P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in hepatic venules after ischemia/reperfusion.

We have recently reported that hepatic ischemia/reperfusion (I/R) is associated with a biphasic increase in the expression of P-selectin in the liver microvasculature, with peak expression levels observed at 20 min and 5 h after reperfusion. This I/R-induced upregulation of P-selectin expression is accompanied by leukocyte-endothelial cell adhesion in terminal hepatic venules (THV). The objective of this study was to determine whether the early expression of P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in THV after liver I/R. Left hepatic lobe ischemia was induced for 30 min in anesthetized C57B1/6 and P-selectin knockout (KO) mice. The number of rolling, saltating, and adherent leukocytes in THV was measured at 0, 15, 30, 60, and 120 min after reperfusion using intravital video microscopy. Hepatic I/R elicited significant increases in the number of rolling, saltating, and adherent leukocytes, with peak values observed at 30 min after reperfusion. All of these responses were absent in P-selectin KO mice and in C57B1/6 mice treated with a blocking antibody to P-selectin. Our findings suggest that P-selectin is the primary determinant of leukocyte-endothelial cell adhesion observed in hepatic venules in the initial period after I/R. Hence, this adhesion molecule may represent a target for therapeutic intervention in liver transplantation and other conditions associated with hepatic I/R.     

The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo

A role for the alpha 4-integrin (alpha 4 beta 1 or alpha 4 beta 7), has been implicated in the recruitment of peripheral blood mononuclear cells (PBMCs) to sites of inflammation. However, the adhesive interactions (i.e., tethering, rolling, and adhesion) mediated by the alpha 4-integrin have not been characterized in vivo. The objective of this study was to establish a model wherein postcapillary venules were chronically inflamed, and then use intravital microscopy to identify the adhesive interactions mediated by the alpha 4-integrin in vivo. Between 4 and 20 d after immunization with Mycobacterium butyricum, animals developed a systemic vasculitis characterized by large increases in the numbers of rolling and adhering leukocytes within mesenteric venules. The selectins could only account for approximately 50% of the leukocyte rolling whereas the remaining cells rolled exclusively via the alpha 4-integrin. Anti-alpha 4 therapy also eliminated the increase in leukocyte adhesion observed in this model, whereas selectin therapies and an anti-CD18 (beta 2-integrin) monoclonal antibody (mAb) did not reduce adhesion. A serum against polymorphonuclear leukocytes (PMNs) was used to confirm that a significant proportion of rolling cells, and most of the adhering cells were PBMCs. Sequential treatment with anti-PMN serum and the anti-alpha 4 mAb demonstrated that alpha 4-dependent rolling was distinct from PMN rolling populations. Initial leukocyte tethering via the alpha 4- integrin could not be demonstrated in this model, whereas L-selectin did support leukocyte tethering. These data suggest that the alpha 4- integrin can mediate both rolling and adhesion in the multistep recruitment of PMBCs in vivo, and these interactions occur independently of the selectins and beta 2-integrins.     

Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin

L-selectin (leukocyte adhesion molecule 1/MEL-14), a member of the selectin family of cell adhesion molecules, mediates leukocyte rolling and leukocyte adhesion to endothelium at sites of inflammation. In addition, L-selectin mediates the binding of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes. The strong amino acid sequence conservation of the cytoplasmic domain of L-selectin between humans and mice suggests an important role for this region. Deletion of the COOH-terminal 11 amino acids from the approximately 17 amino acid cytoplasmic domain of L-selectin eliminated binding of lymphocytes to HEV in the in vitro frozen section assay, and also abolished leukocyte rolling in vivo in exteriorized rat mesenteric venules, but did not alter the lectin activity of L-selectin. Pretreatment of cells with cytochalasin B, which disrupts actin microfilaments, also abolished adhesion without affecting carbohydrate recognition. Therefore, the cytoplasmic domain of L-selectin regulates leukocyte adhesion to endothelium independent of ligand recognition, by controlling cytoskeletal interactions and/or receptor avidity.     

Propofol reduces the migration of human leukocytes through endothelial cell monolayers.

