Gossypol inhibits estrogen binding to rat alpha-fetoprotein

We find that gossypol, a male anti-fertility compound, is a reversible competitive inhibitor of estrogen binding to rat alpha-fetoprotein (AFP). The Kd of gossypol for rat AFP is 1.75 microM, which is similar to gossypol's affinity for lactate dehydrogenase isozyme X, the putative site where gossypol exerts its anti-fertility effects. Reacting sodium cyanoborohydride with gossypol reduces its affinity for AFP, showing that intact aldehyde groups on gossypol are important for binding to rat AFP and indicating that gossypol is specifically inter-acting with a nucleophilic site on AFP that influences estrogen binding.     

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.     

Human adipose tissue macrophages display activation of cancer-related pathways

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.     

Hypoxia modulates lipopolysaccharide induced TNF-alpha expression in murine macrophages

The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-alpha) together with tissue hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-alpha biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known. Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-induced TNF-alpha expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38 MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-alpha. Furthermore, hypoxia inducible factor-1alpha (HIF-1alpha) was detected in vivo after LPS administration but its expression was not affected by SB203580. Our data indicate that LPS induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-alpha secretion. Furthermore, the induction of HIF-1alpha in mice with endotoxemia suggested a synergistic effect on p38 mediated TNF-alpha expression. These findings provide new insights on the pathophysiological effects of hypoxia in sepsis and septic shock.     

ERK-dependent induction of TNFalpha expression by the environmental contaminant benzo(a)pyrene in primary human macrophages

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are toxic environmental contaminants known to enhance production of pro-inflammatory cytokines such as IL-1beta. The present study was designed in order to determine whether TNFalpha, another cytokine acting in inflammation, may also constitute a target for these chemicals. Both TNFalpha mRNA and TNFalpha secretion levels were found to be enhanced in human BP-treated macrophages. Dioxin, a contaminant activating the aryl hydrocarbon receptor (AhR) like PAHs, was also shown to increase TNFalpha expression. BP-mediated TNFalpha induction was however not suppressed by AhR antagonists, making unlikely the involvement of the typical AhR signalling pathway. BP-exposure of macrophages did not enhance NF-kappaB DNA binding activity, but it activated the MAP kinase ERK1/2. In addition, the use of chemical inhibitors of extracellular signal-regulated protein kinase (ERK) activation fully abrogated induction of TNFalpha production in BP-treated macrophages. These data likely indicate that PAHs enhance TNFalpha expression in human macrophages through an ERK-related mechanism. Such a regulation may contribute to confer pro-inflammatory properties to these widely-distributed environmental contaminants.     

Human primary keratinocytes show restricted ability to up-regulate suppressor of cytokine signaling (SOCS)3 protein compared with autologous macrophages

Suppressor of cytokine signaling (SOCS)3 belongs to a family of proteins that are known to exert important functions as inducible feedback inhibitors and are crucial for the balance of immune responses. There is evidence for a deregulated immune response in chronic inflammatory skin diseases. Thus, it was the aim of this study to investigate the regulation of SOCS proteins involved in intracellular signaling pathways occurring during inflammatory skin diseases and analyze their impact on the course of inflammatory responses. Because we and others have previously described that the cytokine IL-27 has an important impact on the chronic manifestation of inflammatory skin diseases, we focused here on the signaling induced by IL-27 in human primary keratinocytes compared with autologous blood-derived macrophages. Here, we demonstrate that SOCS3 is critically involved in regulating the cell-specific response to IL-27. SOCS3 was found to be significantly up-regulated by IL-27 in macrophages but not in keratinocytes. Other STAT3-activating cytokines investigated, including IL-6, IL-22, and oncostatin M, also failed to up-regulate SOCS3 in keratinocytes. Lack of SOCS3 up-regulation in skin epithelial cells was accompanied by prolonged STAT1 and STAT3 phosphorylation and enhanced CXCL10 production upon IL-27 stimulation compared with macrophages. Overexpression of SOCS3 in keratinocytes significantly diminished this enhanced CXCL10 production in response to IL-27. We conclude from our data that keratinocytes have a cell type-specific impaired capacity to up-regulate SOCS3 which may crucially determine the course of chronic inflammatory skin diseases.     

Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.     

