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Background: Little is known about the molecular biology of endothelial cells from different venous vascular beds. As a result, our treatment of deep vein thrombosis and pulmonary artery embolism remain identical. As an initial step in understanding venous thromboembolic disease in the trauma and surgical patients, this study sought to investigate the balance between coagulation and fibrinolysis in the pulmonary and deep venous vascular beds and how trauma might influence this balance., Materials and Methods: Confluent human iliac vein endothelial cells (HIVECs) and human pulmonary artery endothelial cells (HPAECs), were cultured in the absence or presence of tumor necrosis factor (TNFα; 10 ng/mL) for 24 h. The expression of mediators of coagulation and fibrinolysis were determined by Western blot analysis, and plasminogen activator activity was determined by a fibrin clot degradation assay., Results: After TNFα stimulation, there was decreased expression of endothelial protein C receptor and thrombomodulin in both HIVECs and HPAECs. TNFα stimulation increased urokinase plasminogen activator expression in both HIVECs and HPAECs. There was an increase in the expression of tissue plasminogen activator and plasminogen activator inhibitor-1 in response to TNFα in HPAECs, but not in HIVECs. There was significantly greater clot degradation in the presence of both the conditioned media and cell extracts from HIVECs, when compared with HPAECs., Conclusions: HPAECs and HIVECs react differently in terms of fibrinolytic potential when challenged with a cytokine associated with inflammation. These findings suggest that endothelial cells from distinct venous vascular beds may differentially regulate the fibrinolytic pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Ischemia-reperfusion (IR) causes various damages in renal tissues, which is exacerbated by hypoxia-induced excessive inflammation and deteriorates the prognosis of patients after kidney surgery. Celastrol is a potent inflammation inhibitor that has little toxicity. In this report, we investigated whether celastrol protects against IR-induced renal injury in rats., Materials and Methods: Renal IR injury was induced by occlusion of the bilateral renal pedicles for 45 min followed by reperfusion for 6 h. Celastrol or vehicle solution was intraperitoneally injected 30 min before renal ischemia, respectively. Renal histology, function, and pro-inflammatory cytokines and mediators were assessed. The effect of celastrol on nuclear translocation of nuclear factor kappa B (NF-κB) was also measured., Results: Celastrol significantly suppressed elevation of the renal function markers and the lipid peroxidation level, alleviated renal tubular damage, and decreased the levels of tumor necrosis factor-α, interleukin-1β, and monocyte chemotactic protein-1 (MCP-1) messenger RNA in kidney caused by IR. Moreover, celastrol prevented IR-induced expression of pro-inflammatory mediators, which was associated with suppression of nuclear translocation of NF-κB subunit p65., Conclusions: Celastrol ameliorated the acute kidney injury caused by IR, which was associated with inhibiting local NF-κB activation and inflammation. Our findings suggest that celastrol could be useful for preventing IR-induced renal injury., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Hepatic ischemia-reperfusion (I/R) injury is a serious clinical complication that may compromise liver function because of extensive hepatocyte loss. Therefore, the development of novel and effective therapies for hepatic I/R is critical for the improvement of patient outcome. It has been previously shown that administration of milk fat globule-EGF factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, exerts significant beneficial effects under acute inflammatory conditions through multiple physiological processes associated with tissue remodeling., Methods: To determine whether administration of recombinant human (rh) MFG-E8 attenuates liver injury in an animal model of hepatic I/R, male adult rats were subjected to 70% hepatic ischemia for 90 min, followed by reperfusion. At the beginning of reperfusion, rats were treated intravenously with normal saline (vehicle) or rhMFG-E8 (160 μg/kg) over a period of 30 min. MFG-E8 levels and various measurements were assessed 4 h after reperfusion. In addition, survival study was conducted in MFG-E8(-/-) and rhMFG-E8-treated wild-type (WT) mice using a total hepatic ischemia model., Results: Liver and plasma MFG-E8 protein levels were significantly decreased after hepatic I/R. Administration of rhMFG-E8 significantly improved liver injury, suppressed apoptosis, attenuated inflammation and oxidative stress, and downregulated NF-κB pathway. We also noticed that rhMFG-E8 treatment restored the downregulated PPAR-γ expression after hepatic I/R. MFG-E8(-/-) mice showed deterioration on survival and, in contrast, rhMFG-E8-treated WT mice showed a significant improvement of survival compared with vehicle-treated WT mice., Conclusions: MFG-E8-mediated multiple physiological events may represent an effective therapeutic option in tissue injury following an episode of hepatic I/R., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Current blood banking practices allow fresh frozen plasma (FFP) to be thawed and then stored for 5 d between 1 and 6 °C. We hypothesized that aged plasma (d 5 FFP) would be pro-inflammatory to the endothelium compared with fresh plasma (d 0 FFP)., Materials and Methods: Human pulmonary endothelial cells (PECs) were treated with (1) media, (2) media + lipopolysaccharide (LPS), (3) lactated Ringer's (LR), (4) LR + LPS, (5) d 0 FFP, (6) d 0 FFP + LPS, (7) d 5 FFP, and (8) d 5 FFP + LPS. After a 24 h incubation, the PECs were stained with antibodies for I-CAM, V-CAM, P-selectin, and E-selectin. The cells were subsequently analyzed by flow cytometry., Results: In both PEC groups treated with FFP and stimulated with LPS, I-CAM, V-CAM, P-selectin, and E-selectin were significantly up-regulated compared with LR when stimulated by LPS., Conclusion: FFP at both ages significantly increased expression of four different adhesion molecules compared with LR in PECs. This may represent a possible mechanism for increased leukocyte binding on the endothelium as a result of FFP transfusion., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Traumatic brain injury (TBI) can induce a persistent inflammatory response, histopathological changes, and apoptosis in the gut. Progesterone given after TBI has been shown to reduce the cerebral inflammation and neuronal apoptosis in the brain. However, the effects of progesterone on the inflammatory response, structure alterations, and apoptosis in the intestinal mucosa following TBI has not been investigated., Materials and Methods: Right parietal cortical contusion in male rats was made by using the weight-dropping method. Rats were given 0 or 16 mg/kg injections of progesterone at postinjury at 1 and 6 hours and on days 1, 2, 3, 4, and 5. Gut samples were extracted at 5 days after trauma. We measured the concentrations of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay; intercellular adhesion molecule-1 expression by immunohistochemistry; intestinal mucosal morphological changes by histopathological study and electron microscopy; and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining., Results: Administration of progesterone following TBI could decrease the intestinal concentrations of IL-1beta and tumor necrosis factor-alpha, but not IL-6. The level of intercellular adhesion molecule-1 expression in the gut was down-regulated by progesterone. TBI-induced damages of gut structure and apoptosis were attenuated after progesterone injections., Conclusions: The results of the present study suggest that post-TBI progesterone administration could suppress the intestinal inflammation, protect the intestinal mucosal structure, and reduce the mucosa apoptosis.