OBJECTIVE: To test propofol and the solvent of propofol on leukocyte function in the presence of endothelial cell monolayers. The interactions of leukocytes with endothelial cells play a tremendous role during inflammation. Previous studies have investigated the influence of propofol on leukocytes. DESIGN: Prospective, controlled study. SETTING: University research laboratories. SUBJECTS: Seven independent experiments were performed to investigate the influence of propofol (0.4, 4, and 40 ng/mL) on the migration of human leukocytes through human endothelial cell monolayers. Moreover, the authors tested the solvent of propofol on leukocyte migration. INTERVENTIONS: Human endothelial cell monolayers and/or human leukocytes were preincubated with clinically relevant higher and lower concentrations of propofol. The amount of leukocyte migration after 3 hrs was measured with a fluorometer. MEASUREMENTS AND MAIN RESULTS: Human endothelial cells isolated from umbilical veins were cultured on microporous membranes until they formed an endothelial cell monolayer. Leukocytes were separated by standard procedures. The migration of leukocytes through monolayers of endothelial cells using the clinically relevant concentration of propofol was reduced to 93% +- 3.8% (so; p < .05) when the leukocytes but not the endothelial cell monolayers were preincubated with propofol. Leukocyte migration was reduced to 80% - 5.9% (p < .05) when only monolayers of endothelial cells were treated with propofol, and was reduced to 73% + 10.4% (p < .05) when both leukocytes and monolayers of endothelial cells were treated with propofol. The higher and lower concentrations showed a dose-dependent effect. The solvent of propofol had no significant effect. CONCLUSION: The authors investigated the influence of propofol and its solvent on the interaction between both cell systems-leukocytes and endothelial cells. Propofol is able to reduce significantly the migration of leukocytes through endothelial cell monolayers. The use of different doses revealed a dose-dependent effect. The current model allowed treatment of one cell type: leukocyte or endothelial cell. The results of this investigation indicate that the influence of propofol on leukocyte migration affects endothelial cells more than leukocytes.     

Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules.

The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.     

Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo

Macrophage interactions with extracellular matrix and other cells are important in phagocytosis, inflammation, and immunity. To learn more about the surface molecules involved in adhesion we compared the binding of murine macrophages and polymorphonuclear leukocytes (PMN) with artificial substrate in vitro. A distinctive type of adhesion of thioglycollate-elicited peritoneal macrophages (TPM) to bacteriologic plastic (BP) was defined, which was pronase-sensitive, Mg2+-dependent, and required cytoskeletal stabilization. A rat mAb designated 5C6 was isolated because it inhibited TPM attachment to BP, as well as mediating detachment of TPM adherent to that substratum. In addition, it inhibited the attachment of PMN to tissue culture plastic. This antiadhesive property of 5C6 mAb required intact IgG; the F(ab')2 fragment was partially effective and Fab was ineffective. 5C6 recognized the type 3 complement receptor, inhibiting rosetting of EAC3bi to TPM and immunoprecipitating a heterodimer of 160 and 95 kD that comigrated with the M1/70 immunoprecipitate. 5C6 recognized a pronase-stable epitope distinct from that of M1/70. Other mAbs, including M1/70 (CR3) and 2.4G2 (FcR), failed to have any antiadhesive effect in vitro. The inhibitory activity of 5C6 in short-term adhesion assays correlated with its inhibition of recruitment of myelomonocytic cells to a thioglycollate-elicited peritoneal exudate in vivo, after intravenous injection of mAb. 5C6 IgG inhibited recruitment of myelomonocytic cells by 84 +/- 3% at 1 d compared with saline-injected controls. The F(ab')2 fragment and a class-matched control IgG had little effect. Recruitment of TPM at 4 d was also efficiently inhibited by 5C6 IgG. 5C6 IgG was not cytotoxic, had no effect on marrow egress, did not cause increased phagocytic clearance of circulating neutrophils, and had no adverse effect on chemotaxis in vitro. We show that CR3 alone of the LFA-family is necessary for the recruitment of myelomonocytic cells to inflammatory stimuli such as thioglycollate broth. This strategy may be of general use in isolating reagents that inhibit the adhesive function of CR3 and provides a novel approach to antiinflammatory therapy.     

