidly in Stimulated Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Persons

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV−) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV− and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P < 0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-α]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV− PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-α mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P < 0.05) and TNF-α (P < 0.005), compared to HIV− PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-α) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.     

Hypoxia induces expression of the chemokines monocyte chemoattractant protein-1 (MCP-1) and IL-8 in human dermal fibroblasts

Hypoxia is an important factor in the pathophysiology of vascular and inflammatory diseases. Leucocyte infiltration, as a consequence of adhesion molecule up-regulation and chemokine release, is a prominent feature of these diseases. The objective of our study was to investigate the potential role of resident fibroblasts in hypoxia-induced chemotactic responses. We show that MCP-1 and IL-8 mRNA are specifically induced by hypoxia in dermal fibroblasts. This response is paralleled by increased NF-kappaB p65/p50 binding activity, and it is inhibited by pretreatment with N-acetyl-L-cysteine. MCP-1 secreted by fibroblasts is chemotactic for monocytic cells and this activity is significantly increased by hypoxia. Chemotactic index correlates with MCP-1 protein levels and is significantly decreased by neutralizing anti-MCP-1 MoAb. These findings demonstrate the ability of resident fibroblasts to mediate chemotaxis of leucocytes through the release of chemokines in response to hypoxia. Our data point to MCP-1 as an important component in this response, and therefore it may be a potential target in inflammatory responses associated with hypoxia.     

Histamine Blocks Interleukin 2 (IL-2) Gene Expression and Regulates IL-2 Receptor Expression

Histamine inhibited the roliferative response of human peripheral blood mononuclear cells (PBMCT to the T cell mitoFen Phytohema P (PHA-P) in a dose-dependent fashion. This inhibition was mePutiated via the H₂ receptor since cimetidine, a known H₂ antagonist, removed the inhibition, whereas the addition of the H₁ antagonist Diphenhdramine did not. Inhibition occurred durin the inductive phase of the cell cycle, since histamine added 24 hours aBter PHA-P stimulation had no effect on subsequent T cell proliferation, and was attributable to inhibition of interleukin-2 (IL-2) gene expression. Both secreted IL-2 and messenger RNA coding for IL-2 were inhibited by histamine. In contrast, histamne exerted no inhibitory effect on the expression of cell surface receptors for IL-2 as determined by flow cytometry. Furthermore, histamine-treated cells retained full responsiveness to exogenously administered IL-2, which completely reversed the anti-proliferative effect of histamine. In some donors, histamine enhanced the ercentage of IL-2 receptor positive cells. Stimulated PBMC from AIDS KS patients as a group, displayed a lower ercentage of IL-2 receptor bearing cells, which was significantly increased gy the addition of histamine even at concentrations as low as 10^--⁶ M and peaking at 10^--³ M. These findings indicate that histamine exerts its anti-proliferative effects on T cells by inhibiting IL-2 production, via blockade of IL-2 gene expression. In addition, histamine seems to exert immunomodulating effects on IL-2 receptor expression, particularly in those individuals with AIDS-KS.     

氧化型α1-抗胰蛋白酶对HBE细胞释放炎症因子的影响

 目的：探讨氧化型α1-抗胰蛋白酶（Ox-AT）对体外培养人支气管上皮(HBE)细胞炎症因子白细胞介素8（IL-8）和单核细胞趋化蛋白1（MCP-1）释放的影响及可能机制。方法：从人血浆中分离纯化获得天然构型AT（N-AT）,加入氧化剂获得Ox-AT;用不同浓度N-AT和Ox-AT分别作用于体外培养的HBE细胞,用ELISA方法检测不同时段培养上清液中IL-8和MCP-1的含量,同时观察NF-κB抑制剂Bay11-7082对Ox-AT引起HBE细胞炎症因子释放的影响。结果：Ox-AT可促进HBE细胞释放IL-8和MCP-1,其促进作用与Ox-AT的浓度及作用时间呈正相关;用0.5 g/L Ox-AT孵育HBE细胞4、10和24 h,其促进HBE细胞分泌IL-8和MCP-1的作用与10 μg/L肿瘤坏死因子 α（TNF-α）的作用基本一致,而 N-AT无刺激HBE细胞分泌IL-8和MCP-1的作用;Ox-AT 能显著增加NF-κB活性; Ox-AT的促炎症作用能被NF-κB抑制剂Bay11-7082抑制。结论：Ox-AT是人正常支气管上皮细胞的强致炎因子,机制可能与NF-κB信号通路激活有关。     

人单核细胞趋化蛋白－1（MCP－1）cDNA的克隆和序列测定

人单核细胞趋化蛋白－１（ＭＣＰ－１）是由多种细胞产生的一种趋化因子。它不但能趋化单核细胞还能激活单核细胞，在机体防御、炎症恢复和抗肿瘤等方面起重要作用。本文利用ＲＴ－ＰＣＲ方法克隆了人ＭＣＰ－１ｃＤＮＡ，经核苷酸序列测定，除第１２位密码子为ＴＧＴ与国外报道的ＴＧＣ不同、但编码相同的氨基酸半胱氨酸外，其余序列完全相同。     

Differential Induction of Membrane-Associated Interleukin 1 (IL-1) Expression and IL-1α and IL-1β Secretion by Lipopolysaccharide and Silica in Human Monocytes

Bacterial lipopolysaccharide (LPS) and silica dust are known to be effective inducers of interleukin 1 (IL-1) in human cultured monocytes. The data reported here show that although the levels of secreted IL-1 were equally high after in vitro stimulation with an optimal dose of LPS or silica, there were two clear differences: (i) the levels of membrane-associated IL-1 (as detected by the comitogenic effect of paraformaldehyde (PFA)-fixed cells or purified membrane fragments on murine thymocytes) were ca. five times higher after LPS stimulation than after silica stimulation, (ii) the secreted IL-1 after LPS stimulation was mainly of the pI 7 (IL-1 beta) type, while after silica stimulation there were equally high amounts of pI 7 and pI 5 (i.e. IL-1 alpha) forms. In both cases the IL-1 active molecules belonged to the 15 kDa class. These data show that the nature of the activating agent has a clear influence on the distribution of the biologically active IL-1 molecules. Moreover, the finding that after silica stimulation the amount of membrane-associated IL-1 (which was recently shown to be of the IL-1 alpha type) was low, while IL-1 alpha in the culture fluid was clearly elevated, suggests that the IL-1 alpha not attached to the cell membrane (or released from it) significantly contributes to the secreted IL-1 pool.