T Cell Stimulation In Vivo by Lipopolysaccharide (LPS)

Lipopolysaccharide (LPS) from gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other APC. We show here that, under in vivo conditions, LPS also induces strong stimulation of T cells. As manifested by CD69 upregulation, LPS injection stimulates both CD4 and CD8⁺ T cells, and, at high doses, stimulates naive (CD44^lo) cells as well as memory (CD44^hi) cells. However, in terms of cell division, the response of T cells after LPS injection is limited to the CD44^hi subset of CD8⁺ cells. In contrast with B cells, proliferative responses of CD44^hi CD8⁺ cells require only very low doses of LPS (10 ng). Based on studies with LPS-nonresponder and gene-knockout mice, LPS-induced proliferation of CD44^hi CD8⁺ cells appears to operate via an indirect pathway involving LPS stimulation of APC and release of type I (α, β) interferon (IFN-I). Similar selective stimulation of CD44^hi CD8⁺ cells occurs in viral infections and after injection of IFN-I, implying a common mechanism. Hence, intermittent exposure to pathogens (gram-negative bacteria and viruses) could contribute to the high background proliferation of memory–phenotype CD8⁺ cells found in normal animals.     

Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)- anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.     

Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS

Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14- dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R- HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.     

Mice Genetically Hyporesponsive to Lipopolysaccharide (LPS) Exhibit a Defect in Endocytic Uptake of LPS and Ceramide

We have recently shown that monomeric bacterial LPS is rapidly delivered from the plasma membrane to an intracellular site and that agents that block vesicular transport block responses of neutrophils to lipopolysaccharide (LPS) (Detmers, P.A., N. Thiéblemont, T. Vasselon, R. Pironkova, D.S. Miller, and S.D. Wright. 1996. J. Immunol. 157:5589–5596). To examine further the connection between intracellular transport of LPS and signaling, we observed internalization of fluorescently labeled LPS in cells from LPS-hyporesponsive (Lps^d) mice. Binding of fluorescent LPS from LPS–soluble CD14 (sCD14) complexes by peritoneal macrophages from Lps^d and control (Lpsⁿ) mice was quantitatively similar, and confocal images obtained from these cells exhibited an identical appearance immediately after labeling. Incubation of labeled Lpsⁿ macrophages at 37°C caused movement of the fluorescence from the cell perimeter in one or two spots in the perinuclear region. However, in Lps^d cells the fluorescence remained dispersed, suggesting a defect in vesicular transport. LPS resembles ceramide, and Lps^d mice fail to respond to ceramide. As with LPS, we found that binding of fluorescent ceramide by Lps^d and Lpsⁿ macrophages was quantitatively similar, and the label moved rapidly to one to two spots in the perinuclear region in Lpsⁿ mice. However, in Lps^d macrophages the fluorescence remained dispersed. These results show that cells deficient in responses to LPS exhibit defective vesicular transport of LPS and ceramide and point to a role for vesicular transport in responses to these mediators.     

Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.

Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 0111:B4 LPS on movement of 14C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations > or = 0.5 ng/ml, and LPS (10 ng/ml) exposures of > or = 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 micrograms/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of > or = 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required.     

In Vivo Suppression of the Immune Response to Alloantigen by Cholera Enterotoxin

The immune response of C57BL/6 mice to allogeneic (DBA/2) mastocytoma cell suspensions was profoundly suppressed by intraperitoneal administration of 1 mug cholera enterotoxin 4 days after antigenic stimulation. The immune response assayed 11 days after antigen showed decreased cytolytically active thymusderived (T) lymphocytes and markedly depressed serumagglutinating titers. A comparable suppression of the immune response to skin allografts (DBA/2-->C57BL/6) was also effected by cholera toxin administration, although there was no prolongation of allograft survival.The mechanism of the immune suppression is apparently related to the known adenylate cyclase stimulatory activities of choleragen.     

[11C]Gefitinib ([11C]Iressa): Radiosynthesis, In Vitro Uptake, and In Vivo Imaging of Intact Murine Fibrosarcoma

Objective

Gefitinib (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(morpholin-4-yl)propoxy]quinazolin-4-amine, Iressa) is an approved anticancer drug. In this study, we labeled gefitinib with carbon-11 and evaluated [¹¹C]gefitinib to explore its specific binding in intact fibrosarcoma (NFSa)-bearing mice.

