Differential role of E-selectin and P-selectin in T lymphocyte migration to cutaneous inflammatory reactions induced by cytokines

E-selectin and P-selectin are thought to be important in the infiltration of T lymphocytes in inflammation, but their role in cytokine-induced cutaneous inflammatory reactions has not been examined. A technique for quantifying labeled T lymphocyte migration to cytokine-induced dermal inflammation in mice was developed. After i.v. injection, (51)Cr-labeled T lymphocytes migrated to lesions induced by IFN-gamma and tumor necrosis factor (TNF)-alpha, and in even greater numbers to the combination of IFN-gamma + TNF-alpha, and to sites injected with concanavalin A (Con A). In E-selectin mAb-treated and in E-selectin-deficient mice, IFN-gamma-, IFN-gamma + TNF-alpha- and Con A-induced T cell accumulation was inhibited by 45-65%, but TNF-alpha-induced infiltration was unaffected. In P-selectin mAb-treated and P-selectin-deficient mice, T cell accumulation remained unchanged in most of the lesions. Combined, E-selectin and P-selectin mAb treatment inhibited T cell accumulation in all four types of reactions, and significantly more than E-selectin blockade alone in migration to Con A. Results in E-selectin- and P-selectin-deficient mice confirmed these observations, and demonstrated strain-dependent differences in the contributions of the two selectins. In conclusion, T cells migrating to dermal inflammatory reactions utilize both E-selectin and P-selectin, but alternate adhesion pathways also contribute, since blocking both endothelial selectins does not abolish T cell migration. P-selectin plays a less important role than E-selectin, since blocking E-selectin, but not P-selectin, alone decreased T cell accumulation. The relative contribution of the selectins varies depending on the initiating inflammatory stimulus and the genetic background.     

Sialomucin CD43 regulates T helper type 17 cell intercellular adhesion molecule 1 dependent adhesion,apical migration and transendothelial migration

T helper type 17 lymphocytes (Th17 cells) infiltrate the central nervous system (CNS), induce inflammation and demyelination and play a pivotal role in the pathogenesis of multiple sclerosis. Sialomucin CD43 is highly expressed in Th17 cells and mediates adhesion to endothelial selectin (E-selectin), an initiating step in Th17 cell recruitment to sites of inflammation. CD43^−/− mice have impaired Th17 cell recruitment to the CNS and are protected from experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. However, E-selectin is dispensable for the development of EAE, in contrast to intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). We report that CD43^−/− mice have decreased demyelination and T-cell infiltration, but similar up-regulation of ICAM-1 and VCAM-1 in the spinal cord, compared with wild-type (WT) mice, at the initiation of EAE. CD43^−/− Th17 cells have impaired adhesion to ICAM-1 under flow conditions in vitro, despite having similar expression of LFA-1, the main T-cell ligand for ICAM-1, as WT Th17 cells. Regardless of the route of integrin activation, CD43^−/− Th17 cell firm arrest on ICAM-1 was comparable to that of WT Th17 cells, but CD43^−/− Th17 cells failed to optimally apically migrate on immobilized ICAM-1-coated coverslips and endothelial cells, and to transmigrate under shear flow conditions in an ICAM-1-dependent manner. Collectively, these findings unveil novel roles for CD43, facilitating adhesion of Th17 cells to ICAM-1 and modulating apical and transendothelial migration, as mechanisms potentially responsible for Th17 cell recruitment to sites of inflammation such as the CNS.     

Predictors of autoimmune disease: Autoantibodies and beyond

Increased emphasis has been placed in recent years on predictive biomarkers to foretell the onset or future course of disease. In autoimmune diseases, autoantibodies have proven to be valuable biomarkers because of their technical sensitivity, specificity, and stability during storage. Their predictive value is limited, however, by their prevalence. At present, predictive studies have utilized long-time evaluation of stable populations, families with one index case or retrospective investigations where large serum repositories are available. Our increasing knowledge of the steps leading from benign autoimmunity to frank autoimmune disease has suggested ways by which subtle genetic differences combined with assessment of the pattern of critical mediators can trace the progression of disease. The new tools of multiplex testing and information handling open opportunities to identify early signposts of disease.     

Recovery of brain microcirculation during ischemia

Blood pressure in the circle of Willis in rats decreased to about 40 mmHg after ligation of both carotid arteries. Developing ischemia was accompanied by massive adhesion of leukocytes to the walls of brain venules and the smallest veins. Blood pressure in the vessels of the circle of Willis decreased to 16–20 mmHg 2 h after ligation. The number of adhesion events increased sharply. Leukocyte aggregates formed in these vessels, resulting in complete occlusion of the vessels and death of the animals. These processes were found to be reversible. Introduction of 4–4.5 ml of the plasma replacement polyglucin directly into the vessels of the circle of Willis “washed away” the leukocyte aggregates and some of the adhered leukocytes, with restoration of circulation in venules and the smallest veins by 10–15 min. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 4, pp. 420–428, April, 2006.     

Chemokines in autoimmune disease

Growing evidence indicates that structural cells play a crucial role in the chronic inflammation of autoimmunity by their recruitment of chemokine-dependent cells. Members of the two functional classes of chemokines, inflammatory and homeostatic, seem to be involved in lymphocyte recruitment and survival, and in establishing ectopic lymphoid structures in the target organs of autoimmune diseases. Results from animal models suggest that chemokines are reasonable therapeutic targets in autoimmunity.     

Immune cell migration as a means to control immune privilege: lessons from the CNS and tumors

Summary:  Certain organs, such as the brain, eye, and gonads, are particularly sensitive to damage by inflammation. Therefore, these tissues have developed unique immunological properties that curtail inflammatory responses, a phenomenon termed immune privilege. In addition, by co-opting some of the regulatory cues operant in immune privilege in normal organs, tumors can evade immunosurveillance. While many different mechanisms contribute to immune privilege, there is evidence that leukocyte migration is an important checkpoint in its control. This hypothesis is based on the fact that leukocyte entry into these organs is restricted by physical barriers and that the collapse of these obstacles marks a critical step in the development of inflammatory/autoimmune disease at these sites. Numerous studies in a variety of experimental systems have characterized the molecular and cellular mechanisms involved in leukocyte homing to immune-privileged organs. Recently, two-photon microscopy has revealed critical insights into the events occurring in the extravascular space of immune-privileged organs, including locomotion patterns and interactive behavior of leukocytes in the interstitial space. Here, we review our current understanding of immune cell migration to and within immune-privileged organs and highlight how this knowledge may be exploited for immunotherapeutic purposes.     

Effect of specific antigen stimulation on intraepithelial lymphocyte migration to small intestinal mucosa

Migration of intraepithelial lymphocytes (IELs) into intestinal epithelium is not yet well understood. We established an IEL-cell line from ovalbumin (OVA) 23-3 transgenic (Tg) mice and investigated the effect of antigen stimulation on the dynamic process of IEL migration into small intestinal mucosa. The cell line was a T cell receptor (TCR) alphabeta(+) CD4(+) CD8(-) phenotype, expressing alphaEbeta7 integrin in 90% of cells. Under intravital microscopy, the lined IELs adhered selectively to the microvessels of the intestinal villus tip of the Tg mice. The accumulation of IELs was significantly inhibited by an antibody against beta7-integrin and MAdCAM-1. When IELs were stimulated with OVA, the accumulation was attenuated compared to that of resting cells, with decreased expression of alphaEbeta7 integrin. In Tg mice fed with OVA, the number of IELs which migrated in the villus mucosa was significantly smaller than in the non-fed controls. The preferential migratory capacity of IELs to villus mucosa may be altered by specific antigen stimulations.     

Leukocyte-endothelial interactions in pial arterioles and venules on development of cerebral ischemia in rats

In vivo microscopy was used to study the interaction between leukocytes and the pial venular and arteriolar endothelium in rats during cerebral ischemia evoked by bilateral ligation occlusion of the carotid arteries. Specimens were obtained from 40 arterioles and 30 venules (diameter up to 40 μm) of the pia mater from Wistar rats (n = 7) subjected to ischemia for 5 h to respiratory arrest. The experimental data demonstrated significant differences in changes in the adhesion of leukocytes to the endothelium of arterial and venous microvessels during the development of hypoxia. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 1, pp. 45–52, January, 2008.     