Hypertension is an important risk factor associated with development and progression of diabetic retinopathy (DR). The mechanisms by which hypertension increases the risk for DR are poorly understood. As the inflammatory mechanisms play a pivotal role in the pathogenesis of DR, in the present study, we investigated the effects of diabetes, hypertension, and combination of diabetes and hypertension on early inflammatory phenomena in the retina, and the effects of blood pressure control on retinal inflammation. Four-week-old spontaneously hypertensive rats (SHR) and their normotensive counterpart Wistar Kyoto (WKY) rats were rendered diabetic by intravenous injection of streptozotocin. Diabetic SHR rats were randomized to receive no antihypertensive drug (Sd), an antihypertensive drug that acts on renin-angiotensin system (losartan, Sd+Los), or antihypertensive drug that do not affect renin-angiotensin system (triple therapy, Sd+Tri). After 20 days, rats were sacrificed and the retinas were collected. The number of immunohistochemically detected ED1/microglial positive cells and the expression of ICAM-1 in the retina were significantly higher in diabetic SHR than in control SHR (p=0.003). The NF-kappaB p65 levels were higher in SHR compared with WKY groups (p=0.001) and its increment in diabetic SHR was not significant. These abnormalities in diabetic SHR rats were completely prevented by both types of antihypertensive drugs. The concomitance of diabetes and hypertension leads to exuberant inflammatory response in the retina, and the prevention of hypertension abrogates these abnormalities. It is suggested that the inflammatory events may be involved in the mechanism by which hypertension exacerbates retinopathy in patients with diabetes.

Background: Acute lung injury and pulmonary inflammatory responses are important complications most frequently encountered in severely burned patients. Polymorphonuclear leukocyte (PMN) sequestration and the subsequent generation of oxidants and inflammatory mediators play the key roles in the pathogenesis of acute lung injury. In this study, we used CO-releasing molecules (CORM-2) to determine whether the CO-releasing molecules-liberated CO could attenuate leukocytes sequestration and the inflammatory response in the lung of thermally injured mice., Materials and Methods: Fifty-four mice were assigned to three groups in three respective experiments. In each experiment, mice in sham group (n=6) underwent sham thermal injury, whereas mice in the burn group (n=6) received 15% total body surface area (TBSA) full-thickness thermal injury and mice in CORM-2 group (n=6) underwent the same thermal injury with immediate administration of CORM-2 (8 mg/kg, i.v.). PMN accumulation (MPO assay) in mice lungs and tumor necrosis factor-alpha and interleukin-1beta in BAL fluid, pulmonary edema formation, and wet/dry weight ratios of lung were determined. Activation of NF-kappaB and expression level of ICAM-1 in the lung was assessed. In in vitro experiment, PMN adhesion to experimental mice serum-stimulated mouse lung endothelial cells (MLEC) was assessed., Results: Treatment of thermally injured mice with CORM-2 attenuated PMN accumulation and prevented activation of NF-kappaB in the lung. This was accompanied by a decrease of the expression of ICAM-1. In parallel, PMN adhesion to MLEC stimulated by CORM-2-treated thermally injured mice serum was markedly decreased. Also, CORM-2 markedly decreased the production of inflammatory mediators in BAL fluid without suppressing the permeability of pulmonary microcirculation., Conclusions: CORM-released CO attenuates the inflammatory response in the lung of thermally injured mice by decreasing leukocyte sequestration and interfering with NF-kappaB activation, protein expression of ICAM-1, and therefore, suppressing endothelial cells' pro-adhesive phenotype.

Glucosamine is an amine-containing sugar that exhibits immunosuppressive effects in vitro and in vivo, although its mechanism of action is unknown. We investigated whether glucosamine sulfate (GS) modulates the proinflammatory cytokine interleukin (IL)-1beta-induced expression and production of intercellular adhesion molecule (ICAM)-1, the mechanism responsible for this effect, and whether GS inhibits leukocyte adhesion to the monolayer of retinal pigment epithelial (RPE) cells stimulated with various cytokines. We used flow cytometry and an ARPE-19 cell model to determine the effect of GS on the production of ICAM-1 in response to IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha plus IL-1beta, TNF-alpha plus IL-6, and TNF-alpha plus interferon (IFN)-gamma. We also used semiquantitative RT-PCR to determine the effect of GS on IL-1beta-induced expression of the ICAM-1 gene, and immunocytochemistry and western blotting to measure the effect of GS on the activation and nuclear translocation of the nuclear factor NF-kappaB and the degradation of cytoplasmic IkappaB. The functionality of GS-modulated ICAM-1 on leukocyte adhesion was demonstrated in an RPE cell-neutrophil adherence assay. IL-1beta increased the expression of ICAM-1 at the mRNA and protein levels in ARPE-19 cells. GS downregulated the production of ICAM-1 induced by IL-1beta, IL-6, TNF-alpha, and IFN-gamma at the protein level in a dose-dependent manner. GS also inhibited the nuclear translocation of NF-kappaB subunit p65 and partially prevented the degradation of cytoplasmic IkappaB in IL-1beta-stimulated ARPE-19 cells. GS significantly decreased the number of neutrophils adhering to the RPE monolayer in response to cytokines IL-1beta, IL-6, TNF-alpha, and IFN-gamma. GS inhibits the expression of the ICAM-1 gene in ARPE-19 cells stimulated with IL-1beta by blocking NF-kappaB subunit p65 translocation and by partially preventing IkappaB degradation. GS also decreases leukocyte adhesion to the monolayer of ARPE-19 cells stimulated with various cytokines by decreasing ICAM-1 production. Our study demonstrates a potentially important property of GS in reducing ICAM-1-mediated inflammatory mechanisms in the eye.

To study if the endogenous renin-angiotensin system affects diabetic retinal leukostasis, rats with streptozotocin-induced diabetes were treated with an ACE inhibitor (ramipril), an angiotensin II AT(1) receptor antagonist (losartan) and the Ca channel blocker, (nifedipine). In the diabetic rats, these drug treatments reduced systolic blood pressure by approximately 16 mmHg but did not change blood glucose. After 2 weeks, the rats were examined for retinal leukostasis in vivo with a scanning laser ophthalmoscope (SLO). Retinal leukostasis, which was defined as no movement of arrested leukocytes over 2 min, was markedly higher in diabetic rats than normal controls (P<0.01). Leukostasis was significantly decreased by ramipril and losartan (P<0.01 vs. untreated diabetic rats) but was still higher than normal. Retinal leukostasis after nifedipine treatment was not significantly different than in untreated diabetic rats. The same trend was observed when leukostasis was analyzed on retinal flat mounts with concanavalin A and CD45 immunofluorescence; ramipril and losartan treatment, however, decreased leukostasis to values no different than controls. Retinal leukostasis was lowered by nifedipine (P<0.05, untreated diabetes vs. nifedipine-treated) but was still higher than in normal, ramipril-, or losartan-treated rats. Assays of gene expression of retinal intercellular adhesion molecule (ICAM-1) by semi-quantitative RT-PCR indicated that ICAM-1 mRNA was increased in diabetic rats but was decreased markedly by treatment with losartan or ramipril, and modestly by nifedipine. In summary, suppressing the activity of the endogenous renin-angiotensin system markedly decreases, perhaps even normalizes, the retinal leukostasis that accompanies type I diabetes in rats. These effects seem to be partly independent of blood pressure and to be associated with a decrease in ICAM-1 gene expression. Angiotensin II may, thus, mediate retinal leukostasis in early diabetes.

Objective: To study the antigen-presenting cells and co-stimulatory factors (HLA class 2 antigen and adhesion molecule) in different grades of vulval intraepithelial neoplasia (VIN)., Material and Methods: Forty-five histology specimens were obtained from 21 women who had previously undergone vulval biopsies for VIN and included 12 specimens of VIN I, 5 of VIN II and 28 of VIN III. The CD1a (Langerhans cell/antigen-presenting cell marker) and co-stimulatory factors--HLA Class 2 antigens (LN3) and the adhesion molecule (CD54)--were semi-quantitatively analyzed in all the specimens. Pearson Chi-squared test was used for statistical analysis., Results: CD1a was increased in 11/12 (91.6%) biopsies with VIN I, in 3/5 (60%) of VIN II and in 4/28(14.3%) of VIN III. There was thus an inverse correlation between CD1a and severity of VIN (Pearson Chi-squared = 26.876, P = 0.001). Qualitatively, there was a basal location of CD1a-positive cells in normal epithelium but had a haphazard distribution in both low grade and high grade VIN. There was no statistical significance in the distribution of LN3 and CD54 in different grades of VIN., Conclusions: This study shows an alteration in the numbers and spatial arrangement of CD1a-positive Langerhans/antigen-presenting cells in different grades of VIN. There is an increase in the number of cells with CD1a expression in low grade VIN and a decrease in the number of these cells in high grade VIN. Reduction in CD1a expression may reflect the inability of the host to mount an adequate immune response due to reduced antigen presentation in high grade VIN.

Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes which transfer functional MHC-peptide complexes to other DCs. Since immature and mature DCs induce different functional T cell responses (i.e., tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that immature and mature murine DCs secrete morphologically similar exosomes. Extensive proteomic analysis of the two exosome populations showed identical overall protein composition, and provided an exhaustive image of the protein composition of DC-derived exosomes. By quantitative analysis, however, exosomes from mature DCs proved enriched in MHC class II, B7.2, ICAM-1, and depleted in MFG-E8, as compared to immature exosomes. In functional T cell stimulation assays, exosomes secreted by mature DCs were 50- to 100-fold more potent than exosomes from immature DCs, both in vitro and in vivo. MHC class II and ICAM-1 were necessary for the increased immune activity of exosomes secreted by mature DCs. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.

Early detection of synovitis in rheumatoid arthritis (RA) and distinction from osteoarthritis (OA) are important to establish the most appropriate treatment. Increased perfusion over affected joints observed by laser Doppler perfusion imaging was supposed to arise from the underlying joint, because it was detected only by a near-infrared laser and not by a less penetrating red laser source. Using laser Doppler anemometry, this study addressed two questions: (1) whether capillary blood cell velocity (CBV) is increased in the skin over finger joints affected by RA or OA; (2) whether there is a difference between RA and OA in CBV above affected proximal interphalangeal (PIP) joints. Levels of soluble adhesion molecules were measured, because they indicate rheumatoid vasculitis raising flow resistance. Thirty-one patients with RA and 20 with OA were investigated. Compared to 18 controls, CBV (mean+/-SEM) was elevated above PIP joints clinically affected by RA (0.35+/-0.06 mm/s vs. 0.21+/-0.02 mm/s; P<0.05) and over PIP (0.27+/-0.02 mm/s vs. 0.21+/-0.02 mm/s; P<0.05) and distal interphalangeal joints (0.27+/-0.02 mm/s vs. 0.17+/-0.01 mm/s; P<0.001) affected by OA. Levels of soluble adhesion molecules were not correlated with CBV over PIP joints in RA. These observations demonstrated that elevated blood cell velocity is detectable by laser Doppler anemometry in skin capillaries over interphalangeal joints affected by RA or OA and contradict the previous assumption that there is hyperemia only in the affected joint. The lack of a significant difference in CBV over PIP joints between RA and OA patients might be due to some inflammation also occurring in OA rather than to vasculitic processes in RA associated with elevated levels of soluble adhesion molecules.

Purpose: We previously reported that immune-based inflammation occurs on the ocular surface of humans as well as canines with keratoconjunctivitis sicca (KCS). Intercellular adhesion molecule-1 (ICAM-1) was found to be upregulated on lymphocytes and/or vascular endothelial cells resulting in lymphocytic diapedesis to the lacrimal and conjunctival tissues. The purpose of the current study was to demonstrate the role of ICAM-1 in (1) resident epithelial cell response during ocular inflammation, (2) local and/or peripheral lymphocyte activation or accumulation in the ocular tissues, and (3) whether anti-ICAM-1 is effective to attenuate immune-mediated ocular inflammation., Methods: ICAM-1 levels in various ocular tissues of human with KCS and/or MRL/lpr mouse were evaluated by immunohistochemistry and in situ hybridization for protein and messenger RNA (mRNA) expression, respectively. Soluble ICAM-1 concentrations in MRL/lpr mouse plasma over the course of disease development were measured by ELISA. Cell proliferation within ocular tissues was assessed by bromodeoxyuridine (BrdU) incorporation and immunohistochemical detection. The level of T cell activation was determined by IL-2 receptor (CD25, a marker of T cell activation and proliferation) and CD69 (a marker of T cell activation) immunoreactivity using FACS analysis. To examine the effectiveness of anti-ICAM-1/LFA-1 in elimination of lacrimal gland inflammation, MRL/lpr mice were injected intraperitoneally (i.p.) with or without monoclonal antibodies against ICAM-1 and LFA-1 at three or eight weeks of age., Results: Increased endogenous ICAM-1 expression at the level of protein and mRNA was detected in the epithelial cells present in the conjunctival and accessory lacrimal tissues in dry eye patients. In MRL/lpr mice, ICAM-1 expression by lacrimal acinar epithelial cells and conjunctival epithelial cells were detected in addition to inflammatory infiltrates and vascular endothelial cells at 16 weeks of age. Soluble ICAM-1 levels were markedly increased concomitantly with disease progression over time as compared with the controls. No significant lymphocytic proliferation (a lack of BrdU and CD25 immunoreactivities) was detected within lacrimal glands of MRL/lpr mice at the disease onset. However, a population of the infiltrated T cells were CD69 positive, indicating the activation stage of a T cell subset. Treatment using monoclonal antibodies against murine ICAM-1 and LFA-1 resulted in a decrease in the number of inflammatory infiltrates in MRL/lpr mice., Conclusions: Our findings suggest that ICAM-1 upregulation locally and systemically promote lymphocyte activation and migration to the ocular surface (OS). Ocular resident epithelium is an active component of ocular surface and is capable of interacting with invasive lymphocytes by ICAM-1 production in response to immune activation and inflammation. ICAM-1 synthesized by epithelial cells may serve as a signaling molecule for predisposition of ocular surface inflammation and facilitate potential antigen presentation by epithelial cells. Lymphocytic infiltrates in the lacrimal gland of the MRL/lpr mouse appeared to be the result of the accumulation, but not proliferation of circulating lymphocytes diapodesed from the vasculature that had migrated into the local ocular tissues. The potential use of anti-ICAM-1 therapy in treating immune-based inflammatory diseases such as dry eye deserves further investigation.

Background: Pentoxifylline (PTX) attenuates end-organ injury in models of sepsis and hemorrhage. PTX is thought to act by inhibiting phosphodiesterase, thus increasing cAMP and decreasing tumor necrosis factor-alpha (TNF-alpha) synthesis. The effects of PTX on neutrophil and endothelial cell adhesion molecules and, ultimately, organ injury in a chronic endotoxemia model have not been studied. We hypothesized that continuous infusion of PTX reduces acute lung injury (ALI) caused by chronic lipopolysaccharide (LPS) exposure., Materials and Methods: Male Sprague-Dawley rats were given continuous infusion of LPS, PTX + LPS combined, or saline (sham) by implantable pumps. Neutrophil CD11b expression, lung histopathology, lung intercellular adhesion molecule-1 (ICAM-1) expression assessed by immune staining, serum TNF-alpha, serum interleukin-6 (IL-6), and bronchoalveolar lavage (BAL) IL-8 were evaluated at different time points. Lung injury was graded in a blinded fashion from 0 (normal) to 4 (severe) for interstitial inflammation, neutrophil infiltration, congestion, and edema. Total lung injury score (TLIS) was calculated by adding listed categories. White cell count in the peripheral blood and in the BAL was also performed., Results: Animals treated with PTX + LPS showed a significant reduction in lung injury score, a marked decrease in ICAM-1 expression, and a significant decrease in IL-8 levels in the BAL and serum IL-6 levels when compared with LPS-treated animals., Conclusions: Continuous infusion of PTX reduces ALI caused by chronic endotoxemia. The effect seems to be a result of decreased expression of endothelial and epithelial ICAM-1 and modulation of proinflammatory cytokine synthesis.

Alopecia areata is an autoimmune condition directed at hair follicles, which results in loss of hair. We have previously demonstrated that it is possible to transfer hair loss, along with the immunohistologic findings of alopecia areata, to human scalp grafts on Prkdc(scid) (SCID) mice by injection of autologous activated lesional T-cells. This study examines the cytokine profile of T-cells and follicular epithelium following transfer of hair loss. Two consistent findings significantly (P < 0.01) associated with hair loss were production of interferon-gamma-inducible protein-10 kDa (IP-10) by follicular epithelium (13/13), and production of INF-gamma by infiltrating T-cells (10/12). Noninjected control grafts regrew hair, and were generally negative for IP-10 (positive 2/9), and INF-gamma (positive 2/9), but expressed of IL-10 on the follicular epithelium (7/9). These data support an INF-gamma TH1 pathogenesis for hair loss in alopecia areata.