Methods

[¹¹C]Gefitinib was synthesized by the reaction of desmethyl precursor (1) with [¹¹C]CH₃I. In vitro uptake of [¹¹C]gefitinib into NFSa, human-A431 epidermoid carcinoma, and Jurkat T cells was determined. Positron emission tomography (PET) imaging using [¹¹C]gefitinib was performed for NFSa-bearing mice.

Results

[¹¹C]Gefitinib accumulated into NFSa cells with 2.1 uptake ratio (UR)/mg protein in cells. Addition of nonradioactive gefitinib decreased uptake in a concentration-dependent manner. [¹¹C]Gefitinib also had high uptake (2.6 UR/mg protein) into epidermal growth factor receptor/tyrosine kinase (EGFR/TK)-rich A431 cells but low uptake (0.2 UR/mg protein) into EGFR/TK-poor Jurkat cells. In vivo distribution study on NFSa-bearing mice by the dissection method revealed that [¹¹C]gefitinib specifically accumulated into the tumor. The ratio of radioactivity in tumors to that in blood and muscle as two comparative regions increased from 0.4 to 6.0 and from 0.6 to 5.0 during this experiment (0–60 min), respectively. PET for NFSa-bearing mice produced a clear tumor image, although high radioactivity was distributed throughout the body. Treatment with nonradioactive gefitinib (100 mg/kg) decreased uptake in the tumor. In vivo metabolite analysis demonstrated that [¹¹C]gefitinib was stable in the tumor, liver, kidney, and blood.

Conclusion

These results demonstrated the promising potential of [¹¹C]gefitinib to serve as a PET ligand for in vivo imaging of NFSa-bearing mice.     

Analysis of Vasodepressor Responses to Nociceptin and Nociceptin Analogs in the Systemic Vascular Bed of the Anesthetized Rabbit In Vivo

Background: The heptadecapeptide nociceptin, also known as Orphanin FQ, is a recently discovered endogenous ligand for the opioid-like G-protein-coupled receptor ORL(1). Methods and Results: In the present study, responses to nociceptin, [Tyr(1)]-nociceptin, nociceptin-(2-17), nociceptin-(1-11), and nociceptin-(1-7) were compared in the systemic vascular bed of the rabbit. Nociceptin and [Tyr(1)]-nociceptin induced dose related decreases in systemic arterial pressure (SAP) when injected in doses of 1-30 nmol/kg intravenous (IV); in terms of relative vasodepressor activity, [Tyr(1)]-nociceptin and nocicpetin were similar in potency. However, nocicpetin-(2-17), nocicpetin-(1-11), and nociceptin-(1-7) had no effect on SAP when injected in doses up to 30 nmol/kg IV. The decreases in SAP in response to nociceptin and [Tyr(1)]-nociceptin were not altered by the opioid receptor antagonist naloxone at a time when depressor responses to methionine-enkephalin were reduced significantly. Conclusions: The results of the present study show that vasodepressor responses to nociceptin and [Tyr(1)]-nocicpetin are mediated by the activation of a naloxene-insensitive opioid receptor and are not dependent on the presence of Phe at the N-terminus of the nociceptin sequence. Moreover, the present results show that nociceptin-(2-17), nociceptin-(1-11), and nociceptin-(1-7) do not alter SAP in the rabbit, indicating that peptide chain length is important for the expression of vasodepressor activity.     

lrp和mrp基因在预测急性非淋巴细胞白血病耐药的意义

【目的】评价肺耐药相关蛋白（lrp）基因和多药耐药相关蛋白（mrp）基因在预测急性非淋巴细胞白血病（ANLL）患者临床耐药中的意义。【方法】将70例ANLL患者分为临床非耐药组（A组）43人和耐药组（B组）27人．用RT—PCR方法检测患者骨髓中lrp和mrp基因的表达。【结果】lrp基因在A、B组阳性表达分别占4／43和13／27．统计学分析显示差异有显著性（x^2=4．52,P〈0．05）。mrp基因在A、B组阳性表达分别占10／43和17／27,统计学分析显示差异无显著性意义（x^2=0．11,P〉0.05）。27例临床耐药患者中,同时表达两个基因者8例。而临床非耐药组43人中只有1例同时表达两个基因．两组间差异有显著性意义（x^2=41．1,P〈0．01）。【结论】lrp基因单独或与mrp基因联合检测可预测ANLL发生多药耐药。     