Contribution of macrophages to peripheral neuropathic pain pathogenesis

Neuropathic pain pathogenesis is not only confined to changes in the activity of neuronal systems, but also involves neuro-immune interactions mediated by inflammatory cytokines and chemokines. Among the immune cells involved in these interactions, macrophages and their central nervous system counterparts – microglia – are actively involved in the generation of peripheral neuropathic pain. Depending on the type of lesion (traumatic, metabolic, neurotoxic, infections or tumor invasion), the profile of the activated macrophages and microglia in terms of time, place and subtype can substantially vary, due to their remarkable plasticity that allows tuning their physiology according to microenvironmental signals. Knowing what and when specific macrophages activate after a peripheral nerve lesion could help in creating a pattern that can be further used to target the macrophages with cell-specific therapeutics and remit chronicization and complications of neuropathic pain. This minireview summarizes recent findings on the specific contribution of macrophages in different neuropathic pain models.     

Imidocarb,a potent anti-protozoan drug,up-regulates interleukin-10 production by murine macrophages

Interleukin-10 (IL-10), a potent antiinflammatory and immunosuppressive cytokine, plays an important role in the regulation of immune responses. To discover small molecules that stimulate IL-10 production, a cell-based screening assay was performed using a murine macrophage cell line, RAW264.7. Imidocarb, (3,3'-bis-2-imidazolin-2-yl)-carbanilide, which has been used as an anti-protozoan drug for the prevention and treatment of babesiosis in cattle, was thus identified. Imidocarb markedly enhanced LPS-induced IL-10 production not only by RAW264.7 cells but also by murine peritoneal macrophages in a concentration-dependent manner. It also augmented IL-10 production in the presence of zymosan, a yeast cell wall component. In contrast, imidocarb inhibited LPS-induced tumor necrosis factor (TNF)-alpha production by peritoneal macrophages. In mice, intraperitoneal administration of imidocarb significantly increased serum IL-10 levels and lowered TNF-alpha levels. Our results suggest that a novel anti-inflammatory property of imidocarb could lead to new therapeutic approaches in inflammatory conditions.     

γδT细胞相关的趋化因子受体

γδT细胞是一群异质性的免疫活性细胞,根据表面标志和功能可将其分为不同亚群。γδT细胞表达的趋化因子受体各异,趋化因子受体的表达与γδT细胞在抗感染、抗肿瘤和自身免疫中的功能发挥密切相关,现就近年来与γδT细胞相关的趋化因子受体研究进展作一综述。     

Human cytomegalovirus productively infects primary differentiated macrophages.

Monocytes are one of the predominant cell types in the peripheral blood that are infected by human cytomegalovirus (HCMV). Although virus can be detected in these cells in vivo, HCMV replication in cultured monocytes has been unsuccessful. In this study, we demonstrate efficient HCMV replication in cultured monocytes. HCMV permissiveness in these cells was dependent on nonadherent cell-induced stimulation of the monocyte, with subsequent morphological differentiation into macrophages. Approximately 40% of the cells infected by virus were detected by immunofluorescent staining with both immediate-early and late antibodies. In addition, viral plaque assays demonstrated significant productive infection of macrophages. These observations are consistent with the suggestion that the monocyte/macrophage serves as a source of viral amplification and dissemination.     

Human adiponectin binds to bacterial lipopolysaccharide

Adiponectin has anti-inflammatory and anti-atherogenic properties in addition to its acknowledged roles in insulin sensitivity and energy homeostasis. These properties include the suppression of lipopolysaccharide [LPS]-mediated inflammatory events. We demonstrated that both recombinant and native adiponectin directly bind LPS derived from three different bacteria. The interaction occurred at pH 5.0-6.0 and was inhibited by the presence of Ca(2+) and Mg(2+), but enhanced by the sequestration of these cations. Maximal binding occurred at pH 6.0 in the presence of ethylenediaminetetraacetic acid. Lipid A and C1q were not inhibitory, although LPS, heparin, zymosan, and individual sugars all inhibited the reaction. Periodate-mediated deglycosylation of adiponectin, and reduction and alkylation also inhibited binding. Since adiponectin infiltrates into [relatively] acidic sites of inflammation, it may act as a scavenging anti-inflammatory agent in atherosclerosis and vascular damage where LPS [and other pro-inflammatory molecules] are present.     

Human haptoglobin binds to human myoglobin

Human myoglobin, obtained from human heart, was purified to homogeneity by salt precipitation, crystallization and ion-exchange chromatography. Trace contamination by haemoglobin, if any, was removed by repeated adsorption on an immunoadsorbent of anti-haemoglobin antibodies. The interaction between human haptoglobin and human myoglobin was investigated by a solid-phase radioimmunoassay. Myoglobin adsorbents bound 125I-labelled haptoglobin in a specific manner. Linear Scatchard plots of the data indicate that human myoglobin has only one binding site for haptoglobin in terms of the binding affinity (Ka = 8.5 X 10(6) M-1). These results suggest that haptoglobin not only binds haemoglobin but also binds human myoglobin, although with an affinity that is much lower than that of haemoglobin. The physiological significance of this interaction is discussed.     

