Polyinosinic-Polycytidylic Acid Induces the Expression of GRO-α in BEAS-2B Cells

Growth-related oncogene protein-α (GRO-α)/CXCLl is a chemokine that activates neutrophils and plays an important role in inflammatory reactions. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), which is a ligand for Toll-like receptor-3. Poly IC mimics viral infection when applied to cells and induces inflammatory and immune responses. In the present study, we found the induction of GRO-α in BEAS-2B bronchial epithelial cells treated with poly IC. Pretreatment of cells with 2-aminopurine, an inhibitor for dsRNA-dependent protein kinase (PKR), inhibited the expression of GRO-α-induced by poly IC. Overexpression of interferon-regulatory factor-3 (IRF-3) or retinoic-acid inducible gene-I (RIG-I) enhanced the induction of GRO-α by poly IC. PKR, IRF-3, and RIG-I may be involved in the poly IC-induced expression of GRO-α in BEAS-2B cells. Airway viral infection may elicit GRO-α expression in the bronchial epithelium, which may be implicated in inflammatory and immune reactions.     

Double-stranded RNA induces the synthesis of retinoic acid-inducible gene-I in vascular endothelial cells.

Viral infection induces various responses in vascular endothelial cells. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), and treatment of cells with poly IC mimics the viral infection to the cells. Retinoic acid-inducible gene-I (RIG-I) is a protein belonging to the DExH-box family and designated as a putative RNA helicase. RIG-I is considered to play a role in antiviral responses through the regulation of gene expressions. In the present study, the authors treated human umbilical vein endothelial cells (HUVECs) with poly IC and found that poly IC induced the expression of RIG-I. The poly IC-induced RIG-I expression was inhibited by the preincubation of the cells with 2-aminopurine, an inhibitor of dsRNA-dependent protein kinase (PKR). Immunohistochemical examination revealed high levels of RIG-I immunoreactivity in vascular endothelial cells in the thalamus from rats inoculated with hantavirus. Induction of RIG-I by poly IC may be involved in the antiviral responses in endothelial cells.     

Retinoic acid-inducible gene-I mediates RANTES/CCL5 expression in U373MG human astrocytoma cells stimulated with double-stranded RNA

Retinoic acid-inducible gene-I (RIG-I) mediates part of the cell signaling in response to viral infection. Polyinosinic-polycytidilic acid (poly IC) is a synthetic double-stranded RNA (dsRNA) and mimics viral infection when applied to cell cultures. The CC chemokine, RANTES (regulated on activation, normal T-cell expressed and secreted), is a potent attractant for inflammatory cells such as memory T-lymphocytes, monocytes and eosinophils. In the present study, we demonstrated that poly IC enhances the expression of RIG-I in U373MG human astrocytoma cells. The RNA interference of RIG-I resulted in the suppression of the poly IC-induced RANTES expression. Pretreatment of the cells with SB203580, an inhibitor of p38 mitogen-activated protein kinase, and dexamethasone inhibited the poly IC-induced expression of RIG-I. Furthermore, poly IC upregulated RIG-I in normal human astrocytes in culture and the in vivo injection of poly IC into the striatum of the mouse brain induced the expression of RIG-I in astrocytes. We conclude that RIG-I may be involved in immune reactions against viral infection, at least in part, through the regulation of RANTES expression in astrocytes.     

Expression of epithelial neutrophil-activating peptide 78 in cultured human endometrial stromal cells

It has been demonstrated that human endometrial stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of epithelial neutrophil-activating peptide 78 (ENA-78) in the endometrium, concentrations of ENA-78 in cyclic endometrial tissues were measured using enzyme-linked immunosorbent assay. The expression of ENA-78 was also detected in cyclic endometrium by immunohistochemistry. Endometrial tissues in the secretory phase contained higher amounts of ENA-78 protein than did those in the proliferative phase. Immunofluorescence staining revealed that ENA-78 protein was localized mainly in the stroma of endometrium. In addition, to evaluate the involvement of inflammatory mediators and ovarian steroid hormones in the production of ENA-78 by ESC was evaluated by in-vitro studies. Unstimulated ESC constitutively secreted ENA-78. Progesterone, lipopolysaccharide, tumour necrosis factor-alpha, and interleukin-1beta significantly stimulated the expression of ENA-78 by ESC. It is suggested that the production of ENA-78 by ESC is regulated by progesterone as well as by the inflammatory mediators. The modulation of ENA-78 concentration in the local environment by these mediators may contribute to the normal and pathological processes of human reproduction through regulation of leukocyte trafficking into the endometrium.     

