Increased C-C Chemokine Receptor 2 Gene Expression in Monocytes of Severe Obstructive Sleep Apnea Patients and under Intermittent Hypoxia

Background

Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. The chemotaxis and adhesion of monocytes to the endothelium in the early atherosclerosis is important. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the chemotaxis and adhesion of monocytes.

Methods

Peripheral blood was sampled from 54 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of C-C chemokine receptor 2 (CCR2). The effect of intermittent hypoxia on the regulation and function of CCR2 was investigated on THP-1 monocytic cells and monocytes. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. Transwell filter migration assay and cell adhesion assay were performed to study the chemotaxis and adhesion of monocytes.

Results

Monocytic CCR2 gene expression was found to be increased in severe OSA patients and higher levels were detected after sleep. Intermittent hypoxia increased the CCR2 expression in THP-1 monocytic cells even in the presence of TNF-α and CRP. Intermittent hypoxia also promoted the MCP-1-mediated chemotaxis and adhesion of monocytes to endothelial cells. Furthermore, inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of monocytic CCR2 expression by intermittent hypoxia.

Conclusions

This is the first study to demonstrate the increase of CCR2 gene expression in monocytes of severe OSA patients. Monocytic CCR2 gene expression can be induced under intermittent hypoxia which contributes to the chemotaxis and adhesion of monocytes.     

Interleukin-32-induced thymic stromal lymphopoietin plays a critical role in macrophage differentiation through the activation of caspase-1 in vitro

Introduction

Interleukin (IL)-32 is an inflammatory cytokine induced by Mycobacterium tuberculosis and Mycobacterium bovis in a variety of cell types and discovered in the synovial of patients with rheumatoid arthritis (RA). Thymic stromal lymphopoietin (TSLP) play several roles in the pathogenesis of RA. However, the role of IL-32 and TSLP in RA has not been elucidated.

Methods

We evaluated the specific mechanism of between IL-32 and TSLP in RA using human monocyte cell line, THP-1 cells.

Results

Here we documented for the first time that IL-32 highly increased TSLP production in THP-1 cells and human blood monocytes. TSLP expression was induced by IL-32 via activation of caspase-1 and nuclear factor-κB. TSLP produced by IL-32 increased differentiation of monocytes but depletion of TSLP prevented differentiation of monocytes into macrophage-like cells. Chondroprotective drugs such as chondroitin sulfate (CS) and the traditional Korean medicine, BaekJeol-Tang (BT) decrease production of TSLP and activation of caspase-1 and nuclear factor-κB. In addition, CS and BT inhibited IL-32-induced monocytes differentiation.

Conclusions

Taken together, IL-32 and TSLP are important cytokines involved in the development of RA. The effects of CS and BT were associated with the downregulation of TSLP and caspase-1 through negative regulation of IL-32 pathways in RA.     

Rhodomyrtone Modulates Innate Immune Responses of THP-1 Monocytes to Assist in Clearing Methicillin-Resistant Staphylococcus aureus

Background

The increasing resistance of Staphylococcus aureus to conventional antibiotics poses a major health problem. Moreover, S. aureus can survive within phagocytes, thus evading some antibiotics and the innate immune response. Rhodomyrtone, a bioactive compound from the leaves of Rhodomyrtus tomentosa, possesses potent antibacterial activity against methicillin-resistant S. aureus (MRSA). This study was to investigate the immunomodulatory effects of rhodomyrtone on THP-1 monocytes in response to MRSA.

Methods

THP-1 monocytes were stimulated with heat-killed MRSA, followed by treatment with rhodomyrtone. The cell pellets were prepared to detect pro-inflammatory molecules using real-time PCR. The supernatants were collected to assess nitric oxide production using Griess assay. Assays for phagocytosis and bacterial killing by THP-1 monocytes were performed to determine if they were affected by rhodomyrtone.

Results

Expression of pro-inflammatory molecules including IL-1β, TNF-α, IL-6, and iNOS was enhanced in THP-1 monocytes stimulated with high doses of heat-killed MRSA (10⁸ to 10⁹ cfu/ml). In contrast, monocytes stimulated with MRSA at lower doses (10⁶ to 10⁷ cfu/ml) did not induce the expression of these cytokines. However, rhodomyrtone significantly increased the expression of pro-inflammatory mediators, IL-6 and iNOS in monocytes stimulated with heat-killed MRSA at low doses, and displayed some anti-inflammatory activity by reducing TNF-α expression in monocytes stimulated with heat-killed MRSA at high doses. Treatment with rhodomyrtone also significantly up-regulated the expression of the key pattern recognition receptors, TLR2 and CD14, in THP-1 monocytes stimulated with heat-killed MRSA at 10⁶ to 10⁹ cfu/ml, while heat-killed MRSA alone did not induce the expression of these molecules. The ability of rhodomyrtone to eliminate MRSA from the monocytes was observed within 24 h after treatment.

