Prolonged cold preservation augments vascular injury independent of renal transplant immunogenicity and function

BACKGROUND: While prolonged cold ischemia has detrimental effects on graft survival, the mechanisms remain unclear. We tested whether or not cold preservation enhances intragraft inflammatory responses and vascular injury. METHODS: Rat renal grafts were cold preserved in University of Wisconsin solution for 2, 4, 6, 12, 24, and 48 hours, and then transplanted into syngeneic recipients and harvested after 24 hours. Frozen sections were examined histologically and stained for vascular cellular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), major histocompatibility complex (MHC) class II, tissue factor, leukocyte function associated molecule-1 (LFA-1), very late antigen-4 (VLA-4), as well as for inflammatory cells. RESULTS: Function did not differ between isografts preserved for shorter (2 to 6 hours) or longer times (12 to 24 hours). Neutrophil influx and that of LFA-1-positive cells showed similar increases in all groups. Compared with short preservation groups, the long preserved grafts had more VLA-4-positive ED-1+ monocytic infiltrates adjacent to vessels expressing VCAM-1 (P < or = 0.001). Increased preservation duration had no effect on infiltration with recipient ED-2+ macrophages, MHC class II-positive cells, or dendritic cells. Decreased color intensity and continuity of PECAM-1 staining indicated loss of endothelial integrity in grafts preserved for longer than six hours. Intensity in VCAM-1 staining increased progressively in grafts preserved for more than six hours and was localized predominantly on the endothelium of elastic vessels. Endothelial cells, vascular smooth muscle cells, and monocytes expressed increasingly more tissue factor in grafts preserved for more than six hours, revealing enhanced intragraft procoagulant capacity. Furthermore, grafts with preservation times of more than six hours developed more severe vascular endothelial injury and worse tubular necrosis scores (P < or = 0.001) compared with grafts with shorter preservation times. CONCLUSIONS: Because of the prominent vascular injury, strategies for endothelial protection should be attempted in grafts with long preservation times in clinical renal transplantation.     

FTY720-induced lymphocyte homing modulates post-transplant preservation/reperfusion injury

BACKGROUND: A novel immunomodulator, FTY720, modulates lymphocyte migration to injured tissues via enhanced lymphocyte sequestration to secondary lymphoid organs. We tested whether or not single-dose FTY720 (0.5 mg/kg) pretreatment rescues renal grafts from post-transplant preservation/reperfusion injury. METHODS: Rat renal grafts were cold-preserved in University of Wisconsin (UW) solution for 4 hours and then transplanted into syngeneic or allogeneic recipients that received a single dose of FTY720 24 hours before transplantation. Flow cytometry analysis of peripheral blood and lymph nodes was performed to confirm the biologic effect of FTY720. Grafts were harvested after 24 hours. Renal sections were examined histologically and stained for intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), platelet endothelial cellular adhesion molecule-1 (PECAM-1), major histocompatibility complex (MHC) class II, and inflammatory cells. Interleukin-1 (IL-1) production was determined in renal protein extracts. RESULTS: FTY720 pretreatment significantly increased CD3+ T-cell sequestration to lymph nodes in the face of peripheral lymphopenia. Isografts and allografts from the FTY720-treated groups did not develop increased creatinine (0.55 +/- 0.12 in isografts and 0.62 +/- 0.08 mg/dL in allografts), compared with vehicle controls (2.28 +/- 0.20 in isografts and 2.24 +/- 0.18 mg/dL in allografts). Kidneys from FTY720-treated groups also showed lower acute tubular damage scores. Furthermore, FTY720 decreased neutrophil influx, although circulating neutrophils were unchanged. FTY720 also prevented postischemic IL-1 intragraft production not affecting infiltration with recipient ED-1+ macrophages and MHC class II-positive cells. Expression of ICAM-1, VCAM-1, and PECAM did not differ among groups. CONCLUSION: FTY720 ameliorated morphologic and functional consequences of post-transplant reperfusion injury. Thus, FTY720-induced peripheral T-cell absence may influence intragraft IL-1 production and neutrophil infiltration, despite proadhesive endothelial properties. FTY720 may broaden the utility in renal transplantation as a pretreatment strategy against preservation/reperfusion injury.     