Type I interferon negatively controls plasmacytoid dendritic cell numbers in vivo

Plasmacytoid dendritic cells (pDCs) specialize in the secretion of type I interferons (IFN-I) and thus are considered critical mediators of antiviral responses. We recently reported that pDCs have a very early but limited and transient capacity to curtail viral infections. Additionally, pDC numbers are not sustained in human infections caused by Hepatitis B or C viruses (HBV and HCV) and HIV. Thus, the numbers and/or function of pDCs appear to be regulated during the course of viral infection. In this study, we show that splenic pDCs are reduced in vivo during several systemic viral infections and after administration of synthetic toll-like receptor ligands. We demonstrate that IFN-I, regardless of the source, contributes to this decline and mediates pDC death via the intrinsic apoptosis pathway. These findings demonstrate a feedback control mechanism by which IFN-I modulates pDC numbers, thus fine-tuning systemic IFN-I response to viruses. IFN-I-mediated control of pDCs may explain the loss of pDCs during human infections caused by HBV, HCV, or HIV and has important therapeutic implications for settings in which IFN-I is used to treat infections and autoimmune diseases.     

Thiopental inhibits migration of human leukocytes through human endothelial cell monolayers in vitro

Objectives: The interactions between blood and vascular wall cells are essential for the understanding of pathophysiologic processes, e. g. inflammation. The influence of the anesthetic drug thiopental on leukocyte function is well documented. Recently, an inhibitory effect of thiopental on leukocyte chemotaxis in a Boyden chamber assay (i. e. endothelial cells were not included) was demonstrated. In vivo, leukocytes have to interact with endothelial cell monolayers to invade the tissue. The influence of thiopental on a monolayer of endothelial cells has not yet been investigated. The aim of the current study was to investigate the influence of thiopental on the migration of leukocytes through endothelial cell monolayers (ECM). Material and methods: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured on microporous membrane filters to achieve a monolayer. Isolated polymorphonuclear leukocytes (PMNL) as well as ECM were preincubated with different concentrations of thiopental. The rate of leukocyte migration against the chemotactic protein formyl-methyl-leucyl-phenylalanine was measured (n = 7). Thiopental was able to reduce the amount of leukocyte migration through ECM significantly. Conclusion: In conclusion, we could show that thiopental is able to reduce the migration of PMNL through ECM significantly. Received: 1 April 1998 Accepted: 15 June 1998     

Sphingosine-1-phosphate modulates vascular permeability and cell recruitment in acute inflammation in vivo

The sphingosine kinase (SPK)/sphingosine-1-phosphate (S1P) pathway recently has been associated with a variety of inflammatory-based diseases. The majority of these studies have been performed in vitro. Here, we have addressed the relevance of the SPK/S1P pathway in the acute inflammatory response in vivo by using different well known preclinical animal models. The study has been performed by operating a pharmacological modulation using 1) L-cycloserine and DL-threo-dihydrosphingosine (DTD), S1P synthesis inhibitors or 2) 2-undecyl-thiazolidine-4-carboxylic acid (BML-241) and N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide (JTE-013), specific S1P(2) and S1P(3) receptor antagonists. After local injection of carrageenan in mouse paw S1P release significantly increases locally and decreases during the resolution phase. Expression of SPKs and S1P(2) and S1P(3) receptors is increased in inflamed tissues. Administration of L-cycloserine or DTD caused a significant anti-inflammatory effect. By using different animal models we have also demonstrated that the SPK/S1P pathway contributes to changes in vascular permeability and promotes cell recruitment. The S1P effect on cell recruitment results is receptor-mediated because both JTE-013 and BML-241 inhibited zymosan-induced cell chemotaxis without effect on vascular leakage. Conversely, changes in vascular permeability involve mainly SPK activity, because compound 48/80-induced vascular leakage was significantly inhibited by DTD. In conclusion, the SPK/S1P pathway is involved in acute inflammation and could represent a valuable therapeutic target for developing a new class of anti-inflammatory drugs.     

Tumor necrosis factor-dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment.

Monocytes recruited from the blood are key contributors to the nature of an immune response. While monocyte recruitment in a subset of immunopathologies has been well studied and largely attributed to the chemokine monocyte chemoattractant protein (MCP)-1, mechanisms mediating such recruitment to other sites of inflammation remain elusive. Here, we showed that localized inflammation resulted in an increased binding of monocytes to perifollicular high endothelial venules (HEVs) of lymph nodes draining a local inflammatory site. Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG. HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] null mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding. Expression of CXCR3, the receptor for MIG, was detected on a small subset of peripheral blood monocytes and on a significant percentage of recruited monocytes. Most importantly, in both ex vivo and in vivo assays, neutralizing anti-MIG antibodies blocked monocyte binding to inflamed lymph node HEVs. Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.     

自体CIK过继免疫治疗恶性肿瘤的最佳输注时间

摘要：目的:研究恶性肿瘤患者自体细胞因子诱导的杀伤细胞(CIK)的免疫表型与细胞毒活性的变化规律,探讨肿瘤患者CIK过继免疫治疗输注的最佳时间。 方法:采集40例恶性肿瘤患者外周血单个核细胞(PBMC),由IFN-γ、rhIL-1α、rhIL-2等细胞因子和CD3单克隆抗体体外诱导培养成CIK。用流式细胞术动态监测免疫表型,MTT法分析细胞毒活性。 结果：随着诱导时间的延长,PBMC中CD3⁺、CD3⁺CD8⁺、CD3⁺CD56⁺表型细胞所占比例呈上升趋势。CD3⁺CD4⁺细胞在7 d达到峰值,随后缓慢下降。CD25⁺细胞在培养的早期(3～7 d)即达峰值,7～14 d缓慢下降,14～21 d快速下降。HLA-DR+细胞在0～14 d处于上升期,14 d达峰值后快速下降。成熟CIK细胞毒活性［(52.49±7.70)%］较未活化的PBMC［(7.02±2.00)%］显著增高(P<0.01)。 结论:14 d左右能诱导出典型的CIK,CD3⁺CD56⁺细胞处于对数生长期。确立自体CIK过继免疫治疗恶性肿瘤的最佳输注时间为第14天。     

A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature.

A two-step paradigm for leukocyte recruitment has been established in a number of tissues including the mesentery, skin, and muscle, and necessitates an initial rolling step via the selectins before firm leukocyte adhesion via the integrins. In view of the many inflammatory diseases that involve the liver, we investigated the importance of rolling and the selectins in the hepatic microvasculature and compared the responses to that of the commonly used mesentery or cremaster microvasculature. We visualized the liver microvasculature using intravital microscopy and we determined that within the liver the majority of leukocytes adhere within the sinusoids (80%) in response to a chemotactic stimulus such as FMLP (20% in postsinusoidal venules) whereas leukocytes adhere exclusively within postcapillary venules in tissue like the mouse cremaster. In the sinusoids, the adhesive response to FMLP is not dependent upon selectins inasmuch as adhesion was not reduced in the sinusoidal vessels of P-selectin-deficient mice or E-selectin/P-selectin- deficient animals in the presence or absence of L-selectin antibody. No rolling or adhesion was detected in response to FMLP in the selectin-deficient cremaster microvasculature. Immunoneutralization of selectins with fucoidan in wildtype mice eliminated rolling and adhesion in the cremaster but failed to affect adhesion in the liver sinusoids in response to FMLP. More long-term leukocyte recruitment with lipopolysaccharide (4 h) was also impaired in the cremaster but not the liver microvasculature in selectin-deficient animals. Leukocyte adhesion in the sinusoids was reduced in P-selectin-deficient mice also lacking intercellular adhesion molecule-1 (ICAM-1). This study for the first time demonstrates that selectins are not an essential step for leukocyte recruitment into the inflamed liver microvasculature.     