Characterization of coxsackievirus B3 replication in human umbilical vein endothelial cells

After successful invasion of susceptible hosts, systemic distribution of coxsackievirus B3 (CVB3) most likely requires interactions with the endothelial system. Thereby, infection of endothelial cells occurs directly or viruses and/or virus-infected leukocytes migrate through the endothelial barrier. Many of these processes have not been studied so far. In order to analyze viral replication in the endothelium, human umbilical vein endothelial cells (HUVEC) were isolated and infected with CVB3. Time-course experiments revealed maximal viral replication at 10–24 h and viral RNA persistence up to 120 h post-infection (p. i.) without the induction of obvious general cytopathic effects or the loss of cellular viability. However, the application of the EGFP-expressing recombinant virus variant CVB3/EGFP revealed shrinkage and death of individual cells. Using infectious center assays, a noticeable CVB3 replication occurred on an average of 20 % of HUVEC at 10 h p. i. This may be in part due to a higher coxsackievirus/adenovirus receptor expression in a small subgroup of HUVEC (5–7 %) as analyzed by flow cytometry. Interestingly, CVB3 replication escalated and cellular susceptibility increased significantly after reversal of cell cycle arrest caused by serum deprivation indicating that reactivation of cellular metabolism may help to promote CVB3 replication. Finally, CVB3-infected HUVEC cultures revealed increased DNA fragmentation, and inhibition of caspase activity caused an accumulation of intracellular virus particles indicating that apoptotic processes are involved in virus release mechanisms. Based on these observations, it is assumed that CVB3 replicates efficiently in human endothelial cells. But how this specific infection of the endothelium may influence viral spread in the infected host needs to be investigated in the future.     

Endometrial expression of epithelial neutrophil-activating peptide-78 during the menstrual cycle or in progestin-only contraceptive users with breakthrough bleeding and the influence of doxycycline therapy

BACKGROUND: Endometrial breakthrough bleeding is characterized by an inflammatory reaction and increased production of proinflammatory mediators, one of which may be epithelial neutrophil-activating peptide-78 (ENA-78), a chemokine with neutrophil-activating properties. METHODS AND RESULTS: We therefore investigated the endometrial expression of ENA-78 in Norplant users as progestin-only contraceptive with various bleeding patterns (n=35) as compared with non-users with a normal menstrual cycle (n=55). The endometrial stromal cells (ESCs) were the major site of ENA-78 expression with the highest levels found during the secretory phase. The expression of ENA-78 was increased in Norplant users with irregular bleeding as compared with those with regular cycles and amenorrhoea. The levels of ENA-78 detected in uterine washes and sera after the use of Norplant for 3-6 months (n=25) increased compared with baseline (P < 0.05). These levels did not significantly change in Norplant users who received doxycycline (Dox) therapy (25 mg/twice daily for 6 months) when measured midway through or at the conclusion of study when compared with the baseline (n=25). Treatments with medroxyprogesterone acetate (MPA) and tumour necrosis factor-alpha (TNF-alpha) (25 ng/ml), but not 17beta-estradiol (E2) or E2 + MPA (10(-8) M), representing endometrium exposed to contraceptive and inflammatory conditions, respectively, increased the levels of ENA-78 production by ESCs, and this was reduced by co-treatments with Dox (25 microg/ml) (P < 0.05). CONCLUSIONS: The endometrial production of ENA-78 is altered in progestin-only contraceptive users experiencing breakthrough bleeding and is regulated by MPA and TNF-alpha in ESCs. Although Dox therapy did not alter uterine ENA-78 secretion, its suppression in ESCs suggests that Dox, acting site-specifically and through an anti-inflammatory mechanism, may influence the outcome of breakthrough bleeding in contraceptive users.     