Chemoattractant-induced firm adhesion of leukocytes to vascular endothelium in vivo is critically dependent on initial leukocyte rolling

Leukocyte rolling and firm adhesion at the venular endothelium are two discrete events in the cellular inflammatory response mediated via selectin and integrin adhesion molecules, respectively. The dependency of chemoattractant-induced firm leukocyte adhesion on the preceding rolling interaction was investigated in rat mesenteric microvessels through use of intravital microscopy. Leukocyte rolling was dose-dependently inhibited by systemic treatment with the sulphated polysaccharide fucoidin. The firm leukocyte adhesion following stimulation with the chemotactic peptide fMLP was similarly inhibited when fMLP challenge was performed subsequent to inhibition of leukocyte rolling by fucoidin. Thus, based on paired observations in single venules before and after fucoidin treatment, reduced rolling leukocyte flux prior to fMLP challenge was paralleled over a wide range by a proportional decrease in fMLP-induced leukocyte adhesion. The results demonstrate quantitatively a close relationship between the extent of leukocyte rolling and the magnitude of the subsequent firm adhesion response, and, that an initial rolling interaction is a precondition for firm adhesion to occur at physiological blood flow rates in vivo.     

Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune‐mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1‐deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)_35–55‐induced EAE, a model of human MS. The lack of PLD1 reduced chemokine‐mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1‐deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1‐dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.     

Glatiramer acetate attenuates the pro‐migratory profile of adhesion molecules on various immune cell subsets in multiple sclerosis

An altered expression pattern of adhesion molecules (AM) on the surface of immune cells is a premise for their extravasation into the central nervous system (CNS) and the formation of acute brain lesions in multiple sclerosis (MS). We evaluated the impact of glatiramer acetate (GA) on cell‐bound and soluble AM in the peripheral blood of patients with relapsing–remitting MS (RRMS). Fifteen patients treated de novo with GA were studied on four occasions over a period of 12 months. Surface levels of intracellular cell adhesion molecule (ICAM)‐1, ICAM‐3, lymphocyte function‐associated antigen (LFA)‐1 and very late activation antigen (VLA)‐4 were assessed in T cells (CD3⁺CD8⁺, CD3⁺CD4⁺), B cells, natural killer (NK) cells, natural killer T cells (NK T) and monocytes by five‐colour flow cytometry. Soluble E‐selectin, ICAM‐1, ICAM‐3, platelet endothelial cell adhesion molecule (PECAM)‐1, P‐selectin and vascular cell adhesion molecule (VCAM)‐1 were determined with a fluorescent bead‐based immunoassay. The pro‐migratory pattern in RRMS was verified by comparison with healthy controls and was characterized by up‐regulation of LFA‐1 (CD3⁺CD4⁺ T cells, B cells), VLA‐4 (CD3⁺CD8⁺ T cells, NK cells), ICAM‐1 (B cells) and ICAM‐3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA‐1 (CD3⁺CD4⁺) and VLA‐4 (CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NK T, monocytes). Further effects included lowering of ICAM‐1 and ICAM‐3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro‐migratory expression profile of cell‐bound AM is altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects.     

The role of alpha(4) and LFA-1 integrins in selectin-independent monocyte and neutrophil migration to joints of rats with adjuvant arthritis

Monocytes and neutrophils are chronically recruited to joints in rheumatoid arthritis. In the joints of rats with adjuvant arthritis, this is mediated, in part, by selectin-dependent and selectin-independent mechanisms. To define the selectin-independent mechanisms, (51)Cr-labeled blood monocytes, (111)In-labeled neutrophils and function blocking mAb to the selectins and integrins were utilized. Integrins contributed to the selectin-independent monocyte migration to arthritic joints with 58-70% inhibition of this recruitment by anti-alpha(4) or anti-LFA-1 mAb, relative to selectin blockade alone. alpha(4) plus P-selectin blockade was as effective as combined blockade of alpha(4), P-, E- and L-selectin, mediating approximately 83% of the overall monocyte migration to the joints. In contrast, LFA-1 was the predominant selectin-independent mechanism for neutrophil recruitment to the joints. LFA-1 together with P-selectin had essential roles in the talar joint. In dermal inflammation in the arthritic rats, LFA-1 accounted for most (69%) of the selectin-independent monocyte migration to the chemoattractant C5a(desArg) (zymosan-activated serum), whereas LFA-1 and Mac-1 both contributed to selectin-independent neutrophil recruitment to C5a(desArg). alpha(4) integrin and P-selectin in concert mediated monocyte recruitment to lipopolysaccharide and IFN-gamma lesions (81%). Thus: (1) either alpha(4) or LFA-1 can mediate monocyte migration to arthritic joints in the absence of selectin function and alpha(4) together with P-selectin is particularly important; (2) LFA-1 is the predominant mechanism of selectin-independent migration of neutrophils to inflamed joints; and (3) in arthritic rats, selectin-independent migration of monocytes and neutrophils to dermal inflammation is mediated by alpha(4) or LFA-1 or both LFA-1 and Mac-1, depending on the leukocyte type, and inflammatory stimulus.     