热休克反应对内毒素所致小鼠巨噬细胞炎症因子表达的抑制作用与MAPK信号转导通路的关系研究

【目的】探讨热休克反应(HSR)对内毒素(LPS)所致MAPK信号通路的影响。【方法】采用400ng／ml LPS处理小鼠巨噬细胞(RAW264．7),通过免疫印迹,抗磷酸化抗体检测ERK、JNK、p38的磷酸化。热休克反应是将巨噬细胞在42℃的条件下培养1h,随后在37C,5％CO2条件下恢复12h。【结果】ERK、JNK、p38在LPS处理15min后即被活化,30min达高峰并持续至45min,90min后开始恢复。RT—PCR检测发现TNF—α、IL-15 mRNA水平增高。细胞经HSR(42℃ 1h,并恢复12h)后,再经LPS刺激则TNF—α、IL-15 mRNA转录受抑制,但HSR对JNK、p38、ERK等三种MAPK的活化(磷酸化)无影响。【结论】HSR抑制LPS所致的炎症因子表达不涉及MAPK信号通路。     

Influence of Coagulation Factor X on In Vitro and In Vivo Gene Delivery by Adenovirus (Ad) 5, Ad35, and Chimeric Ad5/Ad35 Vectors

The binding of coagulation factor X (FX) to the hexon of adenovirus (Ad) 5 is pivotal for hepatocyte transduction. However, vectors based on Ad35, a subspecies B Ad, are in development for cancer gene therapy, as Ad35 utilizes CD46 (which is upregulated in many cancers) for transduction. We investigated whether interaction of Ad35 with FX influenced vector tropism using Ad5, Ad35, and Ad5/Ad35 chimeras: Ad5/fiber(f)35, Ad5/penton(p)35/f35, and Ad35/f5. Surface plasmon resonance (SPR) revealed that Ad35 and Ad35/f5 bound FX with approximately tenfold lower affinities than Ad5 hexon–containing viruses, and electron cryomicroscopy (cryo-EM) demonstrated a direct Ad35 hexon:FX interaction. The presence of physiological levels of FX significantly inhibited transduction of vectors containing Ad35 fibers (Ad5/f35, Ad5/p35/f35, and Ad35) in CD46-positive cells. Vectors were intravenously administered to CD46 transgenic mice in the presence and absence of FX-binding protein (X-bp), resulting in reduced liver accumulation for all vectors. Moreover, Ad5/f35 and Ad5/p35/f35 efficiently accumulated in the lung, whereas Ad5 demonstrated poor lung targeting. Additionally, X-bp significantly reduced lung genome accumulation for Ad5/f35 and Ad5/p35/f35, whereas Ad35 was significantly enhanced. In summary, vectors based on the full Ad35 serotype will be useful vectors for selective gene transfer via CD46 due to a weaker FX interaction compared to Ad5.     

Expression of the Elm1 Gene, a Novel Gene of the CCN (Connective Tissue Growth Factor, Cyr61/Cef10, and Neuroblastoma Overexpressed Gene) Family, Suppresses In Vivo Tumor Growth and Metastasis of K-1735 Murine Melanoma Cells

We previously isolated a partial cDNA fragment of a novel gene, Elm1 (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells. Here we determined the full-length cDNA structure of Elm1 and investigated the effect of Elm1 expression on growth and metastatic potential of K-1735 cells. The Elm1 gene encodes a predicted protein of 367 amino acids showing ~40% amino acid identity with the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, neuroblastoma overexpressed gene [Nov]) family proteins, which consist of secreted cysteine-rich proteins with growth regulatory functions. Elm1 is also a cysteine-rich protein and contains a signal peptide and four domains conserved in the CCN family proteins. Elm1 was highly conserved, expressed ubiquitously in diverse organs, and mapped to mouse chromosome 15. High-metastatic K-1735 M-2 cells, which did not express Elm1, were transfected with an Elm1 expression vector, and several stable clones with Elm1 expression were established. The in vivo growth rates of cells expressing a high level of Elm1 were remarkably slower than those of cells expressing a low level of Elm1. Metastatic potential of transfectants was reduced in proportion to the level of Elm1 expression. Thus, Elm1 is a novel gene of CCN family that can suppress the in vivo growth and metastatic potential of K-1735 mouse melanoma cells.