Liver fibrosis by diffusion-weighted magnetic resonance imaging and its functional correlates in fontan patients.

OBJECTIVES: The evaluation of Fontan-associated liver disease is often challenging. Diffusion-weighted magnetic resonance imaging can detect hepatic fibrosis from capillary perfusion and diffusion abnormalities from extracellular matrix accumulation. This study investigated its role for evaluation of liver disease in Fontan patients and explored possible diagnostic method for early detection of advanced liver fibrosis. METHODS: Stable adult Fontan patients who could safely receive magnetic resonance examination were enrolled and blood biomarkers, transient elastography were also examined. RESULTS: Forty-six patients received diffusion weighted imaging and 58.7% were with advanced liver fibrosis (severe liver fibrosis 37.0%, and cirrhosis 21.7%). Two parameters of hepatic dysfunction, platelet counts (Spearman's ρ: -0.456, p = 0.001) and cholesterol levels (Spearman's ρ: -0.383, p = 0.009), decreased with increasing severities of fibrosis. Using transient elastography, a cutoff value 14.2 kPa predicted presence of advanced liver fibrosis, but with a low positive predictive value. When we included platelet count, cholesterol, post-Fontan years and TE values as a composite, the prediction capability of advanced liver fibrosis was the most satisfactory (c statistic 0.817 ± 0.071, p < 0.001). A cutoff value of 5.0 revealed a sensitivity of 78% and a specificity of 82%. CONCLUSIONS: In Fontan patients, diffusion-weighted imaging is helpful in detection of liver fibrosis that was correlated with hepatic dysfunction. A simple score was proposed for long-term surveillance and early detection of advanced liver disease in adult Fontan patients. For adult Fontan patients with calculated score > 5.0, we may consider timely diffusion-weight imaging and early management for liver complications.

Short-chain fatty acids ameliorate allergic airway inflammation via sequential induction of PMN-MDSCs and Treg cells.

Background: Reinforcement of the immune-regulatory pathway is a feasible strategy for prevention and therapy of allergic asthma. The short-chain fatty acids (SCFAs) acetate, propionate, and butyrate are pleiotropic microbial fermentation products known to induce regulatory T (Treg) cells and exert an immune-regulatory effect. The cellular mechanism underlying SCFA immune regulation in asthma is not fully understood. Objective: We investigated the role of myeloid-derived suppressor cells (MDSCs) and Treg cells, the immune-regulatory cells of innate and adaptive origin, respectively, in SCFA-elicited protection against allergic airway inflammation. Methods: BALB/c mice were given SCFA-containing drinking water before being rendered asthmatic in response to ovalbumen. When indicated, mice were given a GR1-depleting antibody to investigate the function of MDSCs in allergic inflammation of the airways. MDSCs were sorted to examine their immunosuppressive function and interaction with T cells. Results: The mice receiving SCFAs developed less severe asthma that was accompanied by expansion of PMN-MDSCs and Treg cells. Mice depleted of PMN-MDSCs exhibited aggravated asthma, and the protective effect of SCFAs was abrogated after PMN-MDSC depletion. SCFAs were able to directly induce T-cell differentiation toward Treg cells. Additionally, we found that PMN-MDSCs enhanced Treg cell expansion in a cell contact-dependent manner. Whilst membrane-bound TGF-ß has been shown to induce Treg cell differentiation, we found that MDSCs upregulated surface expression of TGF-ß after coculture with T-cells and that MDSC-induced Treg cell differentiation was partially inhibited by TGF-ß blockage. Conclusions: Although previous studies revealed Treg cells as the effector mechanism of SCFA immune regulation, we found that SCFAs ameliorate allergic airway inflammation by relaying immune regulation, with sequential induction of PMN-MDSCs and Treg cells.

Asthma in the Precision Medicine Era: Biologics and Probiotics.