Mechanisms regulating regional localization of inflammation during CNS autoimmunity

Multiple sclerosis (MS) is a disease of the central nervous system (CNS) characterized by inflammatory, demyelinating lesions localized in the brain and spinal cord. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is induced by activating myelin-specific T cells and exhibits immune cell infiltrates in the CNS similar to those seen in MS. Both MS and EAE exhibit disease heterogeneity, reflecting variations in clinical course and localization of lesions within the CNS. Collectively, the differences seen in MS and EAE suggest that the brain and spinal cord function as unique microenvironments that respond differently to infiltrating immune cells. This review addresses the roles of the cytokines interferon-γ and interleukin-17 in determining the localization of inflammation to the brain or spinal cord in EAE.     

Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis (MS). EAE, induced by immunisation with myelin-associated autoantigens, is characterised by an inflammatory infiltrate in the central nervous system (CNS) associated with axonal degeneration, demyelination and damage. We have recently shown in an experimental mouse model of autoimmune gastritis that methylprednisolone treatment induces a reversible remission of gastritis with regeneration of the gastric mucosa. Here, we examined the effect of oral methylprednisolone on the mouse EAE model of human MS induced by immunisation with myelin oligodendrocyte glycoprotein peptide (MOG_35–55). We examined the clinical scores, CNS pathology and lymphocyte reactivity to MOG_35–55 following treatment and withdrawal of the steroid. Methylprednisolone remitted the clinical signs of EAE and the inflammatory infiltrate in the CNS, accompanied by loss of lymphocyte reactivity to MOG_35–55 peptide. Methylprednisolone withdrawal initiated relapse of the clinical features, a return of the CNS inflammatory infiltrate and lymphocyte reactivity to MOG_35–55 peptide. This is the first study to show that methylprednisolone induced a reversible remission in the clinical and pathological features of EAE in mice accompanied by loss of lymphocyte reactivity to the encephalitogen. This model will be useful for studies directed at a better understanding of mechanisms associated with steroid-induced disease remission, relapse and remyelination and also as an essential adjunct to an overall curative strategy.     

Mechanisms of leukocyte trafficking in allergic diseases: insights into new therapies targeting chemokines and chemokine receptors

Marked inflammatory infiltration by activated leukocytes is a characteristic feature of allergic diseases. Elucidation of the mechanisms of leukocyte trafficking in allergic diseases would identify targets to establish novel anti-inflammatory strategies for treatment of these diseases. Leukocyte trafficking is controlled by tissue-specific expression of chemokines and chemokine receptor expression on the leukocyte surface. Here, we review the role of chemokines and their receptors in leukocyte trafficking to inflammatory sites in allergic diseases and discuss therapeutic strategies targeting chemokine networks for treatment of these diseases.     

活化白细胞黏附分子在宫颈癌中的表达及功能研究

目的：探讨活化白细胞黏附分子（ activated leukocyte celladhesion molecule, ALCAM）在宫颈癌中的表达及在肿瘤转移中的作用。方法利用免疫组织化学法,测定20例正常宫颈组织,20例宫颈上皮内瘤变组织和40例宫颈鳞癌组织中的ALCAM蛋白表达;体外实验中,测定沉默ALCAM基因的表达,利用Trans?Well迁移实验研究HeLa细胞的侵袭能力。结果 ALCAM在正常宫颈组织中低表达（10％）,宫颈上皮内瘤变组织高表达（55％）,宫颈鳞癌组织中高表达（77．5％）,3组比较有显著差异（P＜0．05）。在体外阻断宫颈癌细胞系HeLa细胞的ALCAM基因表达,发现HeLa细胞侵袭力下降。结论 ALCAM在不同恶变程度宫颈癌中的表达存在差异,并在宫颈癌的发生发展过程中表达量呈上升趋势,这对宫颈癌的诊断和预后具有一定的指导意义。     

Leukocyte behaviour and permeability in the rat mesenteric microcirculation following induction of ovulation

