Intercellular Adhesion Molecule-1 Expression In Response Research Articles

548-P: Baicalin Attenuates Fibrosis Process in Human Renal Proximal Tubular Cells in Hyperglycemic Condition

Background: Baicalin, isolated from Scutellaria baicalensis, has been reported to have anti-inflammatory effect by inhibiting expression of cell adhesion molecules and migration of leukocytes in vascular cells. However, there is a limited evidence on its anti-inflammatory effect in hyperglycemia-associated milieu in other cells. Hyperglycemia and its associated factors are known to induce diabetic nephropathy by exacerbating inflammation, especially, in proximal convoluted tubule. Herein, we investigated whether baicalin exerts anti-inflammatory and anti-fibrosis effects on human renal proximal tubular cells in hyperglycemia induced conditions. Method: HK-2 cell was maintained in normal glycemic (5.78mM glucose) or in hyperglycemic (30mM glucose) condition with or without advanced glycation end-products (AGE) and tumor necrosis factor alpha (TNFα). In each condition, baicalin was treated at 10uM. Cell viability was determined by MTT assay. Gene and protein expression of multiple inflammatory and fibrosis markers including toll-like receptor 2 (TLR2), TLR4, fibronectin, collagen IV α1 (ColIVα1), collagen IV α2 (ColIVα2), transforming growth factor β (TGFβ), intercellular adhesion molecule 1 (ICAM1), and vascular adhesion molecule 1 (VCAM1) were evaluated by PCR and WB, respectively. Results: Baicalin had no effect on cell viability. Baicalin significantly decreased gene expression of ColIVα2 in response to TNFα stimulation in normal glycemic condition (p=0.038). In hyperglycemic condition, baicalin decreased gene expressions of ICAM1 (p=0.049) and VCAM1 (p=0.049) significantly in AGE treated cells; and it significantly decreased gene expressions of TGFβ (p=0.038) and ICAM1 (p=0.017) in TNFα treated cells. Baicalin also decreased protein expression of ICAM1 in response to AGE (p=0.048) and TNFα (p=0.040) stimulation in hyperglycemic condition. Conclusion: Baicalin attenuated fibrosis process by hyperglycemia associated factors in HK-2 cell. Disclosure J. Nam: None. J. Kim: None. K. Park: None. S. Lee: None. J. Nam: None. J. Park: None. S. Park: None. Y. Kim: None. C. Ahn: None.

Infection of human intestinal epithelial cells by invasive bacteria activates NF-κB and increases ICAM-1 expression through NOD1.

Background/AimsNucleotide-binding oligomerization domain 1 (NOD1) is required for primary intestinal epithelial cells (IECs) to respond to natural mucopeptides secreted by gram-negative bacteria. Infection of human IECs with invasive bacteria up-regulates intercellular adhesion molecule-1 (ICAM-1) expression. However, the role of NOD family members in host defense has been largely unknown. The aim of this study was to determine whether there is a functional role for NOD1 in the up-regulation of ICAM-1 expression in invasive bacteria-infected IECs.MethodsICAM-1 mRNA expression was compared between controls, Caco-2 or HT29 cells transfected with an empty vector, and IECs stably transfected with a dominant-negative (DN) NOD1. Expression was compared using qualitative reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and flow cytometry after infection with enteroinvasive Escherichia coli O29:NM or Shigella flexneri. Nuclear factor kB (NF-κB) activation was determined by electrophoretic mobility shift assays.ResultsDN NOD1 significantly inhibited the up-regulation of ICAM-1 expression in response to an enteroinvasive bacterial infection. The Caco-2 cells transfected with DN NOD1 manifested marked inhibition of NF-kB activation in response to E. coli O29:NM infection.ConclusionsSignaling through NOD1 may play an essential role in neutrophil trafficking following infection with enteroinvasive bacteria.

Na(+) /H(+) exchanger regulatory factor 1 knockout mice have an attenuated hepatic inflammatory response and are protected from cholestatic liver injury.

