Complementary targeting of liposomes to IL-1α and TNF-α activated endothelial cells via the transient expression of VCAM1 and E-selectin

Inflammation is in part defined by the transient upregulation of cell adhesion molecules on the surface of endothelial cells (ECs) in response to cytokines. We hypothesized that liposomes with a complementary surface presentation of antibodies to the pattern of molecules on the EC surface may enhance targeting. We quantified the expression of vascular cell adhesion molecule-1 (VCAM1) and endothelial leukocyte cell adhesion molecule-1 (E-selectin) on ECs upon exposure to either tumor necrosis factor-α (TNF-α) or interleukin-1α (IL-1α) as a function of time. Liposomes, composed of 95 mol% dioleoyl phosphatidylcholine (DOPC) and 5 mol% dodecanyl phosphatidylethanolamine (N-dod-PE), were prepared by conjugating different molar ratios of antibodies against VCAM1 (aVCAM1) and E-selectin (aE-selectin). Increased binding was observed when immunoliposomes complemented the presentation of VCAM1:E-selectin expressed on TNF-α activated ECs. The 1:1 aVCAM1:aE-selectin liposomes had maximal binding at both 6 and 24 h on IL-1α activated ECs due to differences in molecular organization. The results demonstrate that liposomes targeting to inflamed endothelium may be optimized by exploiting the dynamic expression of VCAM1 and E-selectin on the EC surface.     

Synergistic Effect of Histamine and TNF-α on Monocyte Adhesion to Vascular Endothelial Cells

The histamine level is high during allergic attacks, and patients with allergy may have chronic inflammatory conditions at which tumor necrosis factor (TNF)-α is extensively released by macrophages. Here, in vitro static and microfluidic flow assays were conducted to investigate the combined influence of histamine and TNF-α on adhesion of monocytic THP-1 cells to human umbilical vein endothelial cells (HUVEC). In a static assay, histamine stimulation of TNF-α-activated HUVEC elevated the number of attached THP-1 cells. In a flow assay, the number of crawling and firmly adherent THP-1 cells was higher on TNF-α + histamine activated HUVEC than on HUVEC activated by TNF-α alone. This synergistic effect of histamine and TNF-α is caused by the increased endothelial surface expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Since the exposure of TNF-α-activated endothelium to histamine favors monocyte recruitment, it may be a serious risk factor for atherosclerosis and other inflammatory disorders.     

Adenosine A2A Receptor and TNF-α Regulate the Circadian Machinery of the Human Monocytic THP-1 Cells

Morning stiffness and increased symptoms of inflammatory arthritis are among the most common manifestations of rheumatoid arthritis (RA). Tumor necrosis alpha (TNF-α), an important mediator of inflammation in RA, regulates the circadian expression of clock proteins, and adenosine A_2A receptors (A_2AR) mediate many of the anti-inflammatory and antirheumatic actions of methotrexate, the cornerstone drug in the treatment of RA. We found that A_2AR activation and TNF-α activated the clock core loop of the human monocytic THP-1 cell line. We further observed that interleukin (IL)-10, but not IL-12, mRNA expression fluctuates in a circadian fashion and that TNF-α and A_2AR stimulation combined increased IL-10 expression. Interestingly, TNF-α, but not CGS21680, dramatically inhibited IL-12 mRNA expression. The demonstration that A_2AR and TNF-α regulate the intrinsic circadian clock in immune cells provides an explanation for both the pathologic changes in circadian rhythms in RA and for the adverse circadian effects of methotrexate, such as fatigue.     

Malignant Transformation of Human Embryonic Liver Cells Induced by Hepatitis B Virus and Aflatoxin B_1

Hepatocellular carcinoma (HCC) is a worldwide distribut ed disease accounting for 473 000 newly developed cases ofHCC annually in the world and 235 000 cases in China[1]. Epidemiological and laboratory investigations havedemonstrated that hepatitis…     

Overexpression of the �� Subunit of Human Chorionic Gonadotropin Promotes the Transformation of Human Ovarian Epithelial Cells and Ovarian Tumorigenesis