Monocytes and macrophages play a central role in viral infections, as a target for viruses and in activation of both innate and adaptive immune responses. Epstein-Barr virus (EBV) has evolved elaborate strategies to dampen the immune system and to persist within the host. There is evidence that the product of the BCRF-1 open reading frame of EBV, viral interleukin-10 (vIL-10), inhibits the capacity of monocytes/macrophages to induce T cell activation, but the full mechanism of this effect is unknown. To determine whether this effect might involve modulation of the expression of accessory molecules known to be important in T cell activation, we analyzed by flow cytometry the influence of vIL-10 on the basal as well as on IFN-gamma-induced up-regulation of HLA molecules, ICAM-1, and two members of the B7 family B7.1 (CD80) and B7.2 (CD86) at the surface of human monocytes. Viral IL-10 in a concentration-dependent manner inhibited both basal- and IFN-gamma-induced HLA-class II, ICAM-1 (basal levels of ICAM-2 and ICAM-3 is unaffected), CD80, and CD86 up-regulation when added simultaneously with IFN-gamma. In contrast, complete inhibition of HLA-class I expression on monocytes/macrophages occurred only when vIL-10 was present 2 h prior to the addition of IFN-gamma, implying that vIL-10 affects an early step in the IFN-gamma signaling pathway. As both monocytes and macrophages can be infected by EBV, we propose that vIL-10-mediated impairment of monocyte/macrophage antigen-presenting function could help the virus-infected cells to avoid detection by the host's T cells on one hand and contribute to its immunosuppressive properties on the other., (Copyright 2002 Elsevier Science (USA))

The importance of activated CD8 cells expressing IL-2R in small bowel and other organ rejection has been reported. Some authors even consider that a positive correlation might be demonstrated between the number of apoptotic enterocytes and the degree of graft rejection. In addition, moderate to intense activation of endothelial molecules in small bowel allograft in rats has been reported in chronic rejection. The aim of the present paper is to ascertain, in a heterotopic small bowel transplantation (HSBT) in rats, whether CD3, CD4, CD8, and CD54 cell expression in the allograft infiltrates shows some relationship with allograft enterocyte apoptosis when rejection is present. Wistar Furth male rats were allotted to two groups: group A was the control group without transplantation; group B received a heterotopic small bowel allograft from Fisher rats and an im dose of FK506 (0.25 mg/kg/day). A significant increase of CD8, CD54 cell receptor expression, and apoptosis in the group undergoing HSBT showed rejection. No significant differences have been observed in the variables under study between the control and HSBT without rejection groups or in CD3 and CD4 among the three groups. We observed a significant correlation between apoptosis and rejection, between CD8 and CD54 with apoptosis and with rejection, and between CD8 and CD54. This indicates that the activation of endothelial molecules and cells may play an important role in established HSBT chronic rejection. We consider that this study may contribute to the knowledge of small bowel allograft chronic rejection and its immunomodulation.

Hepatic expression of the proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) occurs in many acute and chronic liver diseases, as well as following exposure to hepatotoxic chemicals, and is believed to help influence both the damage and repair processes that occur following these insults by regulating additional mediators. We examined the role of TNFalpha in transgenic mice deficient in TNF receptors (TNFR) utilizing carbon tetrachloride (CCl(4)) as a model hepatotoxic agent that allowed for the evaluation of necrosis, inflammation, and fibrosis. Hepatocyte damage, as evident by local areas of liver necrosis and elevated levels of serum transaminase, occurred to a similar degree in wild-type and TNFR-deficient knockout (KO) mice following acute exposure to CCl(4). In contrast, the inflammatory response, manifested as an inflammatory cell influx, as well as induction of chemokines and adhesion molecules that occurred in wild-type mice following treatment with CCl(4), was not as evident in TNFR-KO mice. This response was associated primarily with type-1 (TNFR1) rather than type-2 (TNFR2) receptor responses. Liver fibrosis resulting from chronic CCl(4) exposure was also markedly dependent upon TNFalpha as demonstrated by almost a complete histological absence of fibrosis in TNFR-deficient mice. This was further supported by marked reductions in procollagen and transforming growth factor beta synthesis in TNFR-deficient mice. Taken together, these results indicate that TNFalpha is responsible for regulating products that induce inflammation and fibrosis but not direct hepatocyte damage in CCl(4)-induced hepatotoxicity.

Background: Prolonged cold ischemia has been shown to be an important factor in the development of posttransplant renal dysfunction. The exact mechanisms have not been completely defined. The expression of intercellular adhesion molecule-1 (ICAM-1) (CD 54) in rat kidneys stored in University of Wisconsin (UW) solution was studied in an attempt to correlate ischemia time with immunogenicity of the graft., Methods: Kidneys from male Lewis rats were perfused with UW solution, removed, and bathed in UW solution at 4 degrees C for 4, 12, 24, and 48 h. For the evaluation of expression of ICAM-1, immunohistochemical staining, Western blotting, and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were performed., Results: Immunohistochemical staining in normal, nonischemic kidneys revealed that glomerular capillaries expressed ICAM-1 but that tubular cells did not. The preserved kidneys were analyzed by immunohistochemistry, Western blotting, and semiquantitative RT-PCR and showed increased transcription and expression of ICAM-1 in the cortex of the kidney. Expression reached a maximum at 24 h and declined at 48 h. The ICAM-1 protein expression in the preserved kidney cortex relative to control kidneys was increased at 4 h (1.68 +/- 0.60-fold of control kidneys, P = 0.06), 12 h (2.38 +/- 0.90-fold, P = 0.02), 24 h (3.70 +/- 1.29-fold, P = 0.01), and 48 h (2.00 +/- 0.54-fold, P = 0.01). The messenger RNA expression (the ratio of ICAM-1 to glyceraldehyde-3-phosphate dehydrogenase) in preserved kidneys cortex relative to control kidneys was increased at 4 h (1.19 +/- 0.14-fold of control kidneys), 12 h (1.38 +/- 0.16-fold), 24 h (1.77 +/- 0.29-fold), and 48 h (1.19 +/- 0.12-fold) (P < 0.05 for all time points)., Conclusions: We conclude that cold preservation of rat kidneys in UW solution induces increasing levels of ICAM-1 cell surface expression and gene transcription. Further study is necessary to determine if this increase in adhesion molecule expression increases the immunogenicity of the allograft and contributes to the development of posttransplant renal dysfunction., (Copyright 2001 Academic Press.)