Conclusion

Rhodomyrtone enhanced the expression of pattern recognition receptors by monocytes in response to MRSA. Increased expression of these receptors might improve MRSA clearance by modulating pro- and anti-inflammatory cytokine responses.     

CXCL1 Contributes to β-Amyloid-Induced Transendothelial Migration of Monocytes in Alzheimer’s Disease

Background

Bone marrow-derived microglia that originates in part from hematopoietic cells, and more particularly from monocytes preferentially attach to amyloid deposition in brains of Alzheimer’s disease (AD). However, the mechanism of monocytes recruited into the amyloid plaques with an accelerated process in AD is unclear.

Methodology/Principal Findings

Here we reported that monocytes from AD patients express significantly higher chemokine (C-X-C motif) ligand 1 (CXCL1) compared to age-matched controls. AD patient’s monocytes or CXCL1-overexpressing THP-1 cells had enhanced ability of β-amyloid (Aβ)-induced transendothelial migration and Aβ-induced transendothelial migration for AD patient’s monocytes or CXCL1-overexpressing THP-1 cells was almost abrogated by anti-CXCL1 antibody. Furthermore, monocytes derived from a transgenic mouse model of AD also expressed significantly higher CXCL1. CD11b⁺CD45^hi population of cells that were recruited from the peripheral blood were markedly bolcked in APP mouse brain by anti-CXCL1 antibody. Accordingly, in response to Aβ, human brain microvascular endothelial cells (HBMEC) significantly up-regulated CXC chemokine receptor 2 (CXCR2) expression, which was the only identified receptor for CXCL1. In addition, a high level expression of CXCR2 in HBMEC significantly promoted the CXCL1-overexpressing THP-1 cells transendothelial migration, which could be was abrogated by anti-CXCR2 antibody. Further examination of possible mechanisms found that CXCL1-overexpressing THP-1 cells induced transendothelial electrical resistance decrease, horseradish peroxidase flux increase, ZO-1 discontinuous and occludin re-distribution from insoluble to soluble fraction through interacting with CXCR2. ROCK inhibitor, Y27632, could block CXCL1-overexpressing THP-1 cells transendothelial migration, whereas other inhibitors had no effects.

Conclusions/Significance

The present data indicate that monocytes derived from AD patients overexpressing CXCL1, which is a determinant for Aβ-induced transendothelial migration. CXCL1 expressed by monocytes and CXCR2 on HBMEC is involved in monocytes migrating from blood to brain in AD patients.     

Decrease of miR-146b-5p in monocytes during obesity is associated with loss of the anti-inflammatory but not insulin signaling action of adiponectin

Background

Low adiponectin, a well-recognized antidiabetic adipokine, has been associated with obesity-related inflammation, oxidative stress and insulin resistance. Globular adiponectin is an important regulator of the interleukin-1 receptor-associated kinase (IRAK)/NFκB pathway in monocytes of obese subjects. It protects against inflammation and oxidative stress by inducing IRAK3. microRNA (miR)-146b-5p inhibits NFκB-mediated inflammation by targeted repression of IRAK1 and TNF receptor-associated factor-6 (TRAF6). Therefore, we measured the expression of miR-146b-5p in monocytes of obese subjects. Because it was low we determined the involvement of this miR in the anti-inflammatory, antioxidative and insulin signaling action of globular adiponectin.

Methods

miR-146b-5p expression in monocytes of obese subjects was determined by qRT-PCR. The effect of miR-146b-5p silencing on molecular markers of inflammation, oxidative stress and insulin signaling and the association with globular adiponectin was assessed in human THP-1 monocytes.