Ovarian carcinoma is the most lethal gynecologic malignancy, however underlying molecular events remain elusive. Expression of human chorionic gonadotropin β subunit (β-hCG) is clinically significant for both trophoblastic and nontrophoblastic cancers; however, whether β-hCG facilitates ovarian epithelial cell tumorigenic potential remains uncharacterized. Immortalized nontumorigenic ovarian epithelial T29 and T80 cells stably overexpressing β-hCG were examined for alterations in cell cycle and apoptotic status by flow cytometry, expression of proteins regulating cell cycle and apoptosis by Western blot, proliferation status by MTT assay, anchorage-independent colony formation, and mouse tumor formation. Immunoreactivity for β-hCG was evaluated using mouse xenografts and on human normal ovarian, fallopian tube, endometrium, and ovarian carcinoma tissues. T29 and T80 cells overexpressing β-hCG demonstrated significantly increased proliferation, anchorage-independent colony formation, prosurvival Bcl-X_L protein expression, G2-checkpoint progression, elevated cyclins E/D1 and Cdk 2/4/6, and decreased apoptosis. Collectively, these transformational alterations in phenotype facilitated increased xenograft tumorigenesis (P < 0.05). Furthermore, β-hCG immunoreactivity was elevated in malignant ovarian tumors, compared with normal epithelial expression in ovaries, fallopian tube, and endometrium (P < 0.001). Our data indicate that elevated β-hCG transforms ovarian surface epithelial cells, facilitating proliferation, cell cycle progression, and attenuated apoptosis to promote tumorigenesis. Our results further decipher the functional role and molecular mechanism of β-hCG in ovarian carcinoma. β-hCG may contribute to ovarian cancer etiology, which introduces a new therapeutic intervention target for ovarian cancer.Ovarian cancer is the most lethal form of gynecologic cancer in the United States, accounting for an estimated 21,550 new cases and 14,600 deaths in 2009.¹ Survival rates can approach 90% when ovarian cancer is diagnosed at an early stage; however, early detection is challenging, because the relatively nonspecific symptoms of ovarian lesions may be overlooked until abdominal distension by ascites fluid or by large tumor masses becomes unmistakable. Even with extensive surgical debulking and aggressive chemotherapy, the prognosis for women with ovarian cancer currently is not hopeful. Several studies have indicated that different histological subtypes of ovarian carcinoma are associated with different causes and underlying mechanisms, including gene amplification, genetic predisposition, and various carcinogens.^2–5 Nonetheless, the origin and causes of ovarian carcinoma remain to be elucidated.Human chorionic gonadotropin (hCG) has a physiologically significant role during pregnancy. It is produced as a heterodimeric glycoprotein complex by the placenta over the course of the first 3 months of gestation. The heterocomplex consists of an α subunit and a hormone-specific β subunit, which collectively act as a ligand in activating the luteinizing hormone/hCG receptor (LH/hCGR) in gonadal cells to regulate sex hormone synthesis and reproductive processes.⁶ The β subunit of the hCG complex (β-hCG) is an accurate marker for diagnosis and monitoring of trophoblastic tumors and ovarian germ cell tumors.^7,8 Recently, it was shown that elevation in levels of β-hCG in serum, urine, or tumor tissue correlates with patient outcome in a variety of nontrophoblastic tumors of diverse primary tissue origin.^9–15 Moreover, elevated β-hCG was associated with aggressive disease, poor prognosis, and predicted resistance to therapy in bladder cancer patients.¹⁶ Additionally, increased β-hCG expression simultaneously stimulates proliferation and inhibits apoptosis of cancer cells derived from the bladder and the cervix in vitro.^17,18 Furthermore, ovarian carcinoma tissue displays an overexpression not only of the hormone-specific β-hCG subunit, but also the cognate receptor hCGR.^9,19 The functional role and molecular mechanism of β-hCG within ovarian cancer tumorigenesis have yet to be characterized. Recent evidence has strongly implicated epithelial cells derived from the fallopian tube, especially the fimbriated ends, as the likely origin for high-grade serous carcinoma.²⁰ To test whether this molecule plays a direct role in facilitating ovarian epithelial cell activation and tumorigenic potential, we designed a panel of experiments using both in vitro and in vivo methods, including evaluation of β-hCG expression in the normal fallopian tube.     