15-deoxy-delta(12,14)-prostaglandin J2 inhibits IFN-gamma-induced galectin-9 expression in cultured human umbilical vein endothelial cells

BACKGROUND: Galectin-9 is involved in chemotaxis and adhesion of eosinophils, and is induced in vascular endothelial cells by interferon-gamma (IFN-gamma). 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and known to modulate the expression of various genes. METHODS: We have studied the effect of 15d-PGJ(2) on the IFN-gamma-induced galectin-9 expression in human umbilical vein endothelial cells (HUVEC) in culture. RESULTS: 15d-PGJ(2) inhibited the IFN-gamma-induced galectin-9 expression in a PPAR-gamma-independent manner, and also inhibited the adhesion of EoL-1 cells to an HUVEC monolayer treated with IFN-gamma. 15d-PGJ(2) partially inhibited IFN-gamma-induced phosphorylation of STAT-1 in HUVEC. CONCLUSIONS: 15d-PGJ(2) may regulate inflammatory reactions through the inhibition of galectin-9 expression.     

RANTES stimulates cell-mediated transmission of HIV-1 infection.

We wished to determine the effects of the beta-chemokine RANTES in an established system of cell-mediated transmission of HIV-1, that is, normal human umbilical vein endothelial cells (HUVEC) nonproductively infected with HIV-1, cocultivated with CD4+ T cells to rescue productive infection. The results indicate that the addition of RANTES to HUVEC, either before or after HIV-1 infection, stimulates HIV-1 rescue by CD4+ T cells. However, viral DNA is not increased in HUVEC, suggesting that the stimulation exerted by RANTES could be mediated by events following HUVEC infection. The mechanisms of increase seem to be related to the rescue phase, involving membrane interaction of abortively infected HUVEC with permissive T cells. In fact, a strong upregulation and polarization of intercellular adhesion molecule-1 (ICAM-1) is induced in HUVEC by RANTES, and antibodies against ICAM-1 inhibit HIV-1 rescue by T cells. These results indicate that RANTES, similarly to other inflammatory cytokines, may favor HIV-1 spreading and crossing of blood-tissue barriers by indirect mechanisms involving membrane interactions between nonproductively infected and permissive cells.     

Corticosteroids plus Long-Acting Beta(2)-Agonists Prevent Double-Stranded RNA-Induced Upregulation of B7-H1 on Airway Epithelium

Background: Airway viral infections provoke exacerbations of asthma and chronic obstructive pulmonary disease. B7-H1 is a costimulatory molecule that is implicated in an escape mechanism of viruses from host immune systems. This escape may be associated with the persistence of viral infection and lead to exacerbation of underlying diseases. We have shown that an analog of viral double-stranded RNA, polyinosinic-polycytidylic acid (poly IC), upregulated the expression of B7-H1 on airway epithelial cells, an effect which was corticosteroid-resistant. We investigated the effects of corticosteroids plus long-acting β(2)-agonists (LABAs; fluticasone/salmeterol or budesonide/formoterol) on the expression of B7-H1. Methods: BEAS-2B cells and primary airway epithelial cells were stimulated with poly IC or respiratory syncytial virus. The expression of B7-H1 was assessed by flow cytometry. Results: Poly IC upregulated the expression of B7-H1, which was suppressed by high-concentration corticosteroids but not by LABAs. The upregulation was suppressed by very low-concentration corticosteroids when used in combination with LABAs. Their combination also suppressed the virus-induced upregulation of B7-H1. Poly IC stimulation induced the nuclear translocation of nuclear factor ?B (NF-?B). Inhibitors of NF-?B activation prevented the poly IC-induced upregulation of B7-H1. Low-concentration corticosteroids in combination with LABAs enhanced the de novo induction of I?Bα, the endogenous inhibitor of NF-?B activation. Conclusions: Fluticasone/salmeterol or budesonide/formoterol attenuate the virus-associated upregulation of B7-H1 on airway epithelial cells via suppression of NF-?B activation.     

Axl and MerTK receptor tyrosine kinases maintain human macrophage efferocytic capacity in the presence of viral triggers

The requirement to remove apoptotic cells is equally important in homeostasis and inflammatory disease. In particular, during viral infections large quantities of infected cells undergo apoptosis and need to be efficiently cleared by phagocytes to prevent secondary necrosis. Although specific roles of several apoptotic cell sensors, such as the TAM (Tyro3, Axl, MerTK) receptor family, have been characterized in mouse models, little is known about their regulation and involvement in apoptotic cell uptake (efferocytosis) by human macrophages under inflammatory conditions. We show that whereas pro‐inflammatory stimuli consistently downregulated MerTK expression in human monocyte‐derived macrophages (MDMs), stimuli indicative of a viral infection, interferon‐α (IFN‐α) and the TLR3 ligand poly(I:C), specifically induced Axl expression and promoted binding of the bridging molecule Gas6. Axl induction by IFN‐α and poly(I:C) was associated with higher MDM efferocytic capacity compared to cells treated with other pro‐inflammatory stimuli, such as LPS and IFN‐γ. While MerTK blocking antibody uniformly suppressed apoptotic cell uptake by MDMs, Axl blocking antibody significantly reduced efferocytosis by poly(I:C)‐stimulated MDMs, but not by resting MDMs. Our observations demonstrate that Axl induction during viral infections contributes to maintaining macrophage capacity to engulf apoptotic cells, which may have important consequences for resolution of anti‐viral immune responses.     

Fimbria-Dependent Activation of Cell Adhesion Molecule Expression in Porphyromonas gingivalis-Infected Endothelial Cells

Porphyromonas gingivalis is an oral pathogen that has recently been associated with chronic inflammatory diseases such as atherosclerosis. The strength of the epidemiological associations of P. gingivalis with atherosclerosis can be increased by the demonstration that P. gingivalis can initiate and sustain growth in human vascular cells. We previously established that P. gingivalis can invade aortic, heart, and human umbilical vein endothelial cells (HUVEC), that fimbriae are required for invasion of endothelial cells, and that fimbrillin peptides can induce the expression of the chemokines interleukin 8 and monocyte chemotactic protein. In this study, we examined the expression of surface-associated cell adhesion molecules on endothelial cells in response to P. gingivalis infection by fluorescence-activated cell sorting FACS analysis and confocal microscopy. Coculture of HUVEC with P. gingivalis strain 381 or A7436 resulted in the induction in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P- and E-selectins, which was maximal at 48 h postinfection. In contrast, we did not observe induction of ICAM-1, VCAM-1, or P- or E-selectin expression in HUVEC cultured with the noninvasive P. gingivalis fimA mutant DPG3 or when P. gingivalis was incubated with fimbrillin peptide-specific anti-sera prior to the addition to HUVEC. Furthermore, the addition of a peptide corresponding to the N-terminal domain of fimbrillin to HUVEC resulted in an increase in ICAM-1, VCAM-1, and P- and E-selectins, which was maximal at 48 h and similar to that observed for live P. gingivalis. Treatment of P. gingivalis-infected HUVEC with cytochalsin D, which prevented P. gingivalis invasion, also resulted in the inhibition of ICAM-1, VCAM-1, or P- and E-selectin expression. Taken together, these results indicate that active P. gingivalis invasion of HUVEC mediated via the major fimbriae stimulates surface-associated cell adhesion molecule expression. Stimulation of adhesion molecules involved in the recruitment of leukocytes to sites of inflammation by P. gingivalis may play a role in the pathogenesis of systemic inflammatory diseases associated with this microorganism, including atherosclerosis.     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Molecular mechanisms and consequences of cardiac viral infections

Molecular biological methods have confirmed the pathogenetic role of enteroviruses, primarily coxsackieviruses of group B (CVB), in the induction and maintenance of inflammatory cardiomyopathy. More recently, adenoviruses, various herpes viruses, and increasingly parvovirus B19 (B19) have been identified as potential cardiotropic agents. While cardiac myocytes are target cells for enterovirus and adenovirus infections with virus-induced cytolysis, B19-associated inflammatory cardiomyopathy is characterized by infection of intracardiac endothelial cells of small arterioles and veins, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, penetration of inflammatory cells, and secondary myocyte necrosis. Recent observations showed that B19 is involved in intracellular calcium regulation by the viral phospholipase. B19-induced caspase activation can lead to proinflammatory/proapoptotic processes through dysregulation of STAT signaling. These cellular interactions may contribute to mechanisms by which B19 establishes persistent infection in endothelial cells and play a critical role in viral pathogenesis of inflammatory cardiomyopathy.     