Results

miR-146b-5p was downregulated in monocytes of obese persons. Low globular adiponectin decreased miR-146b-5p and IRAK3 in THP-1 monocytes, associated with increased mitochondrial reactive oxygen species (ROS). Intracellular ROS and insulin receptor substrate-1 (IRS1) protein were unchanged. Silencing of miR-146b-5p with an antisense inhibitor resulted in increased expression of IRAK1 and TRAF6 leading to more NFκB p65 DNA binding activity and TNFα. As a response IRAK3 and IRS1 protein increased. Mitochondrial and intracellular ROS production did not increase despite more inflammation. In addition, exposure of miR-146b-5p-depleted THP-1 monocytes to high levels of globular adiponectin resulted in an increased production of TNFα and intracellular ROS. Still, they did not lose their potential to increase IRAK3 and IRS1 protein and to decrease mitochondrial ROS.

Conclusion

miR-146b-5p, decreased in monocytes during obesity, is a major mediator of the anti-inflammatory action of globular adiponectin. It appears not to be involved in insulin signaling possibly by protective response of IRAK3 and lack of mitochondrial ROS production.     

Cytosolic,Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

Rationale

Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.

Methods

IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.

Results

IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.

Conclusions

Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.     

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion,Transendothelial Migration and Phagocytosis

Background

Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β₂ integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis.

Methodology/Principal Findings

THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β₂ integrin cell surface expression and β₂ integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β₂ integrins, in particular CD11b/CD18. SIRPα overexpression reduced β₂ integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β₂ integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression.

Conclusions/Significance

SIRPα negatively regulates β₂ integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis.     

Unlike for human monocytes after LPS activation, release of TNF-α by THP-1 cells is produced by a TACE catalytically different from constitutive TACE

Background

Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α.

Methodology/Principal Findings

Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation.

Conclusions/Significance

On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.     

IFI16 protein mediates the anti-inflammatory actions of the type-I interferons through suppression of activation of caspase-1 by inflammasomes

Background

Type-I interferons (IFNs) are used to treat certain inflammatory diseases. Moreover, activation of type-I IFN-signaling in immune cells inhibits the production of proinflammatory cytokines and activation of inflammasomes. However, the molecular mechanisms remain largely unknown. Upon sensing cytosolic double-stranded DNA, the AIM2 protein forms the AIM2-ASC inflammasome, resulting in activation of caspase-1. Given that the IFI16 and AIM2 proteins are IFN-inducible and can heterodimerize with each other, we investigated the regulation of IFI16, AIM2, and inflammasome proteins by type-I and type-II IFNs and explored whether the IFI16 protein could negatively regulate the activation of the AIM2 (or other) inflammasome.

Methodology/ Principal Findings

We found that basal levels of the IFI16 and AIM2 proteins were relatively low in peripheral blood monocytes (CD14⁺) and in the THP-1 monocytic cell line. However, treatment of THP-1 cells with type-I (IFN-α or β) or type-II (IFN-γ) IFN induced the expression levels of IFI16, AIM2, ASC and CASP1 proteins. The induced levels of IFI16 and AIM2 proteins were detected primarily in the cytoplasm. Accordingly, relatively more IFI16 protein bound with the AIM2 protein in the cytoplasmic fraction. Notably, increased expression of IFI16 protein in transfected HEK-293 cells inhibited activation of caspase-1 by the AIM2-ASC inflammasome. Moreover, the constitutive knockdown of the IFI16 expression in THP-1 cells increased the basal and induced [induced by poly(dA:dT) or alum] activation of the caspase-1 by the AIM2 and NLRP3 inflammasomes.

Conclusions/Significance

Our observations revealed that the type-I and type-II IFNs induce the expression of IFI16, AIM2, and inflammasome proteins to various extents in THP-1 cells and the expression of IFI16 protein in THP-1 cells suppresses the activation of caspase-1 by the AIM2 and NLRP3 inflammasomes. Thus, our observations identify the IFI16 protein as a mediator of the anti-inflammatory actions of the type-I IFNs.     

Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis

Background

Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14⁺ monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis.

Aim

To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.

Methods

CD14⁺ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.

Results

GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.

Conclusion

SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties.     

Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis

Background and Aims

The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys) and secrete substantial amounts of L-Glutamate (L-Glu) via the transport system Xc- (4F2hc+xCT), and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes.

Methods

We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM), and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0–300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha.

Results

The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19), the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio.

Conclusions

A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.     

The CCL2/CCR2 Axis Enhances Vascular Cell Adhesion Molecule-1 Expression in Human Synovial Fibroblasts

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of osteoarthritis (OA). Here, we investigated the intracellular signaling pathways involved in CCL2-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human OA synovial fibroblasts (OASFs).