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS), a highly dangerous and incompletely understood complication of ovulation induction with exogenous gonadotrophins, can include haemoconcentration, hypovolaemia, hypotension, acute renal insufficiency, thromboembolism and ultimately death. Using intravital microscopy, we examined microvascular permeability and leukocyte-endothelial cell interactions in the rat mesenteric microcirculation associated with induction of ovulation. METHODS: In female rats treated with hMG and hCG, mesenteric venules were observed by intravital microscopy assisted by a video imager. Erythrocyte velocity was monitored, and rolling and adhesion of leukocytes were studied by transmission video images. Transvascular leakage of fluorescein isothiocyanate-labelled albumin was assessed by epi-illumination. RESULTS: Administration of hMG and hCG significantly increased vascular protein leakage within a few hours, and also reduced rolling velocities of leukocytes in venules and increased numbers of leukocytes adherent to endothelium at 16 h following hCG injection. The administration of antibodies against intracellular adhesion molecule (ICAM)-1 inhibited these reactions. CONCLUSION: By induction of ovulation, vascular permeability is increased not only at the surface of the ovary but also in the mesentery. Alteration of leukocyte behaviour in the microcirculation through mechanisms involving ICAM-1 is one likely cause of the protein leakage.     

Immune Mechanisms in the Pathogenesis of Demyelinating Diseases

The loss of myelin which characterises many human and experimental demyelinating diseases, among them multiple sclerosis, is thought to be immune mediated, but the precise mechanisms responsible remain unknown despite intense research. Normally, myelin in the central nervous system (CNS) is protected from systemic immune responses by the blood brain barrier, which separates nervous tissue from the peripheral circulation. Here we review evidence suggesting that an understanding of the demyelinating disorders may be helped by considering their immune pathogenesis in two stages. The first is damage to the blood brain barrier; this appears to be cell mediated, and allows infiltration into the CNS of other immune effectors. These include complement and also macrophages, which together may mediate the second stage, injury to the myelin/oligodendrocyte complex.     

胃癌组织活化白细胞黏附分子的表达及其与临床病理的相关性

Objective To investigate the expression of activated leukocyte cell adhesion molecule (ALCAM) in gastric carcinoma and examine its correlation with clinicopathology. Methods The level of ALCAM expression was determined by quantitative real-time PCR and immunohistochemistry in cancer and normal tissues, and its correlation with clinicopathology was analyzed. Results The mRNA level of ALCAM gene in gastric carcinoma tissue [0.008 7(0.005 8-0.016 7) ] was significantly higher compared with normal mucosa [0.001 1 (0.000 1-0.063 5)P＜0.05], especially in poorly differentiated carcinoma [0.012 8(0.008 2-0.016 7) ] and lymph nede-invasive carcinoma [0.017 4 (0.008 6-0.030 7) ]. By immunohistochemistry,expression of ALCAM was stronger in gastric carcinoma tissue tested positive by either cytoplasm or membrane staining as compared with normal tissues. More ALCAM-positive cells were found in lymph nodeinvasive cancer tissues as compared with those without node involvement [ ( 100%(21/21 )vs 66.7%(8/21 ),P＜0.05 ]. Conclusions Over-expression of ALCAM is found in gastric carcinoma tissues and correlated well with lymph node metastasis.     

Identification and characterization of ligands for L-selectin in the kidney. I. Versican, a large chondroitin sulfate proteoglycan, is a ligand for L-selectin

Ligands for a leukocyte adhesion molecule, L-selectin, are expressed not only in the specific vascular endothelium in lymph nodes and Peyer's patches but also in the extravascular tissues such as the brain white matter, choroid plexus and the kidney distal straight tubuli. However, the biological significance of these extravascular ligands is currently unknown. We now report the purification and characterization of a novel extravascular ligand for L-selectin in the kidney using a tubule-derived cell line, ACHN. Binding of L-selectin-IgG chimera (LEC-IgG) to the isolated ligand was specifically blocked with either (i) anti-L-selectin mAb, (ii) EDTA, (iii) fucoidan, (iv) chondroitin sulfate (CS) B or CS E, or (v) treatment with chondroitinases. Partial amino acid sequencing, Western blotting and immunoprecipitation analyses showed that a major ligand for L-selectin in ACHN cells is versican of 1600 kDa. Histochemical as well as biochemical analyses verified that a versican subspecies in the kidney was indeed reactive with L-selectin. Studies with cell lines including those derived from the kidney indicated that a certain glycoform and/or splice form of versican is reactive with L-selectin. Under pathological conditions such as those induced by unilateral ureteral obstruction, versican was shed from the distal straight tubuli and became localized in the adjacent vascular bundles around which a substantial leukocyte infiltration was concomitantly observed. Possible involvement of versican in leukocyte trafficking into the kidney under diseased conditions is discussed.