Cytotoxic reaction and TNF-α response of macrophages to polyurethane particles

Their unique mechanical and biological properties make polyurethanes (PUs) ideal materials for many implantable devices. However, uncertain long-term biostability in the human physiological environment limits their extensive clinical applications. Chronic inflammatory response associated with macrophage activation has been suggested as a prime factor; although the mechanism of macrophage activation in response to biomaterial surfaces and debris is still unknown. The overall objective of this work was to study the response of macrophages to PU materials in vitro by measuring cell viability and activity. The studies were carried out using phagocytozable-size PU particles from three types of commercially-available PUs: Pellethane^® 2363 80ABA (PL); Tecothane^® TT2065 (TC65); and Tecothane^® TT2085 (TC85). These polymers posess the same generic composition but differ in the length of hard and soft segments, as revealed by the FTIR and NMR studies. The results showed that PU particles affected both viability and activity of J774 macrophages. The percentage of mortality ranged from 1 to 15% with 10-100 μg ml^-1 of particles after 24 and 48 h incubation. These three types of particles induced different mortality on the macrophages. Specifically, the mortality with PL particles was 1-4% (p > 0.05), while the mortality with TC85 particles was 2-10% (p < 0.05) and 4-15% with TC65 (p < 0.05). Conversely, these particles also affected cell proliferation. Cell numbers increased by 132 and 167% after 24 and 48 h incubation, respectively, without particles, whereas the cell numbers increased only 46 and 78% with TC65, 66 and 105% with TC85, and 67 and 110% with PL in the presence of 100 μg ml^-1 of particles for the respective incubation times. PU particles also increased TNF-α release from macrophage. After having been incubated for 24 h with 100 μg ml^-1 particles of TC65, TC85, and PL, macrophages release TNF-α 7.4, 5.2, and 4.1 times more than the control. In conclusion, PU particles had cytotoxic effects on J774 macrophage at high concentrations. The order of macrophage response for three types of particles was TC65 > TC85 > PL. PU particles' effect on macrophage viability and activity depends on the concentration of particles and their chemical composition, especially on the ratio of hard to soft segments.     

Differential Sensitivity of Naïve and Memory Subsets of Human CD8+ T Cells to TNF-α-Induced Apoptosis

In this investigation, we have examined the relative sensitivity of human naïve, central memory (T_CM), and two types of effector memory CD8+ T cells (T_EM and T_EMRA) to TNF-α-induced apoptosis. Our data show that naïve and T_CM CD8+ T cells were sensitive, whereas T_EM and T_EMRA CD8+ T cells were relatively resistant to TNF-a-induced apoptosis. The apoptosis profile correlated with the activation of caspase-8 and caspase-3. However, no correlation was observed between relative sensitivity of four CD8 + T cell subsets to apoptosis and the expression of TNFR-I or TNFR-II. T_EM and T_EMRA CD8+ T cells displayed increased phosphorylation of IKKα/β and IκB and increased NF-κB activity as compared to naïve and T_CM CD8+ T cells. Bcl-2, Bcl-x_L and FLIP_L expression was higher and Bax expression was lower in T_EM and T_EMRA CD8+ T cells as compared to naïve and T_CM CD8+ T cells. These data suggest that signaling molecules downstream of TNFRs may be responsible for differential sensitivity among subsets of CD8+ T cells to TNF-α-induced apoptosis.     

Kinetics of LFA-1 Mediated Adhesion of Human Neutrophils to ICAM-1—Role of E-Selectin Signaling Post-Activation

LFA-1 and Mac-1 are the two integrins involved in the arrest and firm adhesion of neutrophils. LFA-1 plays a role in the early stage of cell arrest while Mac-1 stabilizes firm adhesion. Here, we further elucidated the kinetics of LFA-1 activation and its role in mediating neutrophil adhesion to ICAM-1 in the presence of E-selectin interaction. We confirm that LFA-1 activation to high affinity is transient in nature, decaying back to low affinity within 1 min after chemotactic stimulation. However, we show for the first time that this downshift in LFA-1 affinity does not return back to the low affinity state when E-selectin interaction is present and active, but rather E-selectin signals an intermediate LFA-1 conformation through PI3-Kinase that maintains an intermediate level of neutrophil firm adhesion. We further show that this E-selectin signaling is capable of returning LFA-1 to the intermediate affinity conformation outside the 1-min window previously reported for LFA-1 functionality. While our work confirms a role for PI3-Kinase in neutrophil firm adhesion, we show that PI3-Kinase may not be important in the initial transition from rolling to firm arrest (i.e. LFA-1 shift from low to high affinity conformation occurring within seconds of chemotactic stimulation).     

PD-L1 Regulates Inflammation in LPS-Induced Lung Epithelial Cells and Vascular Endothelial Cells by Interacting with the HIF-1α Signaling Pathway

Inflammation - Sepsis-induced lung injury was the most common cause of death in patients. This study aimed to investigate whether PD-L1 regulates the inflammation in LPS-induced lung epithelial...     