Methodology/Principal Findings

Stimulation of OASFs with CCL2 induced VCAM-1 expression. CCL2-mediated VCAM-1 expression was attenuated by CCR2 inhibitor (RS102895), PKCδ inhibitor (rottlerin), p38MAPK inhibitor (SB203580), and AP-1 inhibitors (curcumin and tanshinone IIA). Stimulation of cells with CCL2 increased PKCδ and p38MAPK activation. Treatment of OASFs with CCL2 also increased the c-Jun phosphorylation and c-Jun binding to the AP-1 element on the VCAM-1 promoter. Moreover, CCL2-mediated CCR2, PKCδ, p38MAPK, and AP-1 pathway promoted the adhesion of monocytes to the OASFs monolayer.

Conclusions/Significance

Our results suggest that CCL2 increases VCAM-1 expression in human OASFs via the CCR2, PKCδ, p38MAPK, c-Jun, and AP-1 signaling pathway. The CCL2-induced VCAM-1 expression promoted monocytes adhesion to human OASFs.     

Fucosyltransferase 1 mediates angiogenesis,cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

Introduction

We previously reported that sialyl Lewis^y, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewis^y antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.

Methods

Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.

Results

Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.

Conclusions

These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.     

Effects of Benzalkonium Chloride on THP-1 Differentiated Macrophages In Vitro

Purpose

To characterize the effects of benzalkonium chloride (BAK) in THP-1 differentiated cells in vitro.

Methods

Macrophages were obtained after differentiation of THP-1 cells, a human monocytic leukemia cell line. Macrophages were exposed for 24 h to 33 nM (10⁻⁵%) benzalkonium chloride (BAK), 10 nM dinitrochlorobenzene (DNCB), 100 ng/mL lipopolysaccharide (LPS), 5 ng/mL tumor necrosis factor alpha (TNF-α) or phosphate buffered saline (PBS) as controls. The expression of CD11b, CD11c, CD33 and CD54 was evaluated using immunohistochemistry and flow cytometry (FCM). Phagocytosis function was analyzed using carboxylate-modified fluorescent microspheres and quantified by FCM. Migration was evaluated in cocultures with conjunctival epithelial cells. Cytokine production was detected and quantified in culture supernatants using a human cytokine array.

Results

Stimulation of THP-1-derived macrophages with a low concentration of BAK increased CD11b and CD11c expression and decreased CD33. Macrophages exposed to BAK, LPS and TNF-α had increased phagocytosis. In contrast to LPS, BAK and TNF-α increased macrophage migration. Cytokines in supernatants of macrophages exposed to BAK revealed an increased release of CCL1, CCL4/MIP-1β, TNF-α, soluble CD54/ICAM-1 and IL-1β.

Conclusion

In vitro, BAK has a direct stimulating effect on macrophages, increasing phagocytosis, cytokine release, migration and expression of CD11b and CD11c. Long-term exposure to low concentrations of BAK should be considered as a stimulating factor responsible for inflammation through macrophage activation.     

Cardiopulmonary Bypass during Cardiac Surgery Modulates Systemic Inflammation by Affecting Different Steps of the Leukocyte Recruitment Cascade

Background

It is known that the use of a cardiopulmonary bypass (CPB) during cardiac surgery leads to leukocyte activation and may, among other causes, induce organ dysfunction due to increased leukocyte recruitment into different organs. Leukocyte extravasation occurs in a cascade-like fashion, including capturing, rolling, adhesion, and transmigration. However, the molecular mechanisms of increased leukocyte recruitment caused by CPB are not known. This clinical study was undertaken in order to investigate which steps of the leukocyte recruitment cascade are affected by the systemic inflammation during CPB.

Methods

We investigated the effects of CPB on the different steps of the leukocyte recruitment cascade in whole blood from healthy volunteers (n = 9) and patients undergoing cardiac surgery with the use of cardiopulmonary bypass (n = 7) or in off-pump coronary artery bypass-technique (OPCAB, n = 9) by using flow chamber experiments, transmigration assays, and biochemical analysis.

Results

CPB abrogated selectin-induced slow leukocyte rolling on E-selectin/ICAM-1 and P-selectin/ICAM-1. In contrast, chemokine-induced arrest and transmigration was significantly increased by CPB. Mechanistically, the abolishment of slow leukocyte rolling was due to disturbances in intracellular signaling with reduced phosphorylation of phospholipase C (PLC) γ2, Akt, and p38 MAP kinase. Furthermore, CPB induced an elevated transmigration which was caused by upregulation of Mac-1 on neutrophils.

Conclusion

These data suggest that CPB abrogates selectin-mediated slow leukocyte rolling by disturbing intracellular signaling, but that the clinically observed increased leukocyte recruitment caused by CPB is due to increased chemokine-induced arrest and transmigration. A better understanding of the underlying molecular mechanisms causing systemic inflammation after CPB may aid in the development of new therapeutic approaches.     

Neutrophil adhesion molecules in experimental rhinovirus infection in COPD

Background

COPD exacerbations are associated with neutrophilic airway inflammation. Adhesion molecules on the surface of neutrophils may play a key role in their movement from blood to the airways. We analysed adhesion molecule expression on blood and sputum neutrophils from COPD subjects and non-obstructed smokers during experimental rhinovirus infections.

Methods

Blood and sputum were collected from 9 COPD subjects and 10 smoking and age-matched control subjects at baseline, and neutrophil expression of the adhesion molecules and activation markers measured using flow cytometry. The markers examined were CD62L and CD162 (mediating initial steps of neutrophil rolling and capture), CD11a and CD11b (required for firm neutrophil adhesion), CD31 and CD54 (involved in neutrophil transmigration through the endothelial monolayer) and CD63 and CD66b (neutrophil activation markers). Subjects were then experimentally infected with rhinovirus-16 and repeat samples collected for neutrophil analysis at post-infection time points.

Results

At baseline there were no differences in adhesion molecule expression between the COPD and non-COPD subjects. Expression of CD11a, CD31, CD62L and CD162 was reduced on sputum neutrophils compared to blood neutrophils. Following rhinovirus infection expression of CD11a expression on blood neutrophils was significantly reduced in both subject groups. CD11b, CD62L and CD162 expression was significantly reduced only in the COPD subjects. Blood neutrophil CD11b expression correlated inversely with inflammatory markers and symptom scores in COPD subjects.

Conclusion

Following rhinovirus infection neutrophils with higher surface expression of adhesion molecules are likely preferentially recruited to the lungs. CD11b may be a key molecule involved in neutrophil trafficking in COPD exacerbations.     

The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1β and IL-18 production

Background

The Q705K polymorphism in NLRP3 has been implicated in several chronic inflammatory diseases. In this study we determine the functional role of this commonly occurring polymorphism using an in-vitro system.

Principal Findings

NLRP3-WT and NLRP3-Q705K were retrovirally transduced into the human monocytic cell line THP-1, followed by the assessment of IL-1β and IL-18 levels in the cell culture supernatant. THP-1 cells expressing the above NLRP3 variants were sorted based upon Green Fluorescent Protein (GFP) expression. Cytokine response to alum (one of the most widely used adjuvants in vaccines) in the cells stably expressing NLRP3-WT and NLRP3-Q705K were determined. IL-1β and IL-18 levels were found to be elevated in THP-1 cells transduced with NLRP3-Q705K compared to the NLRP3-WT. Upon exposure to alum, THP-1 cells stably expressing NLRP3-Q705K displayed an increased release of IL-1β, IL-18 and TNF-α, in a caspase-1 and IL-1 receptor-dependent manner.

Conclusions

Collectively, these findings show that the Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to an overactive NLRP3 inflammasome. The option of IL-1β blockade may be considered in patients with chronic inflammatory disorders that are unresponsive to conventional treatments.     

NF-κB repressing factor downregulates basal expression and mycobacterium tuberculosis induced IP-10 and IL-8 synthesis via interference with NF-κB in monocytes

Background

Our previous study showed NF-κB repressing factor (NKRF) downregulates IP-10 and IL-8 synthesis in the peripheral blood mononuclear cells and alveolar macrophages of TB patients with high bacterial loads. However, the mechanism underlying the repressive effect of NKRF is not fully understood.

Results

The levels of IP-10, IL-8 and NKRF were significantly up-regulated in THP-1 cells treated with heated mycobacterium tuberculosis (H. TB). NKRF inhibited NF-κB-mediated IP-10 and IL-8 synthesis and release induced by H. TB. The repressive effect of NKRF is mediated via interference with NF-κB (p65) binding and RNA polymerase II recruitment to promoter sites of IP-10 and IL-8.

Conclusions

We have elucidated that direct contact with MTb induces IP-10, IL-8 and a concomitant increase in NKRF in THP-1 cells. The up-regulated NKRF serves as an endogenous repressor for IP-10 and IL-8 synthesis to hinder host from robust response to MTb infection.