Differential and additive effects of platelet-derived chemokines on monocyte arrest on inflamed endothelium under flow conditions

Platelet-derived chemokines, such as regulated on activation, normal T expressed and secreted (RANTES; CC chemokine ligand 5), platelet factor 4 [PF4; CXC chemokine ligand 4 (CXCL4)], and epithelial neutrophil-activating protein 78 (ENA-78; CXCL5), or precursors, such as beta-thromboglobulin, which can be processed to neutrophil-activating protein-2 (NAP-2; CXCL7), may play an important role in monocyte recruitment during atherogenesis. Platelets can deposit chemokines on inflamed endothelium; however, little is known about differential or additive effects of platelet chemokines on monocyte arrest. Here, we demonstrate that preincubation of activated human microvascular endothelial cells (HMVECs) with RANTES, PF4, or NAP-2 but not ENA-78 dose-dependently increased surface immobilization and subsequent monocyte arrest in flow. RANTES was the most potent and efficient arrest chemokine. Pretreatment of HMVECs with beta-thromboglobulin enhanced monocyte arrest in the presence of cathepsin G generating NAP-2. Combined pretreatment of HMVECs with RANTES and PF4 at suboptimal concentrations synergistically increased arrest, and preincubation with chondroitinase ABC abrogated RANTES- and PF4-induced monocyte arrest. This was associated with reduced expression of chondroitin sulfate, RANTES, and PF4 on the HMVEC surface. Perfusion of HMVECs with platelets known to deposit RANTES and PF4 on the endothelial surface enhanced monocyte arrest, which was inhibited by Met-RANTES, chondroitinase, or a blocking antibody to PF4 but not to ENA-78. The relevance of platelet-derived chemokines was confirmed in adhesion assays with activated whole blood, where Met-RANTES and to a lesser extent, antibodies to PF4 and NAP-2 inhibited arrest of CD14-positive monocytes. Thus, multiple platelet-derived chemokines and processable precursors, which can be presented by specific endothelial proteoglycans, may contribute and cooperate differentially to induce monocyte recruitment.     

CXCR1-binding chemokines in inflammatory bowel diseases: down-regulated IL-8/CXCL8 production by leukocytes in Crohn's disease and selective GCP-2/CXCL6 expression in inflamed intestinal tissue

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) that are characterized by chronic intestinal inflammation and a constant influx of leukocytes mediated by pro-inflammatory cytokines and chemokines. The intestinal expression of the CXCR1-binding chemokines IL-8/CXCL8 and GCP-2/CXCL6 and the participation of immunocompetent cells in IBD were evaluated. IL-8 production by peripheral blood mononuclear cells (PBMC) from IBD patients, stimulated with endotoxin, plant lectin or double-stranded RNA, was significantly lowered in patients with CD, but not in UC patients or healthy subjects. The reduced chemokine production by PBMC from IBD patients was both IL-8 and CD specific, but not inducer dependent. In serum, most chemokines remained undetectable, while the levels of those that were measurable remained unaltered in IBD patients. GCP-2, but not ENA-78/CXCL5, nor IL-8, were highly expressed by endothelial cells in inflamed intestinal tissue of IBD patients. In contrast, stimulated endothelial cell cultures produced more IL-8 than GCP-2. The selective GCP-2 staining of endothelial cells at sites of ulcerations suggests that GCP-2, despite its low production capacity in vitro, plays a role in IBD that is different from that of structurally (ENA-78) and functionally (IL-8) related ELR(+) CXC chemokines. Thus, the chemokine network shows complementarity rather than redundancy.