To understand the lack of protective immunity observed after infection with parainfluenza virus type 3 (PIV3), we tested the effect of the virus on human monocytes and monocyte-derived immature dendritic cells (DCs). Expression of viral antigens on the cell surfaces correlated with replication of the virus, which was marginal in monocytes but extremely efficient in DCs. The virus increased monocyte survival at least in part through the production of granulocyte-macrophage colony-stimulating factor but, in contrast, accelerated DC apoptosis. In addition, PIV3 infection failed to activate monocytes but induced maturation of DCs with increased expression of CD54, HLA-DR, CD86, and CD83 and production of bioactive IL-12. However, PIV3-infected DCs demonstrated low stimulatory properties in DC-T cell cocultures, a finding that could not be attributed to the production of infectious virus or IL-10. These results demonstrate for the first time that PIV3 dramatically modifies the survival and/or the function of antigen-presenting cells and might therefore prevent the development of efficient antiviral immune responses., (Copyright 2001 Academic Press.)

Leukocyte endothelial adhesion (LEA) is the prelude to a complex cascade of reactions following an immunological challenge. Recently, LEA has been implicated in the molecular basis of several dermatological disorders. While the role of proinflammatory cytokines, such as interleukin-1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), in LEA has been investigated using nondermal models, limited data exist regarding their effects on LEA in dermal models. This study shows that cotreatment of cultured human dermal endothelial cells (CADMEC) with IL-1beta and TNF-alpha resulted in a marked increase in the adherence of human promyelocytic leukemia (HL-60) cells to CADMEC and an increase in expression of intercellular adhesion molecule-1 and E-selectin. Pretreatment of CADMEC with dexamethasone, a long-lasting glucocorticoid, resulted in a decrease in both HL-60 cell adhesion to CADMEC and adhesion molecule expression. Taken together, these data demonstrate that LEA may play a role in inflammatory skin conditions and in the mechanisms underlying the potential use of glucocorticoids as a treatment option., (Copyright 2001 Academic Press.)

Primary T cell-mediated immune responses are highly susceptible to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, yet direct effects of TCDD on T cells have been difficult to demonstrate. Since the activation of naive T cells has been shown to be initiated primarily by dendritic cells (DC), these cells represent a potential target for TCDD immunotoxicity. In this report, we have examined the influence of TCDD exposure on splenic DC phenotype and function in the absence of antigenic stimulation. Results showed that DC from TCDD-treated mice expressed higher levels of several accessory molecules including ICAM-1, CD24, B7-2, and CD40, whereas the expression of LFA-1 was significantly reduced. These effects were dose-dependent and persisted for at least 14 days after exposure. The effects were also dependent upon the aryl hydrocarbon receptor (AhR), as similar effects were observed in AhR+/+ C57Bl/6 and Balb/c mice but not in AhR-/- mice. When DC from TCDD-treated mice were cultured with allogeneic T cells, the proliferative response and production of IL-2 and IFN-gamma by the T cells were increased. Production of IL-12 by the DC was likewise enhanced in comparison to cells from vehicle-treated mice. Interestingly, however, the number of DC recovered from TCDD-treated mice was significantly decreased. Taken together, these results suggest that, in the absence of antigen, TCDD provides an activation stimulus to DC that may lead to their premature deletion. Since the survival of DC has been shown to influence the strength and duration of the immune response, these results suggest a possible novel mechanism for TCDD-induced immune suppression., (Copyright 2001 Academic Press.)

Background: Myocardial injury during early reperfusion (R) has been well documented. However, the extent and time course of myocardial injury during late R are still unclear. The purpose of this study was to determine the extent of regional contractile and endothelial dysfunction and myocardial blood flow (MBF) defect as well as extension of infarction in association with neutrophil (PMN) actions during R., Materials and Methods: A total of 29 dogs underwent a protocol of 1 h LAD ischemia followed by 6, 24, 48, and 72 h of R, respectively. Regional contractile function (sonomicrometry), MBF (colored microspheres), infarct size (triphenyltetrazolium chloride staining), and PMN localization (immunohistochemistry) were determined., Results: Percentage segmental shortening at 6, 24, 48, and 72 h of R was significantly blunted (-1.8 +/- 1.2,* - 0.37 +/- 0. 6,* 0.04 +/- 0.2,* and 5.9 +/- 1.2* vs baseline 17.7 +/- 0.8). MBF (ml/min/g) was attenuated at 24 (0.27 +/- 0.03*), 48 (0.46 +/- 0. 07*), and 72 h of R (0.48 +/- 0.06*) vs 6 h of R (0.65 +/- 0.06). Infarct size increased from 6 (27 +/- 2%) to 24 h of R (41 +/- 2%*) with no further increase at 48 and 72 h of R, consistent with a peak of creatine kinase activity. PMN adherence (mm(2) endothelium) to left anterior descending coronary artery (LAD) segments was increased after 6 h of R (63 +/- 3*) vs nonischemic left circumflex coronary artery (LCX) segments (42 +/- 2) with a peak at 48 h of R (111 +/- 5*). Endothelium-dependent vascular relaxation in the LAD was also blunted at 6, 24, and 48 h of R. Immunostaining revealed CD18-positive PMNs were mainly accumulated in intravascular space during 6 h of R with an increase in migration of PMNs seen at 24 h of R, consistent with a peak of myeloperoxidase release. Myeloperoxidase activity in a given area at risk sample was significantly correlated with infarct extension during the first 24 h of R., Conclusions: These results provide pathologic evidence for myocardial injury during the extended R and a basis for exploration of interventions designed to limit myocardial injury after ischemia. (*P < 0.05 vs Baseline, 6 h of R and LCX segments.), (Copyright 2000 Academic Press.)

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species., (Copyright 1999 Academic Press.)

Background: Endothelial cells (EC) are an attractive target for somatic cell gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. Recent evidence, however, suggests that viral transduction may induce unfavorable changes in EC phenotype. We examined the proliferative capacity and cell adhesion molecule (CAM) profile of EC after retroviral gene transfer (GT), employing a clinically relevant ex vivo GT protocol., Methods: Human umbilical vein EC (HUVEC, N = 14 isolates) were exposed to supernatants containing the MFG.nlsLACZ vector, which codes for a nuclear localized beta-galactosidase. Control HUVEC were exposed to empty virus (CRIP) or no virus (NT). Efficiency of GT was quantitated by direct counting of beta-galactosidase-stained cells on a grid. Proliferation was quantitated by a 1-week assay of viable cell counts. Expression of EC activation molecules (Class II major histocompatibility antigen [MHC II], E-selectin, intercellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) was examined using fluorescent cytometry (FACS) at rest and after cytokine stimulation., Results: GT was reproducibly efficient (mean 57%, range 40-77%) using sequential viral exposures without selection. NT, CRIP, and LACZ-transduced HUVEC exhibited identical FACS profiles for E-selectin, ICAM-1, VCAM-1, and MHC II at rest, consistent with a nonactivated state. Upregulation of expression by cytokine was quantitatively similar for all groups. Growth rates were likewise not different between groups., Conclusions: Retroviral vectors may be employed to achieve high percentages of transduced EC for ex vivo GT without the use of selection. Transduced EC generated in this fashion are not activated, demonstrate an unaltered pattern of inducible CAM expression, and exhibit normal cell growth. The effects of GT on target cell phenotype are likely to be both vector and protocol specific and should be carefully assessed in each case prior to in vivo applications., (Copyright 1998 Academic Press.)

Host responses to migrating schistosomula of Schistosoma mansoni were compared in the skin of naive, multiply infected, or vaccinated (with gamma-irradiated cercariae) mice during the first 72 hr after cercarial penetration. Cellular response to the migrating parasite was minimal in the skin of naive mice for up to 72 hr after infection. In sharp contrast, the multiply infected or vaccinated animals exhibited a marked inflammatory response in the skin as early as 8 hr after cutaneous penetration of the challenge cercariae. This early inflammatory response in the skin of sensitized animals was characterized by a significant increase in the number of infiltrating cells, predominantly mononuclear cells and neutrophils. Increased exudation of serum proteins was also present in the skin of sensitized animals in areas of cercarial challenge. A time course of analyses revealed that mononuclear cell numbers increased significantly in the skin of vaccinated animals as early as 60 min after a challenge infection and continued to be present at a significantly higher level up to 72 hr after challenge. Peak neutrophil responses occurred in the skin at 24 hr (in multiply infected animals) and at 48 hr (in vaccinated animals) after a challenge infection. Along with the massive cellular infiltration there was an increased tissue expression of ICAM-1 and mRNA for iNOS in the skin of sensitized animals. Further analysis showed that in sensitized animals increased ICAM-1 expression was predominantly found on endothelial cells lining dermal capillaries, especially in areas around schistosomular migration and on cells that surrounded schistosomula in the dermis. In naive animals, however, a similar infection did not induce any ICAM-1 expression or iNOS production in the skin. Thus, an ICAM-1 mediated early accumulation of mononuclear cells in the skin and local production of nitric oxide may be important for the initial cutaneous inflammatory immune responses to migrating schistosomula of S. mansoni in vaccinated animals. On the contrary, in naive animals a potential parasite-induced suppression of ICAM-1 may play an important role in reducing cellular reaction in the skin and consequently help the parasite evade immune responses in the skin.

The infiltration of pancreatic islets by mononuclear cells is the hallmark of the development of insulin dependent diabetes mellitus (IDDM) in the NOD mouse, an animal model for human IDDM. The aim, of this study was to correlate adhesion molecule expression with the degree of islet infiltration and to compare Th1- and Th2-driven islet inflammation. Cryostat sections of NOD mouse pancreata before and after diabetes development were analysed by semiquantitative immunohistochemistry. NOD mouse islets did not show the expression of ICAM-1, LFA-1, L-selectin and VCAM-1 prior to infiltration by mononuclear cells. Furthermore, islets with early stage insulitis (grade 1, periinsular location of small infiltrates) still were devoid of adhesion molecule expression. ICAM-1 and LFA-1 were first demonstrable in islets with strong periinsular infiltrates (insulitis grade 2) while L-selectin and VCAM-1 were only seen in islets with mild or strong intraislet infiltration (grade 3-4). Adhesion molecules were demonstrable in areas of macrophage and T-lymphocyte infiltrates but not in adjacent endocrine islet tissue. Islets of all infiltration stages contained Th2 lymphocytes (positive for IL-4). Substantial numbers of Th1 cells (positive for IFN-gamma, TNF-alpha, IL-2 and/or IL-2 receptor) were observed only after acceleration of diabetes development by a single injection of cyclophosphamide (250 mg/kg i.p.). Interestingly, the adhesion molecule expression pattern in islets with "Th1' versus "Th2 insulitis' was not different. In conclusion, the expression of adhesion molecules in islets during the development of autoimmune diabetes does not precede mononuclear infiltration but probably occurs in response to the activation of initial small infiltrates. ICAM-1 and LFA-1 expression is seen prior to L-selectin and VCAM-1. However, adhesion molecule expression during Th1 versus Th2 cell infiltration is very similar, suggesting similar adhesion molecule requirements of the two Th subsets.

Experimental autoimmune pigment epithelial protein-induced uveitis (EAPU) is a new type of disease that destroys the retinal pigment epithelium (RPE), and exhibits a hitherto unknown form of progressive chorioretinal dystrophy in which neuroretinal inflammatory foci are absent. The present study was aimed at studying the expression of adhesion molecules, and the kinetics of the appearance of the main types of macrophages and other intraocular immunocompetent cell populations in the various stages of this disease. EAPU was evoked in Lewis rats by immunization with the membrane protein from bovine RPE cells containing PEP-65 as main constituent. In the uvea, increased expression of intercellular adhesion molecule-1, of class II major histocompatibility complex antigen, and of ED2 macrophage reactivity were observed closely before the onset of EAPU. Expression of these reactivities was also slightly elevated by injections of the applied adjuvants alone. The onset of EAPU was mainly characterized by initial uveal infiltrations of ED1+ macrophages and a minor population of CD4 T cells, and an increase in ED3, ED7 and perivascular ED2 reactive macrophages. This was followed by the development of focal accumulations of ED1+ cells at both sides of the Bruch's membrane-RPE layer (Dálen-Fuchs nodules) which was permeated and disintegrated at these sites. The outer choroidal layer, the anterior iridal surface, and the base of the ciliary body more frequently contained active inflammatory cells than the other uveal areas. Lymphoid cells were found scattered through the uvea, aqueous and vitreous. The sites of increased activity of ED2+ and ED3+ cells in the uvea were rather similar to those of ED1 macrophages in the various stages of EAPU. Starting from multiple foci, the process of the formation of plaque-shaped cell accumulations in severe EAPU progressed along the RPE and exhibited a chronic character. The results of this study show that ED1+, ED2+, ED3+ and ED7+ subpopulations of macrophages are actively involved in an immunopathological process in which the RPE is the target. The thickening of the plaque-shaped cell accumulations stops if the integrity of all RPE cells at that site has been affected. We postulate that this is the result of antigen elimination while additional influence of the abrogation of RPE cytokine production is presumed.