黏附分子在大鼠同种异体肝脏移植免疫反应中的作用

目的探讨黏附分子ICAM-1、P-selectin、E-selectin、L-selectin、PECAM-1和VCAM-1在肝脏移植中的表达及意义。方法用免疫组织化学方法和原位杂交法，检测大鼠肝脏移植后不同时间(1、3、5、7d)、不同模型中ICAM-1、P-selectin、L-selectin、E-selectin蛋白、VCAM-1 mRNA、PECAM-1 mRNA的表达情况。结果肝急性排斥组与自发耐受组相比，黏附分子ICAM-1、VCAM-1、PECAM-1、E-selectin表达高；子代组与肝急性排斥组各指标表达类似，而ICAM-1的表达高于肝急性排斥组；半肝组与自发耐受组各指标表达类似，但ICAM-1、VCAM-1表达水平较自发耐受组高。P-selectin、L-selectin表达变化不明显。而正常大鼠肝脏未见黏附分子的表达。结论肝排斥反应可能与黏附分子ICAM-1、VCAM-1、PECAM-1、E-selectin的高表达有关。     

Sequential analysis of adhesion molecules and their ligands in rat renal allografts during the development of chronic rejection

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are important in endothelial cell-leukocyte interactions. In this sequential study, the expression of ICAM-1 and VCAM-1 and their ligands LFA-1 and VLA-4 as well as major histocompatibility complex class II antigens (MHC class II), and interleukin-2-receptor (IL-2R) were investigated during the development of chronic renal allograft rejection in a rat model. The time-related expression of adhesion molecules and their ligands in the graft was correlated to the chronic allograft damage index (CADI). In association with an initial short immune activation, there was a significant ICAM-1 and VCAM-1 induction in the vascular endothelium and the tubular epithelium. In the interstitium, there was infiltration of lymphocytes expressing ligand molecules VLA-4 and LFA-1, as well as activation markers MHC class II and IL-2R. Thereafter, the expression declined together with the increase of CADI-values. In end-stage chronic rejection, there was practically no expression of ICAM-1 and VCAM-1. In the interstitium, there were only few ligand-expressing leukocytes. In conclusion, adhesion molecules and their ligands are involved in the induction phase of the process but no longer in the later stages of chronic rejection. Received: 12 July 1999 Revised: 7 December 1999 Accepted: 27 April 2000     

凋亡相关蛋白及粘附分子在大鼠心脏急性排斥反应中的作用

目的 探讨凋亡及凋亡相关因子Fas／FasL、Bcl-2/Bax，粘附分子ICAM-1、P-selectin、E-selectin、L-Selectin、PECAM-1和VCAM-1在心脏移植中的表达及意义。方法 用免疫组织化学方法和原位杂交法，检测大鼠心脏移植后不同时间(1、3、5、7d)、Fas／FasL、ICAM-1、P-selectin、L-selectin、E-selectin蛋白、Bcl-2/Bax、VCAM-1、PECAM-1 mRNA的表达情况。结果 随着移植后天数的增加，细胞凋亡增多，Fas／FasL、Bax表达增加，粘附分子ICAM-1、VCAM-1、PECAM-1表达也增加，Selectin表达较少。正常大鼠肝脏未见细胞凋亡及粘附分子的表达。结论 细胞凋亡和粘附分子ICAM-1、VCAM-1、PECAM-1表达增加可能与心脏移植后急性排斥反应有关，而Fas／FasL、Bax可能促进了凋亡的发生。     