Role of nitric oxide and peroxynitrite in the cytokine-induced sustained myocardial dysfunction in dogs in vivo.

Studies in vitro suggested that inflammatory cytokines could cause myocardial dysfunction. However, the detailed mechanism for the cytokine-induced myocardial dysfunction in vivo remains to be examined. We thus examined this point in our new canine model in vivo, in which microspheres with and without IL-1beta were injected into the left main coronary artery. Left ventricular ejection fraction (LVEF) was evaluated by echocardiography for 1 wk. Immediately after the microsphere injection, LVEF decreased to approximately 30% in both groups. While LVEF rapidly normalized in 2 d in the control group, it was markedly impaired in the IL-1beta group even at day 7. Pretreatment with dexamethasone or with aminoguanidine, an inhibitor of inducible nitric oxide synthase, prevented the IL-1beta-induced myocardial dysfunction. Nitrotyrosine concentration, an in vivo marker of the peroxynitrite production by nitric oxide and superoxide anion, was significantly higher in the myocardium of the IL-1beta group than in that of the control group or the group cotreated with dexamethasone or aminoguanidine. There was an inverse linear relationship between myocardial nitrotyrosine concentrations and LVEF. These results indicate that IL-1beta induces sustained myocardial dysfunction in vivo and that nitric oxide produced by inducible nitric oxide synthase and the resultant formation of peroxynitrite are substantially involved in the pathogenesis of the cytokine-induced sustained myocardial dysfunction in vivo.     

Somatostatin analogues suppress the inflammatory reaction in vivo.

Somatostatin (Sms) and its agonist analogues inhibit the secretory activities of endocrine and neural cells. Recent studies have suggested that Sms has significant immunomodulatory properties. In this study, we examine the effects of two Sms octapeptide analogues on the inflammatory reaction in vivo. BIM 23014 (Somatulin) and Sandostatin were administered to male Sprague-Dawley rats subject to carrageenin-induced aseptic inflammation, at doses of 2-10 micrograms/rat, given either systemically or locally. Animals were killed 7 h after the induction of the inflammation, and the inflammatory exudates were aspirated and quantitated in terms of volume and leukocyte concentration. Sms analogues, administered via either route, significantly reduced the volume and the leukocyte concentration of the exudate in a time- and dose-dependent fashion. In corroboration of these, immunohistochemical evaluation of the levels of local inflammatory mediators, such as immunoreactive (Ir) TNF-alpha, Irsubstance P, and Ircorticotropin-releasing hormone, was inhibited significantly by Sms analogue treatment. These findings suggest that Sms analogues have significant antiinflammatory effects in vivo, associated with suppression of proinflammatory cytokines and neuropeptides. Furthermore, these data suggest that Sms agonists may be useful in the control of inflammatory reaction.     

Cyclosporine inhibition of calcineurin activity in human leukocytes in vivo is rapidly reversible.

Despite increasing information about the mechanism of action of cyclosporine A (CsA), little is known about the way lymphocytes recover from CsA. Recovery is central to understanding the pharmacodynamics of CsA in vivo. We studied the recovery of calcineurin phosphatase (CN) activity in CsA-treated cells. Single dose kinetics in renal transplant patients showed that inhibition of CN activity in PBL increased and fell concomitant with CsA blood vessels. In vitro, control PBL treated with CsA 100 micrograms/l, washed, and resuspended in CsA-free medium showed little recovery (0-20%) after 24 h. Erythrocytes or anti-CsA Ab added to the recovery medium increased recovery to 50% within 4 h. Similar recovery was seen in the ability of cells to produce IFN-gamma after OKT3 stimulation. Recovery of CN activity was associated with the efflux of [3H]CsA, was not blocked by cycloheximide and was temperature sensitive. A cell line with high expression of surface P glycoprotein (PGP), showed rapid recovery. However, PGP blockade did not prevent recovery in PBL, indicating a different PGP-independent mechanism. In PBL, recovery from CsA is slow and limited in vitro, but rapid in vivo, where CsA equilibrates among a complex set of extralymphocytic binding sites.