The intercellular adhesion molecule 1 (ICAM-1) is induced in mouse liver after bile duct ligation (BDL) and plays a key role in neutrophil-mediated liver injury in BDL mice. ICAM-1 has been shown to interact with cytoskeletal ezrin-radixin-moesin (ERM) proteins that also interact with the PDZ protein, Na(+) /H(+) exchanger regulatory factor 1 (NHERF-1/EBP50). In NHERF-1(-/-) mice, ERM proteins are significantly reduced in brush-border membranes from kidney and small intestine. ERM knockdown reduces ICAM-1 expression in response to tumor necrosis factor alpha. Here we show that NHERF-1 assembles ERM proteins, ICAM-1 and F-actin into a macromolecule complex that is increased in mouse liver after BDL. Compared to wild-type (WT) mice, both sham-operated and BDL NHERF-1(-/-) mice have lower levels of activated ERM and ICAM-1 protein in the liver accompanied by significantly reduced hepatic neutrophil accumulation, serum alanine aminotransferase, and attenuated liver injury after BDL. However, total bile acid concentrations in serum and liver of sham and BDL NHERF-1(-/-) mice were not significantly different from WT controls, although hepatic tetrahydroxylated bile acids and Cyp3a11 messenger RNA levels were higher in NHERF-1(-/-) BDL mice. NHERF-1 participates in the inflammatory response that is associated with BDL-induced liver injury. Deletion of NHERF-1 in mice leads to disruption of the formation of ICAM-1/ERM/NHERF-1 complex and reduction of hepatic ERM proteins and ICAM-1, molecules that are up-regulated and are essential for neutrophil-mediated liver injury in cholestasis. Further study of the role of NHERF-1 in the inflammatory response in cholestasis and other forms of liver injury should lead to discovery of new therapeutic targets in hepatic inflammatory diseases.

Tumour Necrosis Factor α Enhances CCL2 and ICAM-1 Expression in Peripheral Nerve Microvascular Endoneurial Endothelial Cells

Recruitment and trafficking of autoreactive leucocytes across the BNB (blood–nerve barrier) is an early pathological insult in GBS (Guillain-Barré syndrome), an aggressive autoimmune disorder of the PNS (peripheral nervous system). Whereas the aetiology and pathogenesis of GBS remain unclear, pro-inflammatory cytokines, including TNFα (tumour necrosis factor α), are reported to be elevated early in the course of GBS and may initiate nerve injury by activating the BNB. Previously, we reported that disrupting leucocyte trafficking in vivo therapeutically attenuates the course of an established animal model of GBS. Here, PNMECs (peripheral nerve microvascular endothelial cells) that form the BNB were harvested from rat sciatic nerves, immortalized by SV40 (simian virus 40) large T antigen transduction and subsequently challenged with TNFα. Relative changes in CCL2 (chemokine ligand 2) and ICAM-1 (intercellular adhesion molecule 1) expression were determined. We report that TNFα elicits marked dose- and time-dependent increases in CCL2 and ICAM-1 mRNA and protein content and promotes secretion of functional CCL2 from immortalized and primary PNMEC cultures. TNFα-mediated secretion of CCL2 promotes, in vitro, the transendothelial migration of CCR2-expressing THP-1 monocytes. Increased CCL2 and ICAM-1 expression in response to TNFα may facilitate recruitment and trafficking of autoreactive leucocytes across the BNB in autoimmune disorders, including GBS.

447: Egfl7 Suppresses ICAM-1 Expression in Response to H/R Injury and Tacrolimus

Egfl7 is a chemoattractant for endothelial cells and its expression is restricted to endothelial cells. Hypoxia/reperfusion and CNI induced endothelial injury that occurs during and after transplantation incite the subsequent development of allograft vasculopathy. We investigated the effect of Egfl7 on endothelial cell intercellular adhesion molecule-1 (ICAM-1) expression in response to H/R injury and tacrolimus.

Regulation of Bacteria-Induced Intercellular Adhesion Molecule-1 by CCAAT/Enhancer Binding Proteins