Cold ischemia augments allogeneic-mediated injury in rat kidney allografts

BACKGROUND: Some clinical studies demonstrate that kidney grafts with prolonged cold ischemia experience early acute rejection more often than those with minimal ischemia. The mechanism, however, is putative. Therefore, the aim of this study was to unravel the impact of ischemia on the immune response in rat kidney allografts compared with that in isografts. METHODS: To induce ischemic injury, donor kidneys were preserved for 24 hours in 4 degrees C University of Wisconsin solution before transplantation. No immunosuppression was administered. The histomorphology according to the BANFF criteria for acute rejection and infiltrating cells were assessed at days 1, 2, 3, 4, 6, and 8 post-transplantation. RESULTS: In allografts, exposure of the kidney to ischemia led to a significantly earlier onset of interstitial cell infiltration and tubulitis compared with nonischemic allografts. The BANFF score of interstitial cell infiltration was 1 +/- 0 vs. 0.25 +/- 0.29 at day 3 and 2 +/- 0 vs. 1.25 +/- 0.25 at day 4. In contrast, in isografts, the effect of ischemia on the histology was not significant. From day 6, the histologic differences between ischemic and nonischemic grafts disappeared. Ischemia led to a more intense expression of P-selectin (day 1), intercellular adhesion molecule-1 (ICAM-1; day 2), and major histocompatibility complex (MHC) class II on endothelium and proximal tubular cells (day 2) in both allografts and isografts. Concurrently with the up-regulated ICAM-1 and MHC expression, significantly more CD4(+) cells and macrophages infiltrated the ischemic allografts at days 2 and 3 and the ischemic isografts at day 4. Importantly, the influx of these cells after ischemia was significantly greater in allografts than in isografts. CONCLUSIONS: Cold ischemia augments allogeneic-mediated cell infiltration in rat kidney allografts. The earlier onset of acute rejection in 24-hour cold preserved allografts may be prevented by better preservation or treatment using tailored immunosuppression.     

Late graft dysfunction after prolonged cold ischemia of the donor kidney: inhibition by cyclosporine.

BACKGROUND: The present study was devised to elucidate the influence of prolonged cold ischemia on the development of chronic transplant dysfunction (CTD) in kidney isografts (Brown Norway-->Brown Norway; BN-->BN) and in kidney allografts (BN-->Wistar Agouti/ Rij [WAG]) under temporary cyclosporine (CsA) therapy. METHODS: To induce ischemic injury, BN donor kidneys were preserved for 24 hr in 4 degrees C University of Wisconsin solution before transplantation. Renal function (proteinuria), histomorphology according to the BANFF criteria for CTD, and infiltrating cells were assessed. Grafts were examined both early at days 2, 3, 6, and 10, and late at week 26 (allografts) or at week 52 (isografts). RESULTS: Nonischemic isografts preserved a normal function and morphology. Ischemic isografts developed a progressive proteinuria over time and demonstrated significantly more glomerulopathy with macrophage (Me) infiltration and intimal hyperplasia than nonischemic controls at week 52. During the initial 10 days, there was an increased infiltration of MHC class II+ cells, predominantly CD4+ cells and Mphi, coinciding with up-regulated intercellular adhesion molecule-1 expression. CsA treatment in ischemic isografts inhibited infiltration of MHC II+ cells in the early stage, which was accompanied by significantly less renal damage at week 52 compared with untreated controls (proteinuria: 59+/-8 vs. 134+/-19 mg/24 hr; BANFF score: 2.8+/-0.4 vs. 4.3+/-1.0). Under CsA therapy, 24-hr cold ischemia of the allograft affected neither the onset or progress of proteinuria, nor the histomorphology (BANFF score: 7.8+/-2.4 vs. 7.3+/-1.9). In both ischemic and nonischemic allografts, intercellular adhesion molecule-1 expression and mononuclear cell infiltration (CD4, CD8, Mphi was abundantly present during the first 10 days and function deteriorated rapidly. CONCLUSIONS: Prolonged cold ischemia plays a role in the induction of CTD, but its deleterious effect can be successfully inhibited by CsA. Therefore, the alloantigeneic stimulus is the overriding component in the multifactorial pathogenesis of CTD.     

Up-regulation of ICAM-1 and VCAM-1 expression during macrophage recruitment in lipid induced glomerular injury in ExHC rats