Human plasma gelsolin binds to fibronectin

Human plasma gelsolin, a 93,000-dalton actin-binding protein binds to human plasma fibronectin. Qualitative data obtained from experiments employing quasi-elastic light scattering, sucrose gradient sedimentation, gel filtration chromatography, and fibronectin polymerization indicate that gelsolin and fibronectin form a complex in solution. Solid-phase binding studies show that both human plasma and rabbit macrophage gelsolin bind to immobilized fibronectin with a Kd of about 1 microM in a 1:1 complex. The ability of gelsolin to interact with actin was not affected by the presence of fibronectin. Fibronectin also increased the amount of gelsolin binding to fibrin clots. Binding of gelsolin to fibronectin may serve to localize plasma gelsolin in regions where fibronectin is deposited, such as inflammatory sites.     

The HIV-1 co-receptor CCR5 binds to alpha-catenin, a component of the cellular cytoskeleton

The chemokine receptors CCR5 and CXCR4 belong to the family of seven transmembrane-spanning G protein-coupled receptors, which have diverse functions in host cell defense and are associated with numerous diseases. CCR5 and CXCR4 are known as co-receptors for entry of HIV-1. In this study the intracellular carboxy-terminus of CCR5, which is deleted in HIV-infected long-term non-progressors, was shown to interact with the carboxy-terminus of alpha-catenin, a component of the cytoskeleton, in a yeast two-hybrid screen. This interaction was verified in mammalian cells. Furthermore, the interaction of alpha-catenin with CCR5 and CXCR4 at endogenous protein levels was demonstrated in PM1 T-lymphocytes, a host cell line of HIV-1. Our results suggest that alpha-catenin links CCR5 and CXCR4 to the cytoskeleton and is involved in the organization of these receptors at the membrane, thereby possibly affecting HIV-1 infection.     

Role of macrophages in the host response to Lewis lung peritoneal carcinomatosis

Lewis lung (3LL) peritoneal carcinomatosis elicits a complex host response in the peritoneal compartment. The response was delayed, showing few inflammatory cells through day 6 after lethal challenge with 3LL cells. Responses began in about half the mice on day 7 and had appeared in all mice by day 11. On day 7, some mice still showed no detectable 3LL growth in the peritoneal lavage fluid, and no differences in the peritoneal cell populations as compared with the control group. Other tumor-bearing mice, however, had evidence of 3LL cells and hemorrhagic ascites in the peritoneal compartment, with increased numbers of peritoneal macrophages (PM) and polymorphonuclear neutrophils (PMN). By day 11, all tumor-bearing mice had 3LL growth and hemorrhagic ascites. On days 7–11, there was a major influx of macrophages with a later influx of PMN between days 11 and 14. Two distinct PM populations were detected on day 7 in mice that showed detectable 3LL peritoneal carcinomatosis: resident PM, which did not express the Mac-2 antigen, and recruited PM, which were Mac-2⁺. At least some resident PM remained in the peritoneal compartment through day 14. Analysis of the kinetics of the cytotoxic capabilities of PM from tumor-bearing mice showed that by day 7 macrophages were able to kill the B16 melanoma tumor target, but not the 3LL target. The PM, however, were able to be activated further to kill the 3LL target by treatment in vitro with lipopolysaccharide and interferon . No inhibition of PM tumoricidal activity could detected in the peritoneal wash of tumor-bearing mice. A lack of activation of PM from 3LL tumor-bearing mice may be involved in progression of peritoneal carcinomatosis.     

Label-free immunosensing for alpha-fetoprotein in human plasma using surface plasmon resonance

In this study, we attempted to develop a surface plasmon resonance (SPR)-based immunoassay sensor to detect alpha-fetoprotein (AFP) in human plasma at the nanogram level, as is required for clinical diagnosis of hepatocellular tumors. A self-assembled monolayer (SAM) surface of tri(ethylene glycol) (TEG) and carboxyl group-terminated hexa(ethylene glycol) (HEG) was employed to suppress the nonspecific adsorption of plasma components onto the sensor surface. AFP was detected by a sandwich-type immunoassay using two kinds of antibodies, primary and secondary, in this system. The SPR signal shift was further enhanced by applying an antibody (polyclonal) against the second antibody. With this method, the SPR signals were highly intensified, and so nanogram levels (ng/ml) of AFP could be easily detected with a high signal/noise ratio, as is necessary for clinical diagnosis. It is expected that our SPR-based immunoassay method can also be applicable to the detection of several other tumor markers that are present in low concentrations in human blood.