Asthma is a major global health issue. Over 300 million people worldwide suffer from this chronic inflammatory airway disease. Typical clinical symptoms of asthma are characterized by a recurrent wheezy cough, chest tightness, and shortness of breath. The main goals of asthma management are to alleviate asthma symptoms, reduce the risk of asthma exacerbations, and minimize long-term medicinal adverse effects. However, currently available type 2 T helper cells (Th2)-directed treatments are often ineffective due to the heterogeneity of the asthma subgroups, which manifests clinically with variable and poor treatment responses. Personalized precision therapy of asthma according to individualized clinical characteristics (phenotype) and laboratory biomarkers (endotype) is the future prospect. This mini review discusses the molecular mechanisms underlying asthma pathogenesis, including the hot sought-after topic of microbiota, add-on therapies and the potential application of probiotics in the management of asthma.

Multi-Faceted Notch in Allergic Airway Inflammation.

Notch is an evolutionarily conserved signaling family which iteratively exerts pleiotropic functions in cell fate decisions and various physiological processes, not only during embryonic development but also throughout adult life. In the context of the respiratory system, Notch has been shown to regulate ciliated versus secretory lineage differentiation of epithelial progenitor cells and coordinate morphogenesis of the developing lung. Reminiscent of its role in development, the Notch signaling pathway also plays a role in repair of lung injuries by regulation of stem cell activity, cell differentiation, cell proliferation and apoptosis. In addition to functions in embryonic development, cell and tissue renewal and various physiological processes, including glucose and lipid metabolism, Notch signaling has been demonstrated to regulate differentiation of literally almost all T-cell subsets, and impact on elicitation of inflammatory response and its outcome. We have investigated the role of Notch in allergic airway inflammation in both acute and chronic settings. In this mini-review, we will summarize our own work and recent advances on the role of Notch signaling in allergic airway inflammation, and discuss potential applications of the Notch signaling family in therapy for allergic airway diseases.

Increased Plasma Acetylcarnitine in Sepsis Is Associated With Multiple Organ Dysfunction and Mortality: A Multicenter Cohort Study.

OBJECTIVES: Recent metabolomic studies of sepsis showed that increased circulatory acylcarnitines were associated with worse survival. However, it is unknown whether plasma carnitine and acylcarnitines can reflect the severity of sepsis, and the role of specific acylcarnitines in prognostic assessment need further confirmation. This study aimed to clarify these questions. DESIGN: Prospective multicenter cohort studies with derivation and validation cohort design. SETTING: ICUs at two medical centers and three regional hospitals in Taiwan. PATIENTS: Patients with sepsis and acute organ dysfunction were enrolled. Recruitment of the derivation (n = 90) and validation cohorts (n = 120) occurred from October 2010 through March 2012 and January 2013 through November 2014, respectively. INTERVENTIONS: Plasma samples were collected immediately after admission, and the levels of carnitine and acylcarnitines were measured by ultra-high performance liquid chromatography-mass spectrometry. MEASUREMENTS AND MAIN RESULTS: In the derivation cohort, increased plasma levels of short- and medium-chain acylcarnitines were significantly associated with hepatobiliary dysfunction, renal dysfunction, thrombocytopenia, and hyperlactatemia. However, acetylcarnitine is the only acylcarnitine significantly correlating with various plasma cytokine concentrations and also associated with blood culture positivity and 28-day mortality risk. The association between plasma acetylcarnitine and multiple organ dysfunction severity, blood culture positivity, and 28-day mortality, was confirmed in the validation cohort. Patients with high plasma acetylcarnitine (≥ 6,000 ng/mL) had significantly increased 28-day mortality compared with those with plasma acetylcarnitine less than 6,000 ng/mL (52.6% vs 13.9%; hazard ratio, 5.293; 95% CI, 2.340-11.975; p < 0.001 by Cox proportional hazard model). CONCLUSIONS: We confirm that plasma acetylcarnitine can reflect the severity of organ dysfunction, inflammation, and infection in sepsis and can serve as a prognostic biomarker for mortality prediction.

Notch Ligand DLL4 Alleviates Allergic Airway Inflammation via Induction of a Homeostatic Regulatory Pathway.

Notch is a pleiotropic signaling family that has been implicated in pathogenesis of allergic airