Endothelial Cell Chemokine Research Articles

Abnormal expression of PRKAG2-AS1 in endothelial cells induced inflammation and apoptosis by reducing PRKAG2 expression

PRKAG2 is required for the maintenance of cellular energy balance. PRKAG2-AS1, a long non-coding RNA (lncRNA), was found within the promoter region of PRKAG2. Despite the extensive expression of PRKAG2-AS1 in endothelial cells, the precise function and mechanism of this gene in endothelial cells have yet to be elucidated. The localization of PRKAG2-AS1 was predominantly observed in the nucleus, as revealed using nuclear and cytoplasmic fractionation and fluorescence in situ hybridization. The manipulation of PRKAG2-AS1 by knockdown and overexpression within the nucleus significantly altered PRKAG2 expression in a cis-regulatory manner. The expression of PRKAG2-AS1 and its target genes, PRKAG2b and PRKAG2d, was down-regulated in endothelial cells subjected to oxLDL and Hcy-induced injury. This finding suggests that PRKAG2-AS1 may be involved in the mechanism behind endothelial injury. The suppression of PRKAG2-AS1 specifically in the nucleus led to an upregulation of inflammatory molecules such as cytokines, adhesion molecules, and chemokines in endothelial cells. Additionally, this nuclear suppression of PRKAG2-AS1 facilitated the adherence of THP1 cells to endothelial cells. We confirmed the role of nuclear knockdown PRKAG2-AS1 in the induction of apoptosis and inhibition of cell proliferation, migration, and lumen formation through flow cytometry, TUNEL test, CCK8 assay, and cell scratching. Finally, it was determined that PRKAG2-AS1 exerts direct control over the transcription of PRKAG2 by its binding to their promoters. In conclusion, downregulation of PRKAG2-AS1 suppressed the proliferation and migration, promoted inflammation and apoptosis of endothelial cells, and thus contributed to the development of atherosclerosis resulting from endothelial cell injury.

INOS inhibitor S-methylisothiourea alleviates smoke inhalation-induced acute lung injury by suppressing inflammation and macrophage infiltration

ObjectiveWe investigated the effects of the inducible NO synthase (iNOS) inhibitor, S-methylisothiourea (SMT), in a mouse model of smoke inhalation-induced acute lung injury (ALI) and explored the underlying molecular mechanism. Methods and analysisA mouse model of smoke inhalation-induced ALI was established. RNA-sequencing (seq) analysis was conducted to identify the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed for functional annotation of DEGs. Moreover, an immunofluorescence assay using macrophage marker F4/80 was performed to assess macrophage infiltration. A hypoxia-induced HUVEC model was used to mimic smoke inhalation-induced injury in endothelial cells. Finally, a transwell assay was used to analyze the chemoattractive effects of endothelial cells on macrophages. ResultsSMT markedly alleviated the pulmonary pathological symptoms, edema, and inflammatory response in the mouse smoke inhalation-induced ALI model. RNA-seq analysis revealed that SMT may diminish lung injury by regulating the levels of genes associated with inflammatory responses, cell chemokines, and adhesion. In vivo data revealed that the protective effects of SMT against smoke inhalation-induced ALI were partly achieved by inhibiting the production of adhesion molecules and infiltration of macrophages. Furthermore, in vitro data from the hypoxia-induced HUVEC model revealed that SMT reduced macrophage chemotaxis by inhibiting the production of chemokines and adhesion molecules in endothelial cells. ConclusioniNOS inhibitor SMT protects the lungs from smoke inhalation-induced ALI by reducing the production of pro-inflammatory cytokines, adhesion molecules, and chemokines in endothelial cells, thereby inhibiting inflammation and macrophage infiltration.

Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells.

Atherosclerosis is a chronic inflammatory disease and occurs preferentially in arterial regions exposed to disturbed blood flow (d-flow) while the stable flow (s-flow) regions are spared. D-flow induces endothelial inflammation and atherosclerosis by regulating endothelial gene expression partly through the flow-sensitive transcription factors (FSTFs). Most FSTFs, including the well-known Kruppel-like factors KLF2 and KLF4, have been identified from in vitro studies using cultured endothelial cells (ECs). Since many flow-sensitive genes and pathways are lost or dysregulated in ECs during culture, we hypothesized that many important FSTFs in ECs in vivo have not been identified. We tested the hypothesis by analyzing our recent gene array and single-cell RNA sequencing (scRNAseq) and chromatin accessibility sequencing (scATACseq) datasets generated using the mouse partial carotid ligation model. From the analyses, we identified 30 FSTFs, including the expected KLF2/4 and novel FSTFs. They were further validated in mouse arteries in vivo and cultured human aortic ECs (HAECs). These results revealed 8 FSTFs, SOX4, SOX13, SIX2, ZBTB46, CEBPβ, NFIL3, KLF2, and KLF4, that are conserved in mice and humans in vivo and in vitro. We selected SOX13 for further studies because of its robust flow-sensitive regulation, preferential expression in ECs, and unknown flow-dependent function. We found that siRNA-mediated knockdown of SOX13 increased endothelial inflammatory responses even under the unidirectional laminar shear stress (ULS, mimicking s-flow) condition. To understand the underlying mechanisms, we conducted an RNAseq study in HAECs treated with SOX13 siRNA under shear conditions (ULS vs. oscillatory shear mimicking d-flow). We found 94 downregulated and 40 upregulated genes that changed in a shear- and SOX13-dependent manner. Several cytokines, including CXCL10 and CCL5, were the most strongly upregulated genes in HAECs treated with SOX13 siRNA. The robust induction of CXCL10 and CCL5 was further validated by qPCR and ELISA in HAECs. Moreover, the treatment of HAECs with Met-CCL5, a specific CCL5 receptor antagonist, prevented the endothelial inflammation responses induced by siSOX13. In addition, SOX13 overexpression prevented the endothelial inflammation responses. In summary, SOX13 is a novel conserved FSTF, which represses the expression of pro-inflammatory chemokines in ECs under s-flow. Reduction of endothelial SOX13 triggers chemokine expression and inflammatory responses, a major proatherogenic pathway.

Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity and overexpression of CARD8 mRNA was previously identified in atherosclerosis. However, very little is known about the regulation of CARD8 in endothelial cells and atherosclerosis. The aim of this study was to investigate CARD8 in the regulation of cytokine and chemokine expression in endothelial cells. Sections of human atherosclerotic lesions and non-atherosclerotic arteries were immunostained for CARD8 protein. Expression of CARD8 was correlated to mediators of inflammation in atherosclerotic lesions using Biobank of Karolinska Endarterectomies microarray data. The CARD8 mRNA was knocked-down in human umbilical vein endothelial cells (HUVECs) in vitro, followed by quantitative RT-PCR analysis and OLINK Proteomics. Endothelial and smooth muscle cells in arterial tissue expressed CARD8 and CARD8 correlated with vWF, CD163 and the expression of inflammatory genes, such as CXCL1, CXCL6 and PDGF-A in plaque. Knock-down of CARD8 in HUVECs significantly altered proteins involved in inflammatory response, such as CXCL1, CXCL6, PDGF-A, MCP-1 and IL-6. The present study suggest that CARD8 regulate the expression of cytokines and chemokines in endothelial cells and atherosclerotic lesions, suggesting that CARD8 plays a significant role in endothelial activation.

Complement protein C1q modulates endothelial cell functions in atherosclerosis

Abstract Atherosclerosis is a chronic inflammatory disease characterized by the development of plaque in arteries. Interactions between monocytes, endothelial cells and lipoproteins play a major role in the disease pathogenesis. C1q is traditionally known for activation of the complement cascade, leading to inflammation and clearance of foreign targets. However, we previously showed that C1q bound to modified lipoproteins (e.g. oxLDL) plays a protective role by polarizing monocytes and macrophages towards an anti-inflammatory state during engulfment and increases survival and efferocytic function of macrophage foam cells. Additionally, C1q modulates macrophage chemokine production in in vitro and in in vitro models of atherosclerosis. Here we investigate C1q modulation of both endothelial cell chemokine production, and functions affecting monocyte migration. Human aortic endothelial cells (HAEC) were incubated with oxLDL +/− C1q. RNA and supernatants were collected for chemokine gene regulation and protein analysis, respectively. Monocyte adhesion and transendothelial migration were also measured. The data show that C1q modulates chemokine gene and protein expression, increases monocyte adhesion and transendothelial migration and enhances HAEC monolayer permeability. Thus, C1q interactions with HAEC in hyperlipidemic conditions may result in increased availability of phagocytes for clearance in the early atherosclerotic lesion. However in later stages infiltrating cells may contribute to disease progression through an increased proinflammatory environment and complement activation. Understanding timing and molecular mechanisms in atherosclerosis will be vital for the development of novel treatments.

Endothelial-specific deletion of Brahma-related gene 1 (BRG1) assuages unilateral ureteral obstruction induced renal injury in mice

Renal homeostasis is regulated by the interplay among different cell types in the kidneys including endothelial cells. In the present study we investigated the phenotypic regulation of endothelial cells by BRG1, a chromatin remodeling protein, in a mouse model of obstructive nephropathy (ON). We report that endothelial-specific deletion of BRG1 attenuated renal inflammation induced by unilateral ureteral tract obstruction (UUO) in mice, as evidenced by down-regulation of pro-inflammatory cytokines and diminished infiltration of immune cells. Moreover, endothelial BRG1 deficiency suppressed UUO-induced renal fibrosis in mice as measured by expression of pro-fibrogenic genes, picrosirius red staining of collagenous tissues, and quantification of hydroxylproline levels. Mechanistically, BRG1 activated the transcription of adhesion molecules and chemokines in endothelial cells by recruiting histone modifying enzymes leading to macrophage adhesion and chemotaxis. In conclusion, we propose that epigenetic regulation of endothelial function by BRG1 may play an active role in ON pathogenesis.

Chemokines and Relevant microRNAs in the Atherogenic Process.

Chemokines play a significant role in initial and advanced steps of atherogenesis. In early steps, chemokines control the adhesion of leukocytes to endothelial cells (ECs) followed by transmigration of monocytes and their deposition in the intima where they differentiate to proinflammatory macrophages. Except for proinflammatory activity, chemokines are responsible for homeostasis and homing of progenitor cells. Recently, microRNAs (miRs) were found to control expression and activity of chemokines in ECs, vascular smooth muscle cells (VSMCs), and macrophages at different steps of atherogenesis. Expression of the proatherogenic chemokine CXCL1 is suppressed by miR-181 that down-regulates nuclear transcription factor NF-kB stimulation in ECs therefore weakening the adhesiveness of the endothelium for monocytes. MiR-126 activates the endothelial production of a chemokine CXCL12 via self-multiplying feedback loop to promote re-endothelialization and support lesion stability. MiR-155 is proatherogenic by induction of the inflammatory chemokine CCL2 in macrophages. In fact, chemokines, their receptors, and relevant miRs form a complex network that exerts pro- and anti-inflammatory properties in vascular cells during different steps of atherogenic process. Obtaining new information about complicated relations between miRs and chemokines may create prerequisites for the development of novel approaches to treat atherosclerosis.

Fli-1 transcription factor regulates inflammatory mediators in endothelial cells (P6240)

Abstract Fli-1 transcription factor is implicated in the pathogenesis of human and murine lupus. A two-fold overexpression of Fli-1 protein in transgenic mice results in the development of a lupus-like disease. Expression of Fli-1 in lupus patients is higher compared to normal controls. Endothelial cells play a role in a number of biological processes ranging from inflammation to the regulation of blood flow. We have reported that Fli-1 directly regulates the expression of MCP-1 in endothelial cells. In this study we investigated if Fli-1 also regulates other inflammatory chemokines. We transfected an endothelial cell line with Fli-1 specific siRNA and negative control oligo siRNA. There was more than an 80% decrease in Fli-1 expression in cells transfected with Fli-1 siRNA when compared to those transfected with control siRNA. Next, we stimulated these cells with TLR4 ligand lipopolysaccharide and extracted mRNA. An mRNA array was used to screen the levels of 26 chemokines. A number of message levels were decreased, including MCP-1, in the cells transfected with Fli-1 specific siRNA when compared to the control. Expression of MIF was higher in the cells when Fli-1 was silenced. We further confirmed MIF mRNA expression levels by real-time PCR. Our data indicates that Fli-1 is likely involved in regulating the production of several chemokines in endothelial cells.

An Antibody to the Sixth Ig-like Domain of VCAM-1 Inhibits Leukocyte Transendothelial Migration without Affecting Adhesion

VCAM-1 plays a key role in leukocyte trafficking during inflammatory responses. However, molecular mechanisms underlying this function have not been clearly elucidated. In this study, using phage display technology, we developed a rabbit/human chimeric VCAM-1 Ab, termed VCAM-1 domain 6 (VCAM-1-D6), which specifically recognizes aa 511-599 within the sixth Ig-like domain. We report that the VCAM-1-D6 Ab blocked U937 cell transmigration across activated HUVECs but did not alter adhesion of U937 cells to the HUVECs. We also demonstrate that VCAM-1-D6 does not alter TNF-α-stimulated endothelial cell chemokine or cytokine production. Furthermore, through in vivo efficacy testing using a mouse islet allograft model, we demonstrate that VCAM-1-D6 significantly alleviates allograft rejection by blocking leukocyte infiltration to the grafted islets. Taken together, our results suggest that the VCAM-1-D6 Ab may block VCAM-1-mediated inflammation and could be a useful tool in treating inflammatory diseases.

Dose-dependent effect of radiation on angiogenic and angiostatic CXC chemokine expression in human endothelial cells

Blood vessel growth is regulated by angiogenic and angiostatic CXC chemokines, and radiation is a vasculogenic stimulus. We investigated the effect of radiation on endothelial cell chemokine signaling, receptor expression, and migration and apoptosis. Human umbilical vein endothelial cells were exposed to a single fraction of 0, 5, or 20 Gy of ionizing radiation (IR). All vasculogenic chemokines (CXCL1–3/5–8) increased 3–13-fold after 5 or 20 Gy IR. 20 Gy induced a marked increase (1.6–4-fold) in angiostatic CXC chemokines. CXCR4 expression increased 3.5 and 7-fold at 48 h after 5 and 20 Gy, respectively. Bone marrow progenitor cell chemotaxis was augmented by conditioned media from cells treated with 5 Gy IR. Whereas 5 Gy markedly decreased intrinsic cell apoptosis (0 Gy = 16% ± 3.6 vs. 5 Gy = 4.5% ± 0.3), 20 Gy increased it (21.4% ± 1.2); a reflection of pro-survival angiogenic chemokine expression. Radiation induces a dose-dependent increase in pro-angiogenic CXC chemokines and CXCR4. In contrast, angiostatic chemokines and apoptosis were induced at higher (20 Gy) radiation doses. Cell migration improved significantly following 5 Gy, but not 20 Gy IR. Collectively, these data suggest that lower doses of IR induce an angiogenic cascade while higher doses produce an angiostatic profile.

Evolution of the Inflammatory Response in Vertebrates: Fish TNF-α Is a Powerful Activator of Endothelial Cells but Hardly Activates Phagocytes

TNF-alpha is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-alpha are mediated through the activation of endothelial cells. Thus, TNF-alpha promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-alpha, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-alpha in Ag presentation. Lastly, we found that TNF-alpha increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-alpha on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-alpha increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-alpha resulted in increased viral replication. Together, these results indicate that fish TNF-alpha displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.

Role of the Protein C Pathway in the Extraintestinal Thrombosis Associated With Murine Colitis

Chronic inflammatory bowel diseases (IBD) are associated with an increased risk for thromboembolism. Although thrombosis is known to contribute to the morbidity and mortality of patients with IBD, the underlying mechanisms that contribute to the genesis of a hypercoagulable state during intestinal inflammation remain poorly defined. The objective of this study was to determine whether the protein C pathway contributes to the enhanced extraintestinal thrombosis that is associated with dextran sodium sulfate (DSS)-induced colitis in mice. Microvascular thrombosis was induced in cremaster muscle microvessels of normal and colitic mice using a light/dye injury model. DSS colitis enhanced thrombus formation in cremaster arterioles of wild-type mice. The DSS-induced thrombosis response was greatly attenuated in transgenic mice over expressing the endothelial protein C receptor. Activated protein C (APC), administered to colitic WT mice immediately prior to photoactivation, also afforded protection against thrombosis, and an anti-APC antibody enhanced thrombus formation. These findings indicate that elevated APC levels, derived from either endogenous or exogenous sources, confer protection against the extraintestinal thrombosis that accompanies colonic inflammation.

Normal saline resuscitation induces increased sinusoidal MIP‐2 expression in early sepsis

Fluid resuscitation is an integral part of the treatment of patients with severe sepsis. Common fluids administered within the first 6 hours of diagnosis include normal saline and Ringer's lactate, however, currently there is no clear evidence that support the use of one resuscitation fluid over another. Previous work in our lab demonstrated that normal saline increases leukocyte‐endothelial cell interactions. The objective in this study was to determine the mechanisms for saline‐induced hepatic leukocyte recruitment.Sepsis was induced by cecal ligation and perforation in C57Bl/6 mice. Septic, sham‐operated or naïve mice received an IV bolus (20mL/kg) of Ringer's lactate or normal saline (0.9% NaCl). Six hours later the hepatic microcirculation was examined by confocal intravital microscopy to visualize the neutrophil chemokine macrophage inflammatory protein‐2 (MIP‐2) expression using an Alexafluor conjugated monoclonal antibody.MIP‐2 expression, as measured by mean fluorescent intensity, was significantly higher in the hepatic sinusoids of septic mice treated with normal saline compare to septic mice given Ringer's lactate. The use of normal saline as an intravenous resuscitation fluid was associated with increased chemokine expression in the both sinusoids and post‐sinusoidal venules.These data suggest that saline treatment in early sepsis augments neutrophil recruitment by increasing endothelial cell chemokine expression.

Throwing light on DARC

Chemokines play a key role in directing and driving leucocyte trafficking. The efficient regulation of leucocyte recruitment by chemokines requires their appropriate localization in functional micro-anatomical domains, as well as setting limits to their effects in space and time. Both processes are influenced by silent chemokine receptors (interceptors), including DARC (Duffy antigen receptor for chemokines). Increasing experimental evidence suggests that DARC is involved in accumulation of extravascular chemokines in endothelial cells, chemokine transcytosis and presentation on their luminal surface, leading to leucocyte adhesion and emigration. Additionally, DARC is expressed on erythrocytes and can act as a sink for chemokines in blood. This limits the dissemination of chemokines through blood into distant organs and tissues as well as reducing their effects on the circulating leucocytes.

Fibrinogen regulates the expression of inflammatory chemokines through NF-kappaB activation of endothelial cells.

The objective of this study was to characterize the role of fibrinogen in stimulating expression of inflammatory chemokines in endothelial cells through NF-kappaB activation. Human umbilical vein endothelial cells (HUVEC) were exposed to fibrinogen up to 3,000 microg/ml, and NF-kappaB activation was assessed using electrophoretic mobility shift assay (EMSA). Fibrinogen exposure resulted in a concentration dependent increase in NF-kappaB activation that reached a maximum at 1,000 microg/ml after 4 hours and was sustained up to 24 hours. The effect was inhibited by antibodies to alpha(v)beta(3) and alpha(5)beta(1) and by the GRGDS peptide, indicating integrin involvement. Preincubation with Mn(2+) lowered the fibrinogen concentration-dependence, consistent with integrin activation. Supershift assays demonstrated involvement of the p50, p65 and c-Rel components of NF-kappaB. Fibrinogen exposure also resulted in up-regulation of expression of monocyte chemoattractant protein-1 (MCP-1) and of interleukin-8 as shown by RNase protection assays and by real-time RT-PCR. Increased secretion of MCP-1 was confirmed by ELISA. Parthenolide, an IkappaB kinase inhibitor, prevented up-regulation of MCP-1 by fibrinogen, linking this response to NF-kappaB activation. From our findings, we conclude that fibrinogen regulates NF-kappaB activation and expression of inflammatory chemokines in endothelial cells and may be involved in mediating inflammatory processes.

Effect of 15-deoxy-Δ 12,14-prostaglandin J 2 on IL-1 β-induced expression of epithelial neutrophil-activating protein-78 in human endothelial cells

Epithelial neutrophil-activating peptide-78 (ENA-78) is a member of CXC chemokines. It is produced by endothelial cells stimulated with interleukin-1 (IL-1), along with other CXC chemokines such as IL-8 and growth-related oncogene protein- α (GRO- α). IL-1-induced ENA-78 production by endothelial cells may be important for the regulation of neutrophil activation. 15-Deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2) is a natural ligand for peroxisome proliferator-activated receptor- γ (PPAR- γ) and affects the expression of various genes. We examined the effect of 15d-PGJ 2 on the expression of ENA-78 in cultured endothelial cells stimulated with IL-1 β. 15d-PGJ 2 inhibited the IL-1 β-induced expression of ENA-78, but not the expression of IL-8 or GRO- α in response to IL-1. Ciglitazone, another agonist for PPAR- γ, had no effect on the expression of ENA-78, suggesting that 15d-PGJ 2 may inhibit the expression of ENA-78 in a PPAR- γ-independent manner. 15d-PGJ 2 may modulate inflammatory reactions by regulating the balance of CXC chemokines in endothelial cells.

MCSF expression is induced in healing myocardial infarcts and may regulate monocyte and endothelial cell phenotype.

Myocardial infarction is associated with the rapid induction of mononuclear cell chemoattractants that promote monocyte infiltration into the injured area. Monocyte-to-macrophage differentiation and macrophage proliferation allow a long survival of monocytic cells, critical for effective healing of the infarct. In a canine infarction-reperfusion model, newly recruited myeloid leukocytes were markedly augmented during early reperfusion (5-72 h). By 7 days, the number of newly recruited myeloid cells was reduced, and the majority of the inflammatory cells remaining in the infarct were mature macrophages. Macrophage colony-stimulating factor (MCSF) is known to facilitate monocyte survival, monocyte-to-macrophage conversion, and macrophage proliferation. We demonstrated marked induction of MCSF mRNA in ischemic segments persisting for at least 5 days after reperfusion. MCSF expression was predominantly localized to mature macrophages infiltrating the infarcted myocardium; the expression of the MCSF receptor, c-Fms, a protein with tyrosine kinase activity, was found in these macrophages but was also observed in a subset of microvessels within the infarct. Many infarct macrophages expressed proliferating cell nuclear antigen, a marker of proliferative activity. In vitro MCSF induced monocyte chemoattractant protein-1 synthesis in canine venous endothelial cells. MCSF-induced endothelial monocyte chemoattractant protein-1 upregulation was inhibited by herbimycin A, a tyrosine kinase inhibitor, and by LY-294002, a phosphatidylinositol 3'-kinase inhibitor. We suggest that upregulation of MCSF in the infarcted myocardium may have an active role in healing not only through its effects on cells of monocyte/macrophage lineage, but also by regulating endothelial cell chemokine expression.

T-cell mediated induction of allogeneic endothelial cell chemokine expression.

The goal of the current study was to test the ability of T cells to stimulate allogeneic endothelial cells to express chemokines, particularly the T-cell recruiting factors monokine induced by interferon-gamma (Mig) and inducible protein (IP)-10. Lymph node cells from C57BL/6 (H-2b) recipients of C3H (H-2k) skin grafts or from naïve mice were added to monolayers of C3H-derived endothelial cell line 2F-2B. After 5 or 24 hr, the lymph node cells were removed, and RNA was prepared from the endothelial cells and tested by ribonuclease protection assay or Northern blot hybridization for endothelial cell expression of chemokines. Alloantigen-primed T cells induced endothelial cell expression of regulated on activation normal T-cell expressed and secreted (RANTES), IP-10, Mig, monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and macrophage inflammatory protein-1beta within 5 hr of coculture. In vitro chemotaxis assays demonstrated the production of T-cell chemoattractants by the endothelial cells. With the exception of low levels of monocyte chemotactic protein-1 and RANTES, culture with naïve C57BL/6 lymph node T cells did not induce endothelial cell chemokine expression. Alloantigen-primed CD4 T cells induced endothelial expression of IP-10 and RANTES but none of the other chemokines tested, whereas primed CD8 T cells induced all of the chemokines tested. Expression of IP-10 and Mig was not induced when alloantigen-primed T cells from interferon-gamma deficient recipients of C3H skin grafts were cultured with the endothelial cells. This expression was blocked by addition of intercellular adhesion molecule-1 or lymphocyte function-associated antigen-1 specific antibodies to the cultures. CONCLUSIONS These results demonstrate the ability of alloantigen-primed CD8 T cells to quickly and directly stimulate endothelial cells to express and produce chemokines, including those recruiting T cells.

Increased endothelial monocyte chemoattractant protein-1 and interleukin-8 in preeclampsia

OBJECTIVE:To test the hypothesis that preeclampsia is associated with increased endothelial cell chemokine production of monocyte chemoattractant protein-1 and interleukin-8 necessary for monocyte recruitment to the vascular endothelium.METHODS:Plasma was collected from women with severe preeclampsia and normal pregnant women at term and measured for monocyte chemoattractant protein-1, interleukin-8, and lipid peroxide levels by enzyme-linked immunosorbent assays and malondialdehyde assays. Human umbilical vein endothelial cells were cultured with 5% plasma from normal or preeclamptic patients and the media assayed for monocyte chemoattractant protein-1 and interleukin-8 production.RESULTS:In women with severe preeclampsia, plasma levels of monocyte chemoattractant protein-1, interleukin-8, and lipid peroxides were elevated (1.5-fold, 2.5-fold, and 4.5-fold higher, respectively) compared with normal pregnant women. Human umbilical vein endothelial cells cultured with plasma from preeclamptic women significantly increased the production of both monocyte chemoattractant protein-1 (2.3-fold) and interleukin-8 (1.5-fold) compared with plasma from normal pregnant women. Monocyte chemoattractant protein-1 and interleukin-8 production was decreased by the antioxidant vitamin E in human umbilical vein endothelial cells treated with preeclamptic plasma, suggesting that the production of these cytokines may be regulated by signaling mechanisms sensitive to oxidative stress.CONCLUSION:These findings support the hypothesis that circulating factors in the plasma of women with preeclampsia activate endothelial cell monocyte chemoattractant protein-1 and interleukin-8 production, and although not directly examined in this study, may increase monocyte adherence to the vascular endothelium.

Inhibition of human endothelial cell chemokine production by the opportunistic fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated fungal pathogen commonly acquired by inhalation. Extrapulmonary dissemination can lead to infection of the bloodstream and various organs, most commonly resulting in meningoencephalitis. However, infection with C. neoformans is often characterized by a scant inflammatory response. The leukocyte response to infection depends in part upon a gradient of chemotactic factors and adhesion molecules expressed by the host vascular endothelium, yet the inflammatory response of human endothelial cells (EC) to C. neoformans has not been previously investigated. We found that incubation of primary human EC with C. neoformans did not induce chemokine synthesis, and resulted in differential inhibition of cytokine-induced IL-8, IFN-gamma-inducible protein-10, and monocyte chemoattractant protein-1. In contrast, C. neoformans had little effect on EC surface expression of the leukocyte ligand, ICAM-1, as determined by flow cytometry. Modulation of chemokine production was dependent on the chemokine under study, the inoculum of C. neoformans used, fungal viability, and cell-cell contact, but independent of cryptococcal strain or encapsulation. These observations suggest a novel mechanism whereby C. neoformans can affect EC function and interfere with the host inflammatory response.