Direct interaction between bacteria and epithelial cells may initiate or amplify the airway response through induction of epithelial defense gene expression by nuclear factor-kappaB (NF-kappaB). However, multiple signaling pathways modify NF-kappaB effects to modulate gene expression. In this study, the effects of CCAAT/enhancer binding protein (C/EBP) family members on induction of the leukocyte adhesion glycoprotein intercellular adhesion molecule-1 (ICAM-1) was examined in primary cultures of human tracheobronchial epithelial cells incubated with nontypeable Haemophilus influenzae. Increased ICAM-1 gene transcription in response to H. influenzae required gene sequences located at -200 to -135 in the 5'-flanking region that contain a C/EBP-binding sequence immediately upstream of the NF-kappaB enhancer site. Constitutive C/EBPbeta was found to have an important role in epithelial cell ICAM-1 regulation, while the adjacent NF-kappaB sequence binds the RelA/p65 and NF-kappaB1/p50 members of the NF-kappaB family to induce ICAM-1 expression in response to H. influenzae. The expression of C/EBP proteins is not regulated by p38 mitogen-activated protein kinase activation, but p38 affects gene transcription by increasing the binding of TATA-binding protein to TATA-box-containing gene sequences. Epithelial cell ICAM-1 expression in response to H. influenzae was decreased by expressing dominant-negative protein or RNA interference against C/EBPbeta, confirming its role in ICAM-1 regulation. Although airway epithelial cells express multiple constitutive and inducible C/EBP family members that bind C/EBP sequences, the results indicate that C/EBPbeta plays a central role in modulation of NF-kappaB-dependent defense gene expression in human airway epithelial cells after exposure to H. influenzae.

Role of ROS and MAPK in TPA‐induced ICAM‐1 expression in the myeloid ML‐1 cell line

Intercellular adhesion molecule 1 (ICAM-1) has been implicated in playing a key role in the mechanism of inflammatory process initiated in response to environmental agents, and during normal hematopoietic cell differentiation. Though induction of ICAM-1 by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in myeloid cells has been reported, the molecular mechanism by which TPA upregulates ICAM-1 expression remains unclear. In the present study, we investigated the signaling mechanism associated with TPA-induced ICAM-1 expression in ML-1 cells. Herein, our microarray, flow cytometry, and Western blot analysis indicated that ICAM-1 was constitutively expressed at a low level in ML-1 cells, but its expression was further upregulated at both the mRNA and protein levels in response to TPA. ICAM-1 expression in response to TPA was inhibited by pretreatment with GF109203X [a specific inhibitor of protein kinase C (PKC)], or with PD98059 and U0126 (specific inhibitors of MEK), suggesting the importance of PKC, and Erk1/2 signaling cascades in this response. Interestingly, ICAM-1 expression in response to TPA-induced PKC activation was linked to the generation of reactive oxygen species (ROS), as pretreatment with NAC (an ROS scavenger) blocked both ErK1/2 activation and ICAM-1 expression induced by TPA. In addition, TPA-induced ICAM-1 expression was blocked by inhibition of nuclear factor-kappaB (NF-kappaB) activation following pretreatment with BAY11-7085 (a specific inhibitor of NF-kappaB activation). TPA-induced NF-kappaB activation was shown by increased degradation of IkB (NF-kappaB specific inhibitory protein). Together, these observations demonstrated that TPA, a potent activator of PKC, induces ICAM-1 expression via a ROS- and ERK1/2-dependent signaling mechanism in ML-1 cells.

Santonin-related compound 2 inhibits the expression of ICAM-1 in response to IL-1 stimulation by blocking the signaling pathway upstream of IκB degradation

Santonin-related compounds (SRCs) were synthesized from the starting material l-α-santonin and tested for the biological activity on the expression of intercellular adhesion molecule-1 (ICAM-1) in response to IL-1 stimulation on human adenocarcinoma cells. One of the bromoketone derivatives termed SRC2 [(11S)-2α-bromo-3-oxoeudesmanno-13,6α-lactone] strongly inhibited the ICAM-1 expression at an IC50 value of 5.9 μM, whereas l-α-santonin itself was totally inactive up to 100 μM. The blockage of ICAM-1 expression by SRC2 was not due to the direct inhibition of de novo RNA and protein synthesis. The nuclear translocation of NF-κB subunit p65 was markedly prevented by SRC2. Moreover, IκBα degradation upon IL-1 stimulation was strongly inhibited by SRC2. These observations suggest that SRC2 blocks the IL-1 signaling pathway upstream of IκB degradation.

Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression.

Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.

Reduced neointima hyperplasia of vein bypass grafts in intercellular adhesion molecule-1-deficient mice.

Recently, we established a new mouse model of vein graft arteriosclerosis through the grafting of vena cava to carotid arteries. In many respects, the morphological features of this murine vascular graft model resemble those of human venous bypass graft disease. With this model, we studied the role of intercellular adhesion molecule-1 (ICAM-1) in the development of vein graft arteriosclerosis in ICAM-1-deficient mice. Neointimal hyperplasia of vein grafts in ICAM-1 -/- mice was reduced 30% to 50% compared with that of wild-type control animals. Immmunofluorescent analysis revealed that increased ICAM-1 expression was observed on the endothelium and smooth muscle cells (SMCs) of the grafted veins in wild-type, but not ICAM-1 -/-, mice. MAC-1 (CD11b/18)-positive cells that adhered to the surface of vein grafts in ICAM-1 -/- mice were significantly less as identified with en face immunofluorescence, and these positive cells were more abundant in the intimal lesions of vein grafts in wild-type mice. Furthermore, aortic SMCs cultivated from wild-type mice exhibited high ICAM-1 expression in response to tumor necrosis factor-alpha. When tumor necrosis factor-alpha-stimulated SMCs were incubated with mouse spleen leukocytes, the number of cells that adhered to ICAM-1 -/- SMCs was significantly lower than the number that adhered to ICAM-1 +/+ SMCs, which was markedly blocked through pretreatment of leukocytes with the anti-MAC-1 antibody. Taken together, our findings demonstrate that ICAM-1 is critical in the development of venous bypass graft arteriosclerosis, which provides essential information for therapeutic intervention for vein graft disease in patients undergoing bypass surgery.

Invasion of human mucosal epithelial cells by Neisseria gonorrhoeae upregulates expression of intercellular adhesion molecule 1 (ICAM-1).

Infection of the mucosa by Neisseria gonorrhoeae involves adherence to and invasion of epithelial cells. Little is known, however, about the expression by mucosal epithelial cells of molecules that mediate cellular interactions between epithelial cells and neutrophils at the site of gonococcal infection. The aim of this study was to determine the expression of intercellular adhesion molecule 1 (ICAM-1) by epithelial cells during the process of gonococcal invasion. The highly invasive strain FA1090 and the poorly invasive strain MS11 were incubated with human endometrial adenocarcinoma (HEC-1-B) or human cervical carcinoma (ME-180) epithelial cells, after which ICAM-1 expression was measured by flow cytometry. After 15 h of infection with FA1090, expression of ICAM-1 increased 4.7- and 2.1-fold for HEC-1-B and ME-180 cells, respectively, whereas 15 h of infection of HEC-1-B cells with MS11 increased ICAM-1 expression only 1.6-fold. ICAM-1 expression was restricted to the cell surface, since no soluble ICAM-1 was detected. The distribution of staining was heterogeneous and mimicked that seen after treatment of HEC-1-B cells with the ICAM-1 agonist tumor necrosis factor alpha (TNF-alpha) in the absence of bacteria. PCR and dot blot analyses of ICAM-1 mRNA showed no change in levels over time in response to infection. Although TNF-alpha was produced by HEC-1-B cells after infection, the extent of ICAM-1 upregulation was not affected by neutralizing anti-TNF-alpha antiserum. Dual-fluorescence flow cytometry showed that the cells with the highest levels of ICAM-1 expression were cells with associated gonococci. We conclude that epithelial cells upregulate the expression of ICAM-1 in response to infection with invasive gonococci. On the mucosa, upregulation of ICAM-1 by infected epithelial cells may function to maintain neutrophils at the site of infection, thereby reducing further invasion of the mucosa by gonococci.

The participation of proliferative keratinocytes in the preimmune response to sensitizing agents.

The aims of this study were to investigate whether keratinocytes are capable of playing a direct preimmune role in the pathophysiology of allergic contact dermatitis (ACD) and to examine to what extent the degree of differentiation might influence this. We measured the ability of sensitizing agents to up-regulate intercellular adhesion molecule 1 (ICAM-1) expression in cultured normal human keratinocytes (NHK) and in the transformed human keratinocyte HaCaT cell line. In proliferative HaCaT cells, following a 24 h exposure, nickel compounds, para-phenylenediamine (pPD) and 1-chloro-2,4-dinitrobenzene produced a concentration-dependent up-regulation of ICAM-1 expression without reducing cell viability, while K2Cr2O7 led to ICAM-1 up-regulation at cytotoxic concentrations, and CrCl3 was without effect. In NHK, NiSO4 and pPD induced ICAM-1 expression to a significantly greater extent in proliferative cells than in differentiated cells, where involucrin expression was measured to assess the differentiation state. NiSO4- or pPD-pretreatment of proliferative HaCaT cells enhanced T-cell binding, which was abolished by neutralizing antibodies to ICAM-1 or CD18. Our investigations concerning the involvement of oxidative stress in the induction of ICAM-1 expression in response to sensitizing agents were inconclusive. The oxidizing agents FeCl3 and H2O2 up-regulated ICAM-1 expression in HaCaT cells but there was no clear relationship between the ability of agents to induce ICAM-1 expression and their ability to alter the levels of reduced glutathione. Although pPD increased interleukin-1 alpha release from NHK, this cytokine was not capable of inducing ICAM-1 expression in NHK. Tumour necrosis factor-alpha, which does induce ICAM-1 expression in NHK, was not detected in response to pPD, arguing against an autocrine pathway of ICAM-1 induction in response to pPD. In summary, we report the direct interaction of sensitizing agents with keratinocytes leading to the generation of immune signals, particularly by proliferative keratinocytes, suggesting an active role for the proliferative keratinocyte in the pathophysiology of ACD.

Portal hypertension enhances endotoxin-induced intercellular adhesion molecule 1 up-regulation in the rat

BACKGROUND & AIMS: Liver disease or portosystemic shunting enhances th e sensitivity to endotoxin. The aim of this study was to investigate whether intercellular adhesion molecule 1 (ICAM-1) expression in response to endotoxin may be dysregulated in an animal model of portal hypertension. METHODS: Portal hypertension was induced by partial portal vein ligation. Sham-operated animals served as controls. ICAM-1 expression was measured using radiolabeled antibodies under baseline conditions or 5 hours after treatment with either endotoxin or recombinant tumor necrosis factor (TNF). Immunoreactive plasma TNF was also measured. RESULTS: Under baseline conditions, ICAM-1 expression in all organs studied was similar in portal-hypertensive and sham-operated rats. ICAM-1 up-regulation after a high dose of endotoxin (5 mg/kg) was similar in both groups of animals. However, portal-hypertensive animals showed a significantly higher ICAM-1 expression in response to low doses of endotoxin (0.1-10 microgram/kg). The response to a low (but not a high) dose of recombinant TNF was also significantly enhanced in portal-hypertensive animals. In addition, portal-hypertensive rats had higher plasma TNF levels after treatment with endotoxin or recombinant TNF. CONCLUSIONS: Portal hypertension induces an exaggerated ICAM-1 up- regulation in response to endotoxin, which is related to an increased production and decreased clearance of the cytokine. (Gastroenterology 1996 Mar;110(3):866-74)

Cytokine-Induced ICAM-1 Expression in Human Keratinocytes Is Highly Variable in Keratinocyte Strains from Different Donors

Induction of intercellular adhesion molecule 1 (ICAM-1) expression in the epidermis is felt to be an important initiator of leukocyte/keratinocyte interactions in many inflammatory skin diseases. The purpose of this project was to determine the individual variability of cytokine-induced ICAM-1 expression in human keratinocytes obtained from different donors. In 55 different keratinocyte strains, there was significant individual variability in ICAM-1 expression by either tumor necrosis factor alpha (TNF-alpha) or interferon-gamma. There was no correlation (r = 0.266, p = 0.06) in response of the same strain to either TNF-alpha or interferon-gamma. Multiple (n = 22) keratinocyte strains showed no significant induction of ICAM-1 expression to IL-1. The level of ICAM-1 expression in response to TNF-alpha and ultraviolet radiation (UVR) in individual strains was highly correlated in three different comparisons: level of stimulated response versus baseline (TNF-alpha and UVR both p < 0.0001); stimulation index TNF-alpha versus UVR (p = 0.00947); and variability of stimulated response versus variability of baseline (TNF p < 0.001; UVR p = 0.002). UVR-induced release of TNF from keratinocytes also showed variability among different keratinocyte strains. The UVR-induced ICAM-1 response in human keratinocytes and transformed epithelial cell was variably blocked with anti-TNF antibodies. The release of TNF from keratinocytes by UVR and the individually variable but linked characteristics of UVR and TNF-alpha stimulated ICAM-1 expression support the hypothesis that TNF-alpha is a major mediator of UVR-induced ICAM-1 expression.

Failure to detect type 1 interferon production in human umbilical cord vein endothelial cells after viral exposure.

Cytokine production by endothelial cells is known to occur after the reception of signals such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). In the present study, we demonstrate cytokine release and upregulation of adhesion molecule expression of human endothelial cells derived from umbilical cord veins in response to stimulants. The cells were exposed to the plant lectin phytohemagglutinin A (PHA), the viral inducers Newcastle disease virus (NDV) and Sendai virus, and the nucleic acid analog polyinosinic/polycytidylic acid (polyI:C). All stimulants induced expression of IL-1 beta and IL-6. The titers of these cytokines were comparable to those obtained in human leukocytes, whereas no TNF-alpha release was detectable. A further feature of activation was upregulation of intercellular adhesion molecule 1 (ICAM-1), which was analyzed after contact with these stimulants. These experiments revealed an increased expression of ICAM-1 in response to all stimulants tested. A major part of our studies was devoted to the ability of endothelial cells to produce interferons as an important function of a number of cell types in response to viral infections. To ensure stimulation of the cells, the inducers NDV, Sendai virus, and polyI:C were analyzed. Expression of interferon (IFN) was not detected, although viral inducers mediated the release of IL-1 beta and IL-6, suggesting that endothelial cells are quite responsive to these stimulants. In conclusion, these findings show that the ability of endothelial cells to secrete cytokines is not restricted to mediators of the immune system, such as IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.

We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.

Regulation of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human vascular smooth muscle cells.

Vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin are inducible proteins involved in cell-cell adhesion. Immunohistochemical studies have indicated that human atherosclerotic plaques contain smooth muscle cells (SMCs) that express ICAM-1 and VCAM-1. Recently, we demonstrated that SMCs in culture express a functionally active cytokine-inducible ICAM-1. SMCs and mononuclear cells participate in the local accumulation of cytokines and related growth factors in atherosclerotic lesions. Therefore, we determined the effects of different cytokines and growth factors on mRNA content and cell surface expression of VCAM-1, ICAM-1, and E-selectin in cultured human aortic SMCs by Northern blotting, quantitative polymerase chain reaction amplification, and immunofluorescence flow cytometry. Under basal conditions of cultivation, both VCAM-1 mRNA and membrane expression of VCAM-1 were low and were induced very little by interleukin-1 beta (100 U/mL). Platelet-derived growth factor or transforming growth factor-beta decreased VCAM-1 mRNA basal expression. Treatment of SMCs with tumor necrosis factor-alpha (TNF-alpha) led to an increase in both VCAM-1 mRNA and cell surface expression for VCAM-1 in a dose- and time-dependent manner. Interferon-gamma induced a weak increase in VCAM-1 mRNA expression, with no synergistic effect on the stimulation by TNF-alpha. Various differences were noted between the expression of ICAM-1 and VCAM-1 genes, because interleukin-1 beta induced substantial amounts of ICAM-1 but not VCAM-1. The addition of interferon-gamma delays the time at which peak expression of ICAM-1 in response to TNF-alpha stimulation occurs. Under our conditions, we did not detect any expression of E-selectin by SMCs. These results suggest that cytokines regulate VCAM-1 and ICAM-1 expression on arterial SMCs and could play an important role in the pathophysiology of inflammatory and immune processes in atherosclerosis.

In vitro cytokine modulation of intercellular adhesion molecule-1 expression on systemic sclerosis dermal fibroblasts.

Our objective was to study the regulation of intercellular adhesion molecule-1 (ICAM-1) expression by cytokines on cultured fibroblasts obtained from systemic sclerosis and normal skin. ICAM-1 expression on dermal fibroblasts obtained from diffuse systemic sclerosis patients with early disease (< or = 2 years) and normal dermal fibroblasts incubated with and without cytokines was measured by radioimmunoassay and flow cytometry. Systemic sclerosis dermal fibroblasts expressed lower basal levels of ICAM-1 than did normal dermal fibroblasts. Both the normal and systemic sclerosis dermal fibroblasts increased their cell surface expression of ICAM-1 in response to interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in a dose-dependent fashion. Systemic sclerosis dermal fibroblasts appeared to be hyperresponsive to IL-1 beta, TNF-alpha, and IFN-gamma. ICAM-1 expression in response to cytokine stimulation increased to a greater degree on systemic sclerosis compared to normal dermal fibroblasts. The enhanced ICAM-1 expression may play a role in the retention of leukocytes involved in systemic sclerosis skin lesions.