We semiquantitatively monitored the incorporation of host membrane proteins on different strains of human immunodeficiency virus type 1 (HIV-1) grown in several human CD4+ lymphoid cell lines and in primary mitogen-stimulated peripheral blood mononuclear cells. The relative amounts of virally acquired cell proteins were estimated by the ability of HIV-1 to be captured by magnetic beads coated with monoclonal antibodies. Here we report that, among host surface proteins studied, HLA-DR molecules were the most abundant virion-bound host molecules. We have also found that, in contrast to previous studies, HLA-DP and -DQ isotypes were also present on virus progeny. More importantly, we determined that the relative levels of virally acquired host HLA-DR proteins, as estimated by capture with immunomagnetic beads, greatly differed depending on the virus strain and the producer cell. These observations extend beyond already published results and suggest that the process of incorporation of cellular molecules on newly released virus particles is a phenomenon that relies on both the virus strain and producer cell line. These in vitro observations are of prime importance considering that virus-acquired host molecules have been recently shown to affect the biology of HIV.

Human ovarian carcinoma cell lines were genetically engineered to secrete the cytokine interleukin-4 (IL-4) by retroviral-mediated gene transduction. These cells were transduced with the LXSN retroviral vector containing the human IL-4 gene and the neomycin resistance selection marker. Numerous IL-4-secreting clones were isolated from different papillary serous carcinoma cell lines, including SKOV-3, UCI-101, and UCI-107, and one clone derived from UCI-107 extensively characterized. This clone, termed UCI 107E IL-4 GS, was shown to constitutively express high levels of IL-4 (i.e., 900 to 1300 pg/ml/10(5) cells/48 hr) for over 35 passages and 6 months of study. Like the parental cell line (UCI-107), UCI 107E IL-4 GS cells expressed MHC class I and Her-2/neu surface antigens but did not express detectable MHC class II, ICAM 1, CA 125, or IL-4 receptors. No increase in expression of surface proteins was noted between parental and UCI 107E IL-4 GS. The morphology of this clone did not differ from that of the parental or LXSN vector control cells; however, parental cells had a faster growth rates than transductants. UCI 107E IL-4 GS was sensitive to gamma irradiation since as little as 2500 rad killed most of the cells within 10 days of irradiation. However, after irradiation, IL-4 secretion continued until about Day 8. The potential use of these IL-4-secreting ovarian carcinoma cells as vaccines for woman with advanced ovarian cancer will be discussed.

IGF-I, a mitogenic polypeptide hormone, and glutamine (GLN), the preferred enterocyte fuel, singularly improve growth and structure of the small bowel isograft; however, their combined effects on intestinal allografts are unknown. This study examined the effects of IGF-I and GLN, singularly and in combination, on the structure and function of the intestinal allograft. Fifty-nine adult rats underwent resection of the distal 60% of small bowel and received either a 40-cm isograft or an allograft. Either IGF-I (2.4 mg/kg/day) or its vehicle was infused continuously by subcutaneous minipumps. An isocaloric polymeric diet with either 2% GLN or isonitrogenously balanced 2% nonessential amino acids was given continuously by gastrostomy for 10 days. Five groups were studied: isograft (ISO) alone, allograft (ALLO) alone, ALLO and GLN, ALLO and IGF-I, and ALLO and IGF-I with GLN. All recipients received Cyclosporine A (15 mg/kg, im) daily. Mucosal villus height, surface area, crypt depth, IgA, IgG, IgM, and intercellular adhesion molecule-1 (ICAM-1) plasma cells in intestinal tissue, glucose and water absorption of intestinal graft, bacterial translocation (BT) to mesenteric lymph nodes, and body weight were determined. IGF-I increased villus height, surface area (P < 0.001), crypt depth (P < 0.01), and glucose absorption (P < 0.05) compared to the ISO and ALLO groups. GLN increased only crypt depth when compared to the ALLO group (P < 0.01). Both IGF-I and GLN independently decreased BT to MLN (P < 0.05) and, in combination, enhanced water absorption (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Reperfusion injury involves the adhesion and activation of neutrophils (PMN) both in affected tissues and distant organs. Cell adhesion molecules (CAM) such as endothelial-leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1) are known to mediate, at least in part, the adherence of activated PMN to the endothelium. To characterize the cellular mechanisms of this phenomenon, we exposed cultured human umbilical vein endothelial cells (HU-VEC) to hypoxia and reoxygenation (H/R) using an incubator chamber purged of oxygen with 100% nitrogen. Confluent monolayers of HUVEC were subjected to 60 min of hypoxia followed by variable periods of reoxygenation (120 and 240 min). Flow cytometry was utilized to assess the expression of ELAM-1 and ICAM-1, expressed as percent shift from baseline expression. To determine what role endothelium-derived cytokines such as IL-1 play in the expression of CAM after H/R, we performed additional experiments in the presence of recombinant IL-1 receptor antagonist (IL-1RA). ICAM-1 was present on unstimulated HUVEC while ELAM-1 was not constitutively expressed. Following exposure of cells to hypoxia and reoxygenation, significant increases in ELAM expression were seen (8.4 +/- 2.4% at 120 min; 19.1 +/- 7.4%, P < 0.05). While there was similar trend in ICAM expression, this did not achieve statistical significance (0.10 < P < 0.05). The addition of IL-1RA (10 ng/ml) to hypoxic HUVEC consistently attenuated ELAM-1 upregulation during reoxygenation (0.8 +/- 0.7% at 120 min and 5.9 +/- 4.1% at 240 min) such that expression was not significantly greater than baseline.(ABSTRACT TRUNCATED AT 250 WORDS)