Summary: A number of studies have demonstrated an important role for macrophages (Mo) in lipid induced glomerular injury; however, little is known of the mechanisms which facilitate Mo infiltration in this disease. the present study examined the expression of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) during the development of glomerular Mo infiltration in ExHC rats; a strain which is susceptible to lipid induced glomerular injury. Twenty-five male 6 week old ExHC rats were placed on a normal diet supplemented with 3% cholesterol, 0.6% sodium cholate and 15% olive oil (high-cholesterol diet, HCD). Groups of five rats were killed prior to the beginning of the HCD or after 3 days, 1, 2 and 6 weeks on a HCD. A group of five matched ExHC rats on a normal diet served as a control. ExHC rats fed a HCD showed marked hypercholesterolaemia in the absence of any increase in plasma triglyceride levels from day 3 (190 ± 14 vs 42 ± 2 mg/dL in control; mean ± s.e.m., P<0.01), and developed mild proteinuria (21.9 ± 2.7 vs 5.2 ± 0.5 mg/24 h in control; P<0.01) and segmental glomerular lesions at week 6. Immunoperoxidase staining identified a significant increase in glomerular ED1⁺Mo at week 1 (2.0 ± 0.2 vs 1.0 ± 0.1 ED1⁺Mo/glomerular cross-section in control, P<0.01) which was further increased at week 6 (6.9 ± 0.4 ED1⁺Mo/gcs). There was also a significant increase in glomerular cells expressing the adhesion molecule ligands lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4). Coincident with Mo infiltration, there was an increase in the intensity of glomerular ICAM-1 protein expression as shown by antibody staining. In addition, northern blot analysis of cortical RNA and in situ hybridization demonstrated an increase in glomerular ICAM-1 and VCAM-1 mRNA expression from day 3 onwards. In conclusion, these results suggest that both ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions play an important role in Mo recruitment and accumulation during the development of lipid induced glomerular injury.     

Functional binding of human adipose-derived stromal cells: effects of extraction method and hypoxia pretreatment

Human adipose-derived stromal cells (hASCs) were evaluated in vitro for their ability to bind vascular adhesion and extracellular matrix proteins to arrest (firmly adhere) under physiological flow conditions. hASCs were flowed through a parallel plate flow chamber containing substrates presenting immobilized type I collagen, fibronectin, E-selectin, L-selectin, P-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) under static and laminar flow conditions (wall shear stress = 1 dyn/cm). hASCs were able to firmly adhere to type I collagen, fibronectin, VCAM-1, and ICAM-1 substrates, but not to any of the selectins. Pretreatment with hypoxia increased the ability of hASCs isolated by liposuction to adhere to VCAM-1 and ICAM-1, but this effect was not seen in cells isolated by tissue excision. These results indicate that hASCs possess the ability to adhere key adhesion proteins, illustrate the importance of hASC harvest procedure, and suggest mechanisms for homing in a setting where interaction with inflamed or injured tissue is necessary.     

Islet Allograft Rejection in Rats: A Time Course Study Characterizing Adhesion Molecule Expression,MHC Expression,and Infiltrate Immunophenotypes

Wistar Furth (RT1^u) islets transplanted under the renal capsules of streptozotocin-diabetic Lewis (RT1^l) rats reject after 5–6 days of normoglycemia. Hand-picked WF islets (1500–2000) were transplanted under the kidney capsules of diabetic Lew or WF rats. Rats bearing iso- or allografts were killed on posttransplant days 2, 4, and 6. Serial frozen sections of grafts and controls were stained by immunoperoxidase for rat MAC-1, class II MHC, CD2, CD4, CD8, B-cells, VLA-4, LFA-1, L-selectin, ICAM-1, and VCAM-1. Infiltrating cells, parenchymal cells, and endothelial cells in five distinct compartments (i.e., peritoneal reflection, subcapsular perivascular space, islet grafts, graft–kidney interface, and kidney) were evaluated for expression of the various markers at each interval. Significant infiltrates arrived in three distinct waves in both iso- and allografts. First, macrophages blanketed the peritoneal capsular reflection and infiltrated by day 2. Second, the first wave of lymphocytes arrived in the edematous subcapsular soft tissue via capsular vessels by day 2 (allo > iso). Third, the second wave of lymphocytes arrived from the renal parenchyma to form a dense band at the graft–kidney interface and around grafts by days 4 and 6 (allo >>> iso); CD4+ cells vastly outnumbered CD8+ cells, with CD4+ cells being mobilized first and from interstitial vessels throughout the entire kidney. CD8+ cells emigrated only from renal interstitial vessels adjacent to the graft. Large numbers of L-selectin+, VLA-4+, and LFA-1+ cells were seen in the infiltrates with the most intensely staining cells being intravascular. B-cells composed a very small proportion of infiltrating cells in both allo- and isografts. Endothelial staining for ICAM-1 and VCAM-1 was prominent throughout. Both class II MHC and ICAM-1 expression were induced on renal tubular epithelial cells, but neither was found on islet parenchymal cells. In conclusion, this study shows that islet allograft rejection is more complex than previously realized.     