Effects of Transforming Growth Factor-β1 on Proliferation of Smooth Muscle Cells in Human Aortic Intima and Human Promonocytic Leukemia THP-1 Cells

We studied the effects of transforming growth factor on proliferation of cultured smooth muscle cells from human aortic intima and proliferation and differentiation of human leukemia THP-1 promonocytes. Transforming growth factor inhibited proliferation of these cells, but stimulated differentiation of THP-1 cells. Therefore, transforming growth factor probably modulates proliferation and differentiation of smooth muscle cells and monocytes/macrophages involved in the pathogenesis of atherosclerotic damages.     

Levels of Circulating Th17 Cells and Regulatory T Cells in Ankylosing Spondylitis Patients with an Inadequate Response to Anti−TNF-α Therapy

Purpose

To investigate the effects of TNF-α blockage on levels of circulating Th17, Treg and their related cytokines in ankylosing spondylitis (AS) patients with different response to anti-TNF-α therapy.

Methods

The frequencies of circulating Th17 and Treg and serum levels of related cytokines were determined using flow cytometry analysis and ELISA, respectively, in 222 AS patients both before (baseline) and 6 months after anti-TNF-α therapy. Therapeutic response was defined according to ASAS (Assessment in Spondyloarthritis International Society) response criteria.

Results

Significantly higher baseline circulating Th17 and serum TNF-α, IL-6, IL-17, IL-23 were observed in active AS patients than in healthy controls. After anti-TNF-α therapy, 168 patients (75.7 %) were responders and 54 (24.3 %) were non-responders. Frequencies of Th17 significantly decreased in responders, but significantly increased in non-responders. Treg increased significantly in responders but decreased significantly in non-responders. Levels of TNF-α, IL-6, IL-17, and IL-23 were significantly decreased in responders. In contrast, IL-17 and IL-23 significantly increased in non-responders. TGF-β were significantly increased only in responders, whereas no significant changes were seen in IL-10 in either responders or non-responders. Spearman correlation analysis showed that frequencies of Th17 and levels of TNF-α, IL-6, IL-17, and IL-23 were positively correlated with BASDAI score. They were also positively correlated with BASFI score except for IL-6. Treg were found to be negatively correlated with BASDAI score.

Conclusions

The beneficial effect of anti-TNF-α therapy in AS might not only neutralize the effects of TNF-α but also down-regulate Th17 and Th17-related cytokines accompanied by up-regulating the Treg/TGF-β axis in responders.     

Lipopolysaccharide Could Be Internalized into Human Peripheral Blood Mononuclear Cells and Elicits TNF-α Release,but not via the Pathway of Toll-Like Receptor 4 on the Cell Surface

Lipopolysaccharide (LPS), the principal component of the outer membrane of Gram-negative bacteria, stimulates various cell types to release numerous proinflammatory mediators such as TNF-alpha, IL-6 and IL-12, which may damage cells and lead to organ injury, even sepsis and septic shock. Toll-like receptor 4 (TLR4) has been identified as the receptor involved in the recognition of LPS, but the role of LPS uptake in activating signal transduction remains controversial. In the present study, TNF-alpha was used as a marker of macrophages/ monocytes activated by LPS, and CQ was used as an inhibitor of endosome mature in order to definitude what stage of the signal transduction elicited by LPS was interrupted. We found that there indeed existed internalization of LPS and internalization partially participated in LPS signaling since CQ inhibited cytokine release, and decreased accumulation of FITC-LPS in hPBMCs. In contrast, anti-hTLR4 antibody could decrease cytokine release, but had no inhibition on accumulation of FITC-LPS. This result revealed that inhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells. But TLR4 on the cell surface couldn't participate in internalization of LPS. Thus, LPS signaling and internalization couldn't be viewed as mutually independent processes.     

Lipopolysaccharide Could Be Internalized into Human Peripheral Blood Mononuclear Cells and Elicit TNF-α Release,but not via the Pathway of Toll-Like Receptor 4 on the Cell Surface

Lipopolysaccharide(LPS),the principal component of the outer membrane of Gram-negative bacteria,stimulatesvarious cell types to release numerous proinflammatory mediators such as TNF-α,IL-6 and IL-12,which maydamage cells and lead to organ injury,even sepsis and septic shock.Toll-like receptor 4(TLR4) has beenidentified as the receptor involved in the recognition of LPS,but the role of LPS uptake in activating signaltransduction remains controversial.In the present study,TNF-α was used as a marker of macrophages/monocytes activated by LPS,and CQ was used as an inhibitor of endosome mature in order to definitude whatstage of the signal transduction elicited by LPS was interrupted.We found that there indeed existedinternalization of LPS and internalization partially participated in LPS signaling since CQ inhibited cytokinerelease,and decreased accumulation of FITC-LPS in hPBMCs.In contrast,anti-hTLR4 antibody coulddecrease cytokine release,but had no inhibition on accumulation of FITC-LPS.This result revealed thatinhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells.But TLR4 on thecell surface couldn't participate in internalization of LPS.Thus,LPS signaling and internalization couldn't beviewed as mutually independent processes.Cellular & Molecular Immunology.2004;1(5):373-377.     

Kinetics of Receptor-Mediated Uptake and Processing of Interferon-α2a and Tumor Necrosis Factor-α by Human Tumor Cells

Abstract

The kinetics of uptake and processing of recombinant human interferon-a₂a (IFN) and recombinant human tumor necrosis factor-a (TNF) were studied in human epithelial rumor cell lines differing in sensitivity to growth inhibition by IFN and TNF. Concentrations of [¹²⁵I]IFN or [¹²⁵I]TNF at the cell surface and internalized by confluent cell monolayers incubated at 37°C were measured as a function of time. Cells incubated with [¹²⁵I]IFN exhibited transient maxima of surface-associated and internalized [^I25I]IFN after 1-2 hr of incubation followed by a steady-state attained after approximately 6 hr of exposure to [¹²⁵I]IFN. Concentration of [¹²⁵I]TNF associated with the plasma membrane displayed a maximum within 20 min of incubation. Internalized radioactivity increased with time and did not achieve a steady state. We analyzed the time-dependent concentrations of membrane-associated and internalized cytokines by use of a compartmental model. This model describes changes in concentrations of free surface receptors, of ligand-receptor complex at the membrane and of internalized ligand and contains a term for receptor recycling. The rate constants for concentration changes were evaluated by fitting model functions to data. The best fits for IFN were obtained without receptor recycling. The best-fit values for the endo-cytotic rate constant (k_tlFN) varied among cell lines from 2.4x10^?4 to 7.8 x10^?4 sec^?1. To obtain fits to time-dependent concentrations of surface-associated and internalized [¹²⁵I]TNF, introduction of a term for receptor recycling was required. Best-fit values for k, _TNF ranged among cell lines from 8.4x10^?4 to 2.5x10^?3 sec^?1. For every cell line, the value of k_lTNF was larger than the value of k_clFN. We tested the significance of these differences by substituting K.IFN ^tor K.TNT ^as fixed parameters in fits to data for TNF and v.v., respectively. Under these conditions, fits were significantly worse. Our data indicate that recycling is insignificant in kinetics of Type-I IFN receptor; recycling is necessary to explain the kinetics of TNF receptor. Upon binding, TNF is taken up by cells faster than IFN and is eliminated from cells more slowly than IFN.     

Signaling via the kinase p38α programs dendritic cells to drive TH17 differentiation and autoimmune inflammation

Dendritic cells (DCs) bridge innate and adaptive immunity, but how DC-derived signals regulate T cell lineage choices remains unclear. We report here that the mitogen-activated protein kinase p38α programmed DCs to drive the differentiation of the T(H)17 subset of helper T cells. Deletion of p38α in DCs protected mice from T(H)17 cell-mediated autoimmune neuroinflammation, but deletion of p38α in macrophages or T cells did not. We also found that p38α orchestrated the expression of cytokines and costimulatory molecules in DCs and further 'imprinted' signaling via the receptor for interleukin 23 (IL-23R) in responding T cells to promote T(H)17 differentiation. Moreover, p38α was required for tissue-infiltrating DCs to sustain T(H)17 responses. This activity of p38α was conserved in mouse and human DCs and was dynamically regulated by pattern recognition and fungal infection. Our results identify p38α signaling as a central pathway for the integration of instructive signals in DCs for T(H)17 differentiation and inflammation.     

Production of TNF-α and IL-1β by Peripheral Blood Mononuclear Cells in Carriers of Different Allele Variants of the Gene

Associations of cytokine production by mononuclear cells and the TNF-α genetic polymorphism in positions -238, -308, -376, -857, -1031, and of IL-1β in positions -31 and +3954 were studied. The data on distribution of allele and genotype incidence and on the level of spontaneous and mitogen-induced production of these cytokines by donor mononuclear cells demonstrated a statistically significant association of TNF-α production by mononuclear cells with polymorphic variants of the gene promoter regions -238 > A (rs361525) and -857C > T (rs1799724). Carriers of -238GG/-308GG/-857CC/-1031NC genotype were characterized by low production of TNF-α, while carriers of -31TT/+3954CT genotype were characterized by low IL-1β stimulation index in response to mitogen in comparison with carriers of other genotype combinations.