Induction Of Intercellular Adhesion Molecule-1 Research Articles

Botulinum Toxin: A Novel Adjuvant for Giant Cell Tumor

To the Editor Giant cell tumor (GCT) is a locally aggressive, benign bone lesion that shows extensive bone destruction that often leads to pain and pathologic fracture. It usually occurs in the epiphysis and metaphysis of bone near the joint. The destruction of subchondral bone impairs joint function. In rare instances, the tumor can spread to the lungs or can present as multifocal disease. On histologic examination, the tumor shows a mixture of cells consisting of osteoclastlike multinucleated giant cells, mononuclear cells, and stromal cells. This unique composition of different cell types has been the subject of the study of osteoclast biology. The preferred treatment options include surgical treatment such as curettage, in conjunction with chemical or physical adjuvant therapy, or extensive resection followed by major reconstructive surgery. The outcome after surgical resection has been affected negatively by tumor recurrence and additional bone destruction. Many topical adjuvant therapies such as phenol, hydrogen peroxide, or liquid nitrogen have been used in an attempt to decrease local recurrence by inducing physical or chemical damage to the remaining GCT cells after curettage, but a specific adjuvant therapy that directly targets osteoclasts and GCT cells is needed. In GCT, the mesenchymal stromal cells are the neoplastic cells and induce recruitment and formation of osteoclasts. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic preosteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). RANKL is an osteoclastogenesis factor released by osteoblasts, stromal cells, and activated T cells. RANK is a receptor that is present on the cell membrane of osteoclasts, monocytes, and osteoblasts. Studies have shown the role ofRANKL inGCT as evidenced by the inhibitory effect of osteoprotegerin (a decoy receptor of RANKL) on RANKL-mediated tumoral osteoclastogenesis. Cell adhesion of osteoblasts and osteoclastic precursors of hematopoietic origin is a prerequisite for osteoclast maturation. Several studies have demonstrated that interaction of RANKL on osteoblast and RANK on osteoclast precursor provides an essential signal to osteoclast precursors for their maturation into resorbing cells. b1 integrin/focal adhesion kinase (FAK)-mediated signaling is involved in intercellular adhesion molecule 1 (ICAM-1) and RANKL induction of osteoblasts and affects cellular adhesion between osteoblasts and osteoclast precursors through the ICAM-1 andRANKL/RANKpathways and leads to differentiation of osteoclast progenitors to osteoclasts. Therefore, b1 integrin/FAK-mediated signaling is involved in ICAM-1 and RANKL induction of osteoblasts. Small GTPaseRho signaling is involved in b1 integrin-mediated upregulation of ICAM-1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation. Clostridium botulinum, producing two classes of enzymes that have very specific protein targets, the neurotoxin A-G and the ADP-ribosyltransferases C2, C3 bot 1, and C3 bot 2. C2 and C3 bot are part of a larger family of ADP-ribosylating toxins, including diphtheria toxin and cholera toxin, which cleave Published online December 29, 2007. Address correspondence and reprint requests to: Hamid Namazi; E-mail: namazih@sums.ac.ir

G Protein-Coupled Receptor Ca2+-Linked Mitochondrial Reactive Oxygen Species Are Essential for Endothelial/Leukocyte Adherence

Receptor-mediated signaling is commonly associated with multiple functions, including the production of reactive oxygen species. However, whether mitochondrion-derived superoxide (mROS) contributes directly to physiological signaling is controversial. Here we demonstrate a previously unknown mechanism in which physiologic Ca(2+)-evoked mROS production plays a pivotal role in endothelial cell (EC) activation and leukocyte firm adhesion. G protein-coupled receptor (GPCR) and tyrosine kinase-mediated inositol 1,4,5-trisphosphate-dependent mitochondrial Ca(2+) uptake resulted in NADPH oxidase-independent mROS production. However, GPCR-linked mROS production did not alter mitochondrial function or trigger cell death but rather contributed to activation of NF-kappaB and leukocyte adhesion via the EC induction of intercellular adhesion molecule 1. Dismutation of mROS by manganese superoxide dismutase overexpression and a cell-permeative superoxide dismutase mimetic ablated NF-kappaB transcriptional activity and facilitated leukocyte detachment from the endothelium under simulated circulation following GPCR- but not cytokine-induced activation. These results demonstrate that mROS is the downstream effector molecule that translates receptor-mediated Ca(2+) signals into proinflammatory signaling and leukocyte/EC firm adhesion.

Sustained activation of nuclear factor-κB by reactive oxygen species is involved in the pathogenesis of stress-induced gastric damage in rats

Stress ulceration is a common complication in critically ill patients, but the mechanisms involved are poorly understood. In this study we investigated the temporal activation of the redox-sensitive transcription factor nuclear factor-kappaB and its roles in an experimental model of cold immobilization stress-induced gastric mucosal lesions. Prospective, controlled, and randomized animal study. University research laboratory. Male Sprague-Dawley rats. The rats were subjected to cold immobilization stress for a total of 6 hrs. The temporal profiles of nuclear factor-kappaB activation and expression of tumor necrosis factor-alpha, interleukin-1beta, cytokine-induced neutrophil chemoattractant-1 (CINC-1), intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS) were determined in the gastric corpus mucosa of stressed rats. To study the roles of nuclear factor-kappaB activation, rats received an intravenous bolus of a specific nuclear factor-kappaB inhibitor Bay 11-7082 (20 mg/kg) 1 hr before stress. For antioxidant administration, rats were treated with intravenous injection of a free radical scavenger pyrrolidine dithiocarbamate (50, 100, and 200 mg/kg) 1 hr before stress. Exposure of rats to 6 hrs of stress led to a rapid and persistent activation of nuclear factor-kappaB, which was associated with transient degradation of inhibitory protein IkappaBalpha and slower but sustained degradation of IkappaBbeta. Nuclear factor-kappaB activation preceded the induction of tumor necrosis factor-alpha, interleukin-1beta, CINC-1, ICAM-1, and iNOS messenger RNAs, all of which were linearly increased with the duration of stress. Bay 11-7082 selectively blocked the stress-induced nuclear factor-kappaB activation and up-regulation of tumor necrosis factor-alpha, interleukin-1beta, CINC-1, ICAM-1, and iNOS messenger RNAs. Inhibition of expression of these proinflammatory genes prevented the increases in myeloperoxidase activity (an indicator of neutrophil infiltration) in gastric mucosa and the development of gastric damage. Pyrrolidine dithiocarbamate dose-dependently inhibited the stress-induced nuclear factor-kappaB pathway activation and consequential proinflammatory gene expression, neutrophil infiltration, and gastric damage, suggesting the involvement of reactive oxygen species in these processes. Sustained activation of nuclear factor-kappaB by reactive oxygen species is an important in vivo mechanism mediating stress-induced gastric inflammatory damage in rats.

Carbamylated Low-Density Lipoprotein Induces Monocyte Adhesion to Endothelial Cells Through Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1

Carbamylated low-density lipoprotein (LDL), the most abundant modified LDL isoform in human blood, has been recently implicated in causing the atherosclerosis-prone injuries to endothelial cells in vitro and atherosclerosis in humans. This study was aimed at testing the hypothesis that carbamylated LDL acts via inducing monocyte adhesion to endothelial cells and determining the adhesion molecules responsible for the recruitment of monocytes. Exposure of human coronary artery endothelial cells with carbamylated LDL but not native LDL caused U937 monocyte adhesion and the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 adhesion molecules as measured by cell enzyme-linked immunosorbent assay. Silencing of intercellular adhesion molecule-1 by siRNA or its inhibition using neutralizing antibody resulted in decreased monocyte adhesion to the endothelial cells. Similar silencing or neutralizing of vascular cell adhesion molecule-1 alone did not have an effect but was shown to contribute to intercellular adhesion molecule-1 when tested simultaneously. Taken together, these data provide evidence that intercellular adhesion molecule-1 in cooperation with vascular cell adhesion molecule-1 are essential for monocyte adhesion by carbamylated low-density lipoprotein-activated human vascular endothelial cells in vitro.

Bioactive Pregnanes from Nerium oleander

Three new pregnanes, 21-hydroxypregna-4,6-diene-3,12,20-trione (1), 20R-hydroxypregna-4,6-diene-3,12-dione (2), and 16beta,17beta-epoxy-12beta-hydroxypregna-4,6-diene-3,20-dione (3), were isolated from Nerium oleander, together with two known compounds, 12beta-hydroxypregna-4,6,16-triene-3,20-dione (neridienone A, 4) and 20S,21-dihydroxypregna-4,6-diene-3,12-dione (neridienone B, 5). The structures of compounds 1-3 were established on the basis of their spectroscopic data. The anti-inflammatory activity in vitro of compounds 2-4 was examined on the basis of inhibitory activity against the induction of intercellular adhesion molecule-1 (ICAM-1), and compound 4 was active. The cytotoxic activity of compounds 1-5 was evaluated against four human cell lines, normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), human liver tumor cells (HepG2), and human lung carcinoma cells (A-549). Compound 4 showed significant cell growth inhibition of VA-13 and HepG2 cells. The MDR-reversal activity of compounds 1-5 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 1, 2, and 5 showed significant effects on calcein accumulation.

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.

Fever-range thermal stress promotes lymphocyte trafficking across high endothelial venules via an interleukin 6 trans-signaling mechanism

Fever is an evolutionarily conserved response during acute inflammation, although its physiological benefit is poorly understood. Here we show thermal stress in the range of fever temperatures increased the intravascular display of two 'gatekeeper' homing molecules, intercellular adhesion molecule 1 (ICAM-1) and CCL21 chemokine, exclusively in high endothelial venules (HEVs) that are chief portals for the entry of blood-borne lymphocytes into lymphoid organs. Enhanced endothelial expression of ICAM-1 and CCL21 was linked to increased lymphocyte trafficking across HEVs. A bifurcation in the mechanisms controlling HEV adhesion was demonstrated by evidence that the thermal induction of ICAM-1 but not of CCL21 involved an interleukin 6 trans-signaling pathway. Our findings identify the 'HEV axis' as a thermally sensitive alert system that heightens immune surveillance during inflammation by amplifying lymphocyte trafficking to lymphoid organs.

Signal Transduction Pathways of Inflammatory Gene Expressions and Therapeutic Implications

Intercellular adhesion molecule-1 (ICAM-1), an inducible cell adhesion glycoprotein of the immunoglobulin supergene family and cyclooxygenase-2 (COX-2), an inducible prostaglandin G/H synthase, are overexpressed by proinflammatory mediators in a wide variety of cell types. These stimuli increase ICAM-1 or COX-2 expression primarily through activation of ICAM-1 or COX-2 gene transcription. The architecture of the ICAM-1 or COX-2 promoter is complex, containing a large number of binding site for inducible transcription factors, the most important of which is NF-kB. NF-kB acts in concert with other transcription factors or transcriptional coactivators which facilitate the assembly of distinct stereospecific transcription complexes on the ICAM-1 or COX-2 promoter. These transcription complexes presumably mediate the induction of ICAM-1 or COX-2 expression in different cell types and in response to different stimuli. In this review, I summarize the current understanding of ICAM-1 and COX-2 gene regulation with a particular emphasis on the transcription factors or coactivators, and signal transduction pathways critical for their expression. A PKC-dependent c-Src pathway activating NF-kB or GAS to enhance ICAM-1 or COX-2 gene expression is discussed. Furthermore, natural products and novel agents targeting on the transcription factor with potential anti-inflammation and anti-tumor activity are also discussed.

Inhibition of ICAM-1 expression by garlic component, allicin, in gamma-irradiated human vascular endothelial cells via downregulation of the JNK signaling pathway

Ionizing radiation used in cancer therapy frequently exerts damaging effects on normal tissues and induces a complex response including inflammation. Since the upregulation of adhesion molecules on endothelial cell surface has been known to be associated with inflammation and our previous data showed that irradiation enhanced adhesion molecules expression, interfering with the expression of adhesion molecules may be an important therapeutic target of inflammatory diseases. We examined the effect of allicin, a major component of garlic, on the induction of intercellular adhesion molecule-1 (ICAM-1) by gamma-irradiation (γIR) and the mechanisms of its effect in gamma-irradiated human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated for 20 h with allicin (0.01–1 μg/ml) and then exposed to 8 Gy radiation. Allicin significantly inhibited γIR-induced surface expression of ICAM-1 and ICAM mRNA in a dose-dependent manner. In addition, pretreatment with allicin resulted in the decrease of AP-1 activation and phosphorylation of the c-Jun NH2-terminal kinase (JNK) induced by γIR. These results suggest that allicin downregulates γIR-induced ICAM-1 expression via inhibition of both AP-1 activation and the JNK pathway and may be considered in therapeutic strategies for the management of patients treated with radiation therapy.

Anti-inflammatory effects of 8-hydroxydeoxyguanosine in LPS-induced microglia activation: suppression of STAT3-mediated intercellular adhesion molecule-1 expression

To elucidate the roles of 8-hydroxydeoxyguanosine (oh(8)dG), the nucleoside of 8-hydroxyguanine (oh(8)Gua), we examined the effects of oh(8)dG upon LPS-induced intercellular adhesion molecule-1 (ICAM-1) expression and the underlying mechanisms in brain microglial cells. We found that oh(8)dG reduces LPS-induced reactive oxygen species (ROS) production, STAT3 activation, and ICAM-1 expression. oh(8)dG also suppresses pro-inflammatory cytokines, such as TNF-alpha, IL-6 and IFN-gamma. Overexpression of dominant negative STAT3 completely diminshed STAT3-mediated ICAM-1 transcriptional activity. Chromatin immunoprecipitation studies revealed that oh(8)dG inhibited recruitment of STAT3 to the ICAM-1 promoter, followed by a decrease in ICAM-1 expression. Using mice lacking a functional Toll-like receptor 4 (TLR4), we demonstrated that, while TLR4+/+ microglia were activated by LPS, TLR4-/- microglia exhibited inactivated STAT3 in response to LPS. Evidently, LPS modulates STAT3-dependent ICAM-1 induction through TLR4-mdiated cellular responses. Oh(8)dG apparently plays a role in anti-inflammatory actions via suppression of ICAM-1 gene expression by blockade of the TLR4-STAT3 signal cascade in inflammation-enhanced brain microglia. Therefore, oh(8)dG in the cytosol probably functions as an anti-inflammatory molecule and should be considered as a candidate for development of anti-inflammatory agents.

Taraxasterane- and Ursane-Type Triterpenes from Nerium oleander and Their Biological Activities

Two new taraxasterane-type triterpenes, 20beta,28-epoxy-28alpha-methoxytaraxasteran-3beta-ol (1) and 20beta,28-epoxytaraxaster-21-en-3beta-ol (2), were isolated from an ethyl acetate extract of the leaves of Nerium oleander, together with ursane-type triterpenes, 28-nor-urs-12-ene-3beta,17beta-diol (3) and 3beta-hydroxyurs-12-en-28-aldehyde (4). The structures of 1 and 2 were established on the basis of their spectroscopic data. Anti-inflammatory activity of 1-4 was examined on the basis of inhibitory activity against the induction of intercellular adhesion molecule-1 (ICAM-1). Cytotoxic activity of 1-4 was evaluated against four human cell lines, A-549, WI-38, VA-13, and HepG2 cells.

CML-1 inhibits TNF-α-induced NF-κB activation and adhesion molecule expression in endothelial cells through inhibition of IkBα kinase

CML-1 is a purified extract from a mixture of 13 oriental herbs (Achyranthis Radix, Angelicae Gigantis Radix, Cinnamomi Cortex Spissus, Eucommiae Cortex, Glycyrrhizae Radix, Hoelen, Lycii Fructus, Paeoniae Radix, Rehmanniae Radix Preparata and Atractylodis Rhizoma, Zingiberis Rhizoma, Zizyphi Semen, Acori Graminei Rhizoma) that have been widely used for the treatment of inflammatory diseases in Asia. Since our previous study has been shown to have the anti-inflammatory activity of CML-1 in vivo and the upregulation of adhesion molecules in response to numerous inducing factors is associated with inflammation, this study examined the effect of CML-1 on the expression of adhesion molecules induced by TNF-α in cultured human umbilical vein endothelial cells (HUVECs). Preincubation of HUVECs for 20 h with CML-1 (1–100 μg/ml) dose-dependently inhibited TNF-α (10 ng/ml)-induced adhesion of THP-1 monocytic cells, as well as mRNA and protein expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). CML-1 was also shown to inhibit NK-kB activation induced by TNF-α. Furthermore, CML-1 inhibited TNF-α-induced IkB kinase activation, subsequent degradation of IkBα, and nuclear translocation of NK-kB. Evidence presented in this report demonstrated that CML-1 inhibited the adhesive capacity of HUVEC and the TNF-α-mediated induction of E-selectin, ICAM-1 and VCAM-1 in HUVEC by inhibiting the IkB/NF-kB signaling pathway at the level of IkB kinase, which may explain the ability of CML-1 to suppress inflammation and modulate the immune response.

12/15 Lipoxygenase Mediates Monocyte Adhesion to Aortic Endothelium in Apolipoprotein E–Deficient Mice Through Activation of RhoA and NF-κB

12/15 lipoxygenase (12/15LO) has been implicated as a mediator of inflammation and atherosclerosis. In the current study, we identified mechanisms through which 12/15LO mediates monocyte:endothelial interactions in vivo in apolipoprotein E-deficient mice (apoEKO), a well-characterized mouse model of atherosclerosis. In apoEKO mice that are also deficient in 12/15LO (doubleKO), monocyte adhesion to aorta in vivo was reduced by 95% in doubleKO mice compared with apoEKO mice. Inhibition of 12/15LO in apoEKO mice in vivo using CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate) prevented monocyte adhesion to aortic endothelium in apoEKO mice. Aortic endothelium of apoEKO mice had significant activation of rhoA compared with doubleKO aortic endothelium. Further, apoEKO aorta displayed significant activation of NF-kappaB. DoubleKO aorta displayed little nuclear localization of NF-kappaB. Finally, we found significant upregulation of intercellular adhesion molecule-1 (ICAM-1) on apoEKO aortic endothelium compared with doubleKO endothelium. Inhibition of rhoA and PKCalpha significantly reduced NF-kappaB activation, ICAM-1 induction, and monocyte adhesion to aorta. We conclude that 12/15LO products activate endothelial rhoA and PKCalpha. Activation of rhoA and PKCalpha cause activation and translocation of NF-kappaB to the nucleus, which, in turn, results in induction of ICAM-1. Induction of ICAM-1 on aortic endothelium stimulates monocyte:endothelial adhesion in vivo in apoEKO mice.

The Synthetic Cannabinoid R(+)WIN 55,212-2 Inhibits the Interleukin-1 Signaling Pathway in Human Astrocytes in a Cannabinoid Receptor-independent Manner

R(+)WIN 55,212-2 is a synthetic cannabinoid that controls disease progression in models of multiple sclerosis. This is associated with its ability to reduce migration of leukocytes into the central nervous system. Because leukocyte migration is dependent on induction of adhesion molecules and chemokines by pro-inflammatory cytokines, we examined the effects of R(+)WIN 55,212-2 on their expression. Using 1321N1 astrocytoma and A-172 glioblastoma as cell models we show that R(+)WIN 55,212-2, but not its inactive chiral form S(-)WIN 55,212-2, strongly inhibits the interleukin-1 (IL-1) induction of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the chemokine IL-8. This inhibition is not mediated via the CB1 or CB2 cannabinoid receptors, because their selective antagonists and pertussis toxin failed to affect the inhibitory effects of R(+)WIN 55,212-2. Furthermore reverse transcription-PCR analysis did not detect the expression of either receptor in 1321N1 cells. R(+)WIN 55,212-2 was shown to inhibit adhesion molecule and chemokine expression at the level of transcription, because it strongly inhibited the IL-1 induction of ICAM-1, VCAM-1, and IL-8 mRNAs and blocked the IL-1 activation of their promoters. The NFkappaB pathway was then assessed as a lead target for R(+)WIN 55,212-2. NFkappaB was measured by expression of a transfected NFkappaB-regulated reporter gene. Using this assay, R(+)WIN 55,212-2 strongly inhibited IL-1 activation of NFkappaB. Furthermore R(+)WIN 55,212-2 inhibited the ability of overexpressed Myd88, Tak-1, and IKK-2 to induce the reporter gene suggesting that R(+)WIN 55,212-2 acts at or downstream of IKK-2 in the IL-1 pathway. However R(+)WIN 55,212-2 failed to inhibit IL-1-induced degradation of IkappaBalpha, excluding IKK-2 as a direct target. In addition electrophoretic mobility shift and chromatin immunoprecipitation assays showed that R(+)WIN 55,212-2 does not regulate the IL-1-induced nuclear translocation of NFkappaB or the ability of the latter to bind to promoters regulating expression of ICAM-1 and IL-8. These data suggest that R(+)WIN 55,212-2 blocks IL-1 signaling by inhibiting the transactivation potential of NFkappaB.

Redox Regulation of p38 MAPK Activation and Expression of ICAM-1 and Heme Oxygenase-1 in Human Alveolar Epithelial (A549) Cells

We have explored the potential role of redox events in p38 mitogen-activated protein kinase (MAPK) activation and their relevance to the inducible expression of intercellular adhesion molecule-1 (ICAM-1) and heme oxygenase-1 (HO-1) in A549 cells. Tumor necrosis factor-alpha (TNFalpha) and hydrogen peroxide (H2O2) both activated p38, but only TNFalpha activated nuclear factor-kappaB (NF-kappaB). N-Acetyl-L-cysteine (20 mM) inhibited both H2O2- and TNFalpha-induced p38 phosphorylation (14 +/- 7 and 37 +/- 4% of control, respectively). The mitochondrial complex I and III inhibitors, rotenone and antimycin A, and allopurinol partially inhibited H2O2- but not TNFalpha-induced p38 activation. However, rotenone and antimycin A augmented intracellular oxidative stress measured by dichlorofluorescein fluorescence. TNFalpha, but not H2O2, induced ICAM-1 in A549 cells, which was attenuated by a proteasome inhibitor, but not by the p38 MAPK inhibitor SB203580. In contrast, hemin and hemoglobin, but neither TNFalpha nor H2O2, caused efficient HO-1 expression. However, hemin had no effect on p38 activation and SB203580 did not influence hemin-induced HO-1 protein expression. Collectively, these data suggest that p38 is a cytokine- and oxidative stress-responsive pathway in A549 cells. Whereas NF-kappaB appears crucial in ICAM-1 induction, p38 activation itself is not sufficient to confer HO-1 expression and may not be involved in HO-1 and ICAM-1 induction in A549 cells.

Melanocortins in fibroblast biology – current update and future perspective for dermatology

The skin is a target organ and source for proopiomelanocortin (POMC)-derived peptides, such as alpha-melanocyte-stimulating hormone (alpha-MSH), which acts by binding to melanocortin receptors (MC-Rs). Recent progress in our understanding of the cutaneous POMC system has demonstrated that human dermal fibroblasts (HDFs) are a novel target for alpha-MSH. MC-1R is expressed by HDFs in vitro and in situ. MC-1R expression is also detectable in human connective tissue sheath fibroblasts (CTSFs) and in dermal papilla cells (DPCs) of the hair follicle, the latter concomitantly expressing MC-1R and MC-4R in vitro and in situ. Both HDFs and DPCs are capable of generating POMC-derived peptides, although cell-specific differences exist in the expression of prohormone convertases and the amounts of POMC-derived peptides generated. Functional studies have shown that alpha-MSH exerts anti-inflammatory actions in human fibroblastic skin cells by suppressing interleukin-1 (IL-1)-induced IL-8 production, activation of the transcription factor activator protein-1 (AP-1) and induction of intercellular adhesion molecule-1 by interferon-alpha. In addition, alpha-MSH antagonizes the effect of transforming growth factor-beta1 (TGF-beta1) on collagen synthesis in HDFs in vitro and exerts antifibrogenic activity in a mouse model of cutaneous fibrosis. These findings indicate that fibroblastic cells participate in the cutaneous POMC system in which alpha-MSH appears to act as a modulator of inflammatory and fibrogenic responses. The biological activities of alpha-MSH in fibroblastic cells of the skin point towards novel clues in our understanding of the pathophysiology of fibrotic skin disorders and inflammatory diseases of the hair follicle and, finally, suggest innovative therapeutic options for the treatment of these conditions.

Gene Expression Profiling of Inflamed Human Endothelial Cells and Influence of Activated Protein C

During systemic inflammation, activation of vascular endothelium by proinflammatory cytokines leads to hypotension, microvascular thrombosis, and organ damage. Recent data suggest a link between coagulation and inflammation through the activated protein C (APC) pathway. We studied gene expression profiles in human coronary artery endothelial cells (HCAECs) exposed to proinflammatory stimuli and the influence of APC on expression of candidate genes regulated by these stimuli. HCAECs were stimulated with interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha. In gene expression profiling, 400 of 8400 genes were regulated >2-fold. Verification of selected candidate genes was achieved by measuring expression of mRNA species by real-time polymerase chain reaction, cytokine secretion by ELISA, and metabolites of tetrahydrobiopterin (BH4) biosynthesis by high-performance liquid chromatography. BH4 synthesis, interleukin-6, interleukin-8, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were downregulated by APC at the transcriptional and protein level. Endothelial nitric oxide synthase, endothelial adhesion molecule, and vascular cell adhesion molecule-1 were not affected by APC. Activities of transcription factors c-Fos, FosB, and c-Rel were inhibited by APC in inflamed HCAECs. Our study revealed a novel antiinflammatory mechanism of APC-dependent gene regulation in HCAECs since c-Fos-dependent induction of MCP-1 and ICAM-1 was suppressed. APC downregulates expression and activity of genes related to inflammation, most pronounced under intermediate or mild inflammatory conditions.

TNFα suppresses human colonic circular smooth muscle cell contractility by SP1- and NF-κB-mediated induction of ICAM-1

Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn's disease. We investigated the roles of transcription factors Sp1 and nuclear factor kappa B (NF-kappaB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFalpha. Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. TNFalpha treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-kappaB inhibition before, but not after, 1 hour of TNFalpha-stimulation blocked the expression of ICAM-1. TNFalpha significantly enhanced Sp1/DNA binding. Sp1 bound to the 3' flanking region of a variant kappaB site in the -192/-172 region of ICAM-1 promoter. Mutation of this region abolished the response to TNFalpha. The treatment of HCCSMCs with Sp1 antisense oligonucleotides (ODNs) blocked the expression of ICAM-1, but sense ODNs had no effect. Protein kinase C zeta (PKCzeta) inhibition before or 3 hours after stimulation with TNFalpha also blocked the expression of ICAM-1. TNFalpha treatment of circular muscle strips pretreated with ICAM-1 sense ODNs or control medium significantly reduced their response to acetylcholine, whereas pretreatment with antisense ODNs blocked this effect. The expression of ICAM-1 on HCCSMCs in response to TNFalpha is regulated by transcription factors Sp1 and NF-kappaB binding independently to the -192/-172 region of the ICAM-1 promoter. The expression of ICAM-1 plays a critical role in the suppression of cell contractility in response to TNFalpha.

Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells.

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.

Induction of Intercellular Adhesion Molecule-1 on Human Brain Endothelial Cells by HIV-1 gp120: Role of CD4 and Chemokine Coreceptors

Central nervous system dysfunction is commonly observed in children with HIV-1 infection, but the mechanisms whereby HIV-1 causes encephalopathy are not completely understood. We have previously shown that human brain microvascular endothelial cells (HBMEC) from children are responsive to gp120 derived from X4 HIV-1 by increasing expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule-1. However, the mechanisms involved in gp120-mediated up-regulation of cell adhesion molecule expression is unclear. In the present study, we found that gp120 derived from both X4 and R5 HIV-1 induced increased expression of ICAM-1 on HBMEC, but the degree of this up-regulation differed among the various HBMEC isolates. The up-regulation of ICAM-1 was inhibited by anti-CD4 antibodies as well as by specific antibodies directed against chemokine receptors and small-molecule coreceptor inhibitors. Anti-CD4 antibodies inhibited the increase in ICAM-1 expression mediated by gp120 derived from X4 and R5 HIV-1, whereas antibodies against chemokine receptors displayed a differential inhibition depending on the source of gp120. Both X4 and R5 gp120-induced ICAM-1 expression was sensitive to pertussis toxin and involved the nuclear factor-kB pathway. These findings indicate a direct involvement of CD4 and a differential involvement of chemokine receptors in the activation of pediatric HBMEC by X4 and R5 gp120. The activation of brain endothelium of children by HIV-1 protein gp120 by way of CD4 and chemokine receptors may have implications for the pathogenesis of HIV-1 encephalopathy in the pediatric population.