Rat cytomegalovirus infection in kidney allograft recipients is associated with increased expression of intracellular adhesion molecule-1 vascular adhesion molecule-1, and their ligands leukocyte function antigen-1 and very late antigen-4 in the graft

BACKGROUND: Cytomegalovirus (CMV) infection is suggested to be a risk factor for chronic rejection. We have recently shown that rat CMV (RCMV) increases the inflammatory response and accelerates chronic rejection in a model of rat kidney allograft. In this study, the early inflammatory response and time-related expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and their ligands, leukocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), in the grafts were investigated in RCMV-infected rats and compared to noninfected rats developing chronic rejection. METHODS: Transplantations were performed in a rat strain combination of DA (RT1a)->BN (RT1n) receiving triple drug immunosuppression. One group of rats was infected with RCMV, and the other was left uninfected. The grafts were harvested at different time points after transplantation. The adhesion molecules, their ligands and activation markers, MHC class II antigens and interleukin-2-receptors (IL-2-R), were demonstrated by monoclonal antibodies and immunoperoxidase staining from frozen sections of the grafts. Graft histology was evaluated according to the Banff criteria. RESULTS: RCMV caused a significant, prolonged increase of VCAM-1 and ICAM-1 expression in the vascular endothelium compared to the noninfected grafts. Also, the number of cells expressing activation markers, LFA-1 and VLA-4 was significantly enhanced in these animals. Significantly enhanced histological changes of chronic rejection were seen in the RCMV-infected group. CONCLUSIONS: Prolonged, increased expression of ICAM-1 and VCAM-1, and increased numbers of inflammatory cells expressing their ligands in the CMV infected grafts, were associated with accelerated chronic allograft nephropathy.     

Characterisation of adhesion receptors mediating lymphocyte adhesion to bronchial endothelium provides evidence for a distinct lung homing pathway

BACKGROUND: The lung is an important tertiary lymphoid organ and many lung diseases are associated with disordered lung immunity. Unlike the gut (alpha4beta7 binding to MAdCAM-1) and skin (CLA+ve T cells binding to E-selectin) where the adhesion receptors involved in organ specific homing of T cells have been identified, the molecular pathways controlling lymphocyte migration to the lung are unclear. Using a modified version of the Stamper-Woodruff assay we have investigated the receptors mediating adhesion of peripheral blood lymphocytes to airway endothelium. METHODS: Longitudinal frozen sections of bronchus (8 micro m) obtained from lung resection specimens were incubated with T cell enriched peripheral blood mononuclear cells for 30 minutes under shaking conditions in the presence of a fluorescently labelled polyclonal anti-von Willebrand antibody to identify blood vessels. After fixation the percentage of blood vessels supporting adhesion was measured. Blocking monoclonal antibodies were used to determine the role of individual adhesion receptors in lymphocyte binding. RESULTS: Specific cation dependent binding of lymphocytes to bronchial endothelium was observed which was significantly inhibited by antibodies against P-selectin, PSGL-1, L-selectin, LFA-1, ICAM-1 and ICAM-2 but not E-selectin, VLA-4, VCAM-1 or Mac-1. This was consistent with the pattern of endothelial expression of these receptors with strong expression of P-selectin and ICAM-1, but negligible expression of E-selectin on bronchial endothelium. CONCLUSION: This study suggests an important role for PSGL-1/P-selectin in T cell migration into the bronchi and provides further evidence for a pattern of recirculation for respiratory tract homing T cells distinct from the gut and skin.     

Expression of adhesion molecules and their ligands in liver allografts during cytomegalovirus (CMV) infection and acute rejection

Abstract  Vascular adhesion molecules and their ligands are important in leukocyte-endothelial cell interactions and in T-cell activation of rejection cascade. Also, cytomegalovirus (CMV) infection is suggested to be involved in the mechanisms of rejection. In this study, the expression of vascular adhesion molecules ICAM-1, VCAM-1 and ELAM-1 in the liver allografts, the number of leukocytes positive for their ligands LFA-1, VLA-4 and SLex, and activation markers (class II, IL2-receptor) were investigated in liver allografts during CMV infection and acute rejection and compared to grafts with normal function and histology. The adhesion molecules, their ligands and activation markers were demonstrated from liver biopsy frozen sections by the immunoperoxidase technique and monoclonal antibodies. A significant induction of ICAM-1 and VCAM-1 was seen in vascular and sinusoidal endothelium associated with both CMV and rejection, and induction of ELAM-1 in vascular endothelium in rejection only. In both cases, the number of leukocytes expressing LFA-1 was significantly increased, but VLA-4-positive cells were more characteristic for CMV and SLex-positive cells more for rejection. IL2-receptor positivity was practically seen in rejection only, but class II-expressing cells were increased during both CMV infection and rejection. In conclusion, adhesion molecules were induced and the infiltrating cells expressed their ligands both in liver rejection and during CMV infection, although the expression pattern was slightly different.     

Trigger for intercellular adhesion molecule-1 expression in rat lungs transplanted from non-heart-beating donors

BACKGROUND: Lung transplantation from non-heart-beating donors causes ischemia-reperfusion injury. We sought to determine the trigger for expression of intercellular adhesion molecule-1 (ICAM-1) caused by ischemia-reperfusion injury. METHODS: Thirty-six Sprague-Dawley rats underwent left lung transplant (six groups of 6). Lungs were transplanted immediately after arrest, or from non-heart-beating donors after 2 hours of oxygen-ventilation or no ventilation. Recipients were reperfused for 4 or 6 hours, then lungs were stained with a mouse anti-rat ICAM-1 monoclonal antibody, developed with avidin-biotin peroxidase to a biotinylated anti-mouse immunoglobin G antibody. Intercellular adhesion molecule-1 expression was graded by two masked observers as 0 = absent, 1 = weak, or 2 = strong in alveoli, arterioles, and venules. Explanted recipient left lungs served as negative controls, and positive controls were generated 6 hours after intraperitoneal injection of endotoxin. Intercellular adhesion molecule-1 expression above baseline among groups was compared by Fisher's exact test. RESULTS: Constitutive expression of ICAM-1 was present in rat lung alveoli, with 24 of 35 controls staining weakly and 4 of 35 strongly positive in alveolar areas. Intercellular adhesion molecule-1 expression was not increased in transplanted lungs evaluated after 4 hours of reperfusion, even lungs retrieved from non-heart-beating donors. But when non-heart-beating donor lungs were assessed 6 hours after onset of reperfusion, ICAM-1 expression was significantly more apparent in alveolar and arteriolar areas, compared with controls and lungs transplanted immediately after arrest. CONCLUSIONS: Lungs transplanted immediately after circulatory arrest do not sustain sufficient ischemia-reperfusion injury to upregulate ICAM-1. Onset of reperfusion is the signal for ICAM-1 expression, not the onset of ischemia or the total duration of ischemic and reperfusion time together. Strategies at reperfusion may minimize ICAM-1 expression.     

Attenuation of experimental subarachnoid hemorrhage-induced increases in circulating intercellular adhesion molecule-1 and cerebral vasospasm by the endothelin-converting enzyme inhibitor CGS 26303

OBJECT: Adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, are important mediators of inflammation, and their levels are elevated in the serum of patients following aneurysmal subarachnoid hemorrhage (SAH). The investigators previously found that CGS 26303 is effective in preventing and reversing arterial narrowing in a rabbit model of SAH. The purpose of the present study was to examine whether levels of adhesion molecules are altered after treatment with CGS 26303 in this animal model. METHODS: New Zealand White rabbits were each injected with 3 ml of autologous blood in the cisterna magna, and intravenous treatment with CGS 26303 (30 mg/kg) was initiated 1 hour later. The compound was subsequently administered at 12, 24, and 36 hours post-SAH. Blood samples were collected at 48 hours post-SAH to measure ICAM-1, VCAM-1, and E-selectin levels. After the rabbits had been killed by perfusion-fixation, the basilar arteries (BAs) were removed and sliced, and their cross-sectional areas were measured. Treatment with CGS 26303 attenuated arterial narrowing after SAH. Morphologically, corrugation of the internal elastic lamina of BAs was prominently observed in the SAH only and vehicle-treated SAH groups, but not in the CGS 26303-treated SAH group or in healthy controls. There were no significant differences in the levels of VCAM-1 among the four groups. The levels of E-selectin were increased in all animals subjected to SAH (those in the SAH only, SAH plus vehicle, and SAH plus CGS 26303 groups) compared with healthy controls (no SAH); however, the levels of ICAM-1 in the SAH only and SAH plus vehicle groups were significantly elevated (p < 0.001), and treatment with CGS 26303 reduced ICAM-1 to control levels following SAH. CONCLUSIONS: These results show that ICAM-1 may play a role in mediating SAH-induced vasospasm and that a reduction of ICAM-1 levels after SAH may partly contribute to the antispastic effect of CGS 26303.     

Effect of leukocyte depletion on endothelial cell activation and transendothelial migration of leukocytes during cardiopulmonary bypass

Background

Although leukocyte depletion from systemic circulation during cardiopulmonary bypass (CPB) has been studied, the effect of leukocyte depletion on the leukocyte-endothelial cascade remains poorly understood. So far, there has been no published work on the effects of leukocyte filters during cardiac operations from the viewpoint of endothelial activation and transendothelial neutrophil migration.

Methods

Thirty-two patients undergoing elective heart operations were randomly allocated to a leukocyte-depletion (LD) group or a control group. Blood samples were collected at seven time points: before sternotomy, at 30 minutes and at 60 minutes of CPB, at 5 minutes after coronary reperfusion, at the end of CPB, and at 2 hours and 24 hours after the cessation of CPB. The plasma concentrations of P-selectin, intercellular adhesion molecule-1 (ICAM-1), interleukin-8, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were measured using enzyme-linked immunosorbent assays. Plasma malondialdehyde (MDA) concentration was determined by measurement of thiobarbituric acid-reactive substances in plasma. In addition, blood samples collected at intervals before and after operation were used for arterial blood gases.

Results

Our studies show significant increases of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA during and after CPB in the control group. Interestingly, a significant decrease of plasma levels of P-selectin, ICAM-1, interleukin-8, PECAM-1, and MDA, and better preservation of lung function could be found in the LD group compared with the control group.

Conclusions

Our results demonstrate a rationale for using a leukocyte filter in patients undergoing cardiac surgery to attenuate the endothelial-mediated component of the CPB-induced inflammatory response by reducing endothelial activation and neutrophil transmigration.     

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.     

Hypoxia and reoxygenation do not upregulate adhesion molecules and natural killer cell adhesion on human endothelial cells in vitro

Objectives: Ischemia/reperfusion injury is characterized by endothelial cell activation leading to increased expression of adhesion molecules such as inter-cellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, endothelial- and platelet-selectin (E- and P-selectin), and to the subsequent recruitment of leukocytes. The aim of the present study was to investigate the respective effects of a proinflammatory cytokine (tumor necrosis factor alpha , TNF-), hypoxia and/or reoxygenation on adhesion molecule expression and natural killer (NK) cell adhesion in an in vitro model of I/R. Methods: Human aortic endothelial cells (HAEC) were stimulated in vitro for 8h with TNF- (1000 U/ml) and exposed to hypoxia (1% O₂), reoxygenation (21% O₂) or different combinations thereof. Cell surface expression of ICAM-1, VCAM-1 and E-/P-selectin on HAEC was analyzed by flow cytometry, and culture supernatants were tested for soluble adhesion molecules by ELISA. Rolling adhesion of NK cells on HAEC was determined using a rotating assay. Results: Untreated HAEC constitutively expressed ICAM-1 on their surface but neither expressed E-/P-selectin, VCAM-1, nor shedded soluble adhesion molecules. Exposure of HAEC to hypoxia or hypoxia and reoxygenation did not upregulate cell surface expression or shedding of adhesion molecules. In contrast, TNF- significantly upregulated cell surface expression of ICAM-1, VCAM-1, and E-/P-selectin and led to the shedding of ICAM-1 and E-selectin. Combined treatment of HAEC with TNF-, hypoxia and reoxygenation reduced E-/P-selectin surface expression and enhanced E-selectin shedding, but did not further influence ICAM-1 and VCAM-1. Soluble VCAM-1 was not detected. NK cell adhesion on HAEC increased 4-fold after TNF- stimulation, but was not affected by hypoxia or hypoxia and reoxygenation. Conclusions: Both the expression of endothelial adhesion molecules and rolling NK cell adhesion was upregulated by TNF- but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injury.