Increase In Intercellular Adhesion Molecule-1 Research Articles

Infection of human endothelial cells with Staphylococcus aureus induces the production of monocyte chemotactic protein-1 (MCP-1) and monocyte chemotaxis.

Bacterial infection coincides with migration of leucocytes from the circulation into the bacterium-infected tissue. Recently, we have shown that endothelial cells, upon binding and ingestion of Staphylococcus aureus, exhibit proinflammatory properties including procoagulant activity and increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression on the cell surface, resulting in hyperadhesiveness, mainly for monocytes. The enhanced extravasation of monocytes to bacterium-infected sites is facilitated by the local production of chemotactic factors. From another study we concluded that the locally produced chemokine MCP-1 is important in the recruitment of monocytes to the peritoneal cavity in a model of bacterial peritonitis. In the present study we investigated whether cultured human endothelial cells after infection with bacteria produce and release MCP-1, which in turn stimulates monocyte chemotaxis. We observed that endothelial cells released significant amounts of MCP-1 within 48 h after ingestion of S. aureus. This was dependent on the number and the virulence of the bacteria used to infect the endothelial cells. The kinetics as well as the amount of MCP-1 released by S. aureus-infected endothelial cells differed markedly from that released by endothelial cells upon stimulation with IL-1beta. Supernatant from S. aureus-infected or IL-1beta-stimulated cells promoted monocyte chemotaxis which was almost entirely abrogated in the presence of neutralizing anti-MCP-1 antibody, indicating that most of the chemotactic activity was due to the release of MCP-1 into the supernatant. Our findings support the notion that endothelial cells can actively initiate and sustain an inflammatory response after an encounter with pathogenic microorganisms, without the intervention of macrophage-derived proinflammatory cytokines.

Tumor necrosis factor α-induced activation of downstream NF-κB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion: Involvement of PKCα, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-α induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-α-induced ICAM-1 expression. TNF-α produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-α-induced response. 12- O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-α resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-α-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C 2 ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-κB DNA–protein binding and ICAM-1 promoter activity were enhanced by TNF-α and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-κB DNA–protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-α- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCα or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-α and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-α activates PC-PLC to induce activation of PKCα and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-κB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.

Reduction of inflammatory response in composite flap transfer by local stress conditioning-induced heat-shock protein 32

Background. The failure of composite flaps despite anastomotic patency is thought to be mediated by the inflammatory response within the microvasculature, which results from unavoidable surgical trauma and transfer-related ischemia-reperfusion. Evidence suggests that stress conditioning may improve flap survival; however, the molecular mechanisms of protection are far from being clear. Therefore, we analyzed whether stress conditioning-induced heat-shock protein 32 is effective to prevent the inflammatory response in transferred osteomyocutaneous flaps. Methods. In a rat model, leukocyte-endothelial cell interaction and endothelial integrity disruption as early indicators of the inflammatory response were quantitatively analyzed in muscle, subcuticular tissue, and periosteum of microvascularly transferred osteomyocutaneous flaps by using intravital fluorescence microscopy. Twenty-four hours before flap transfer, stress conditioning was induced by local heating of the left hindlimb up to 42.5°C for 30 minutes. In additional animals, stress conditioning-induced activity of heat-shock protein 32 was inhibited by tin protoporphyrin-IX. Unconditioned flaps served as controls. Results. In all tissues analyzed, control flaps showed significant leukocyte adherence in postcapillary venules, increased intercellular adhesion molecule-1 (ICAM-1) expression, and endothelial integrity disruption, but a lack of heat-shock protein 32. In contrast, stress conditioning induced marked heat-shock protein 32 expression, which was associated with a significant reduction (P <.05) of leukocyte adherence, ICAM-1 expression, and endothelial hyperpermeability. The inhibition of heat-shock protein 32 by tin protoporphyrin-IX completely abolished the stress conditioning-induced amelioration of the inflammatory response in all tissues analyzed. Conclusions. Stress conditioning by local heat-shock priming reduces the inflammatory response in osteomyocutaneous flaps. The protective effect is predominantly mediated by the induction of heat-shock protein 32. (Surgery 2001;129:292-301.)

Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: findings from brain and renal microvascular cell culture and patients with active disease

Endothelial injury is believed to be a key initiating event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and clinical assays to rapidly and reliably monitor endothelial damage are not readily available. Using flow cytometry, we measured endothelial microparticles (EMPs) generated from cultured renal and brain microvascular endothelial cells (MVECs) during activation and apoptosis, and evaluated the effect of TTP plasma on them. EMPs were measured using positivity for monoclonal antibodies (mAbs) CD31 and CD51, and their procoagulant activity was assessed using a Russell viper venom assay. Both cell lines generated procoagulant EMPs when cultured with inducers of activation (tumour necrosis factor alpha; TNF-alpha) or apoptosis (mitomycin C). TTP plasma induced a five- to sixfold increase of EMP generation and a two- to threefold increase of procoagulant activity in cultured brain and renal MVECs. TTP plasma induced a threefold and 13-fold increase of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, respectively, on renal MVECs. Procoagulant activity tended to parallel EMP numbers. The effect of TTP plasma on cell viability was similar to that of TNF-alpha, implying that it induced activation rather than apoptosis. Control plasma and idiopathic thrombocytopenic purpura (ITP) plasma had little effect. In the clinical study, EMP assay of blood from acute TTP patients showed levels markedly elevated compared with normal controls, but values returned to normal in remission. In conclusion, TTP plasma activated and induced injury to MVECs in culture, judged by production of EMP and expression of activation markers. Released procoagulant EMP may play a role in the pathogenesis of TTP. Assay of EMP may be a useful marker of disease activity and endothelial injury in TTP and possibly other thrombotic disorders.

Endotoxin-specific NF-kappaB activation in pulmonary epithelial cells harboring adenovirus E1A.

Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-kappaB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-kappaB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c-jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-kappaB induction in these cells.

Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that also induces vascular permeability and macrophage migration. VEGF expression is weak in normal adult brain, but is strongly upregulated in glioma cells and reactive astrocytes, suggesting that chronic overexpression of VEGF in the brain contributes to blood-brain barrier (BBB) breakdown. We examined the effects of chronic VEGF overexposure on the integrity of the BBB using the following approaches: 1) continuous intracerebral infusion of VEGF via miniosmotic pump; and 2) intracerebral injection of an adenoviral vector encoding the VEGF165 gene (AdCMV.VEGF). After 6 days both treatments produced approximately 10-fold breakdown of the BBB (as measured by transport of 14C-aminoisobutyric acid (AIB) from blood into brain) compared with the respective controls (albumin infusion or AdCMV.beta gal virus). BBB disruption in AdCMV.VEGF-treated brains was accompanied by a severe inflammatory response not observed in brains receiving AdCMV.beta gal or VEGF protein infusion, indicating that neither VEGF nor viral particles alone were responsible for the inflammatory response. However, injection of AdCMV.beta gal followed by VEGF infusion to the same site also elicited inflammation. Chronic overexposure of normal brain to VEGF also increased intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class I and II expression. Although VEGF itself is not inflammatory, VEGF may modulate immune responses in the central nervous system (CNS) by opening the BBB, altering the immunoprivileged status of the brain, and allowing contact between normally sequestered CNS antigens and blood-borne immune mediators.

Alteration of cellular adhesion molecule gene expressions in the lungs of newborn mice exposed to hyperoxia. † 1569

Hyperoxic lung injury is a frequent and unfortunate consequence of supraphysiologic oxygen therapy. Relative to adults, newborns of most species are resistant to lung O2 toxicity. However, the development of newborn lung exposed to hyperoxia is profoundly affected with a marked inhibition of normal alveolization. We have previously shown increases in intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule 1(PECAM-1), and P-Selectin in adult mouse lungs exposed to hyperoxia that coincided with a rapid deterioration of respiratory status. In the present study, we will compare the gene expression of ICAM-1, PECAM-1, and P-Selectin in the lungs of newborn mice exposed to 100% O-2 for 4, 7 or 10 days. Age-matched animals exposed to room air will serve as control. Cellular adhesion molecule (CAM) mRNA expression was assessed by in situ hybridization. In control animals, only PECAM-1 mRNA was detected in the endothelium of large arteries with a slight increase at 10 days. ICAM-1 mRNA was not detected before 7 d of hyperoxia, whereas it was abundantly expressed in the alveoli and airway epithelium. A marked increase in PECAM-1 mRNA expression was also seen in endothelial cells, and alveoli after 7 d of hyperoxia. In contrast, only barely detectable P-Selectin mRNA was observed in the endothelial cells of large vessels of the same animals. Both ICAM-1 and PECAM-1 expression appeared maximum at 7 days of hyperoxia. In contrast to the adult mouse, the increase in CAMs expression occurred later and was not associated with lethal lung damage. Moreover, the increase in PECAM-1 mRNA was higher than in the adults, but P-Selectin increase was smaller. We speculate that the induction of some CAMs during hyperoxia could be beneficial by promoting the healing process.

The role of cytokines, adhesion molecules, and chemokines in interleukin-2-induced lymphocytic infiltration in C57BL/6 mice.

IL-2 mediates the regression of certain malignancies, but clinical use is limited because of associated toxicities, including parenchymal lymphocytic infiltration with multiple organ failure. Secondarily induced cytokines are important mediators of IL-2 toxicity and IL-2-induced lymphocyte-endothelial adherence and trafficking. The recently discovered C-C chemokines, RANTES (regulated on activation, normal T expressed and secreted) and macrophage inflammatory protein-1alpha, have also been implicated in lymphocytic migration. We hypothesized that IL-2 alters cytokine, C-C chemokine, and adhesion molecule expression in association with parenchymal lymphocytic infiltration. C57BL/6 mice were injected with 3x10(5) IU of IL-2 or 0.1 ml of 5% dextrose intraperitoneally every 8 h for 6 d, then killed. IL-2 induced massive lymphocytic infiltration in the liver and lung and moderate infiltration in the kidney in association with organ edema and dysfunction. Immunostaining showed increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in association with this organ-specific lymphocytic infiltration. Flow cytometry showed increased expression of the corresponding ligands (lymphocyte function-associated antigen-1 and very late antigen-4) on splenocytes. IL-2 increased TNF-alpha mRNA and protein expression in the liver. Organs infiltrated by lymphocytes had increased TNF-alpha mRNA, whereas RANTES mRNA was increased in all organs, regardless of lymphocytic infiltration. IL-2 toxicity involves organ-specific TNF-alpha and RANTES production with increased ICAM-1 and VCAM-1 expression as potential mechanisms facilitating lymphocytic infiltration and organ dysfunction.

P-selectin is upregulated early in the course of hyperoxic lung injury in mice

While treatment with supplemental oxygen is often essential in patients with lung disease, prolonged therapy may cause lung injury by itself. Although the mechanisms responsible for initiating hyperoxic lung damage almost certainly involve primary oxidative transformations, the possible contributions of inflammation to the tissue injury have been attracting increasing research activity. Increases in intercellular adhesion molecule-1 (ICAM-1) coincide with the inflammation, but in other models of inflammation transient adhesion mediated by members of the Selectin gene family was found to be essential before ICAM-1/β 2 interactions could occur. We, therefore, wondered whether a similar sequence of initial transient adhesion followed by subsequent responses would be observed in hyperoxic lung inflammation. We, therefore, determined the effects of hyperoxia exposure on lung mRNA for P-and E-Selectin in mouse lungs. We found that there was no detectable mRNA for E-Selectin through 72 h of hyperoxia exposure by Northern blotting, but that mRNA for P-Selectin was detectable as early as 48 h after initiation of hyperoxia. To determine the location of P-Selectin upregulation we examined hyperoxia-exposed mouse lungs by in situ hybridization and found that the upregulation of P-Selectin at 48 h was localized to large muscularized vessels, at 72 h expression was detected in some medium size muscularized vessels, and at 96 h abundant expression was observed also on nonmuscularized small vessels. In conclusion, increases in mRNA for P-Selectin early in the course of hyperoxia exposure suggest that P-Selectin expression in hyperoxic lungs increases in parallel with upregulation of ICAM-1, leading to the accumulation of neutrophils in hyperoxic lungs, and that interventions targeting these two adhesion molecules may lead to a diminution in hyperoxic lung inflammation and lung injury.

Interactions of leukocyte integrins with intercellular adhesion molecule 1 in the production of inflammatory vascular injury in vivo. The Shwartzman reaction revisited.

We have investigated the role of leukocyte-endothelial cell interactions in a rabbit model of hemorrhagic vasculitis. Microvascular injury was produced in the skin by intradermal injection of Salmonella typhosa endotoxin followed 20 h later by intravenous zymosan, which activates complement. Hemorrhagic necrosis develops in the "prepared" skin sites which is characterized by microthrombi, neutrophil aggregation, platelet and fibrin deposition, and massive extravasation of erythrocytes. Hemorrhage in these Shwartzman-like lesions was quantitated by 99mTc-labeled autologous erythrocytes. Inhibition of the hemorrhagic response was obtained with mAb reactive with ICAM-1 as well as mAb against the leukocyte CD18 when either was administered intravenously just before intravenous zymosan challenge. This observation suggests that an intravascular event occurring in response to complement activation is required for the development of hemorrhagic vasculitis. We hypothesize that agents which successfully prepare the skin for the Shwartzman response after their intradermal injection do so by promoting increased intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Activation of complement then induces CD11/CD18 expression on circulating leukocytes thus producing an intravascular CD11/CD18-ICAM-1 (leukocyte-endothelium) adhesion event. Inhibition of intravascular leukocyte-leukocyte aggregation with mAb against CD11b (Mac-1) showed partial inhibition of hemorrhage, while mAb against CD11a (LFA-1) showed no inhibitory activity. This type of cytokine-primed, neutrophil-dependent vascular damage may be a model of human vasculitic processes where microvascular damage is produced in the absence of immune-complex deposition.

IFN-gamma enhances endothelial activation induced by tumor necrosis factor but not IL-1.

Previous studies have established that different cytokines induce distinct patterns of activation in cultured endothelial cells (EC). Treatment of EC with either TNF or IL-1 causes transient induction of endothelial leukocyte adhesion molecule-1 (ELAM-1) and a sustained increase in intercellular adhesion molecule-1 (ICAM-1) expression. TNF but not IL-1 also increases class I MHC Ag expression. IFN-gamma, which by itself increases EC class I MHC and ICAM-1 but does not induce ELAM-1 expression, has been found to act synergistically with TNF to increase class I expression. In our study, we have further examined IFN-gamma effects on both TNF and IL-1 beta responses. In contrast to IFN-gamma plus TNF cotreatment, IFN-gamma up-regulation of class I MHC molecules is not augmented by cotreatment with IL-1 beta. IFN-gamma plus TNF cotreatment synergistically increases ICAM-1 expression by 24 h of cotreatment, whereas IFN-gamma plus IL-1 beta cotreated EC show at most additive increases. IFN-gamma increases TNF-induced ELAM-1 expression such that a greater number of EC express ELAM-1 at both 4 and 24 h in the presence of IFN-gamma plus TNF compared to cultures treated with TNF alone, although the maximal level of surface expression on individual cells is not increased. Inasmuch as these times represent pre- and post-peak expression time (6 h), respectively, IFN-gamma appears both to accelerate and to prolong transient ELAM-1 expression. Although similar interactions are seen with IL-1 beta, IFN-gamma has a consistently greater effect on TNF-induced compared to IL-1-induced ELAM-1 expression. We next explored the possible mechanism(s) of the synergy between IFN-gamma and TNF. IFN-gamma and TNF do not cooperatively enhance total protein synthesis. Unexpectedly, IFN-gamma and IL-1 beta combine to depress protein synthesis, which may contribute to the failure of these cytokines to positively interact. IFN-gamma and TNF cooperatively increase ELAM-1 mRNA at 6 h and ICAM-1 mRNA at both 6 and 24 h, whereas IFN-gamma and IL-1 show markedly less cooperative augmentation of these transcripts. We conclude that IFN-gamma enhances TNF-induced EC activation by selectively and synergistically increasing synthesis of specific surface molecules, whereas IL-1-induced EC activation is largely unaffected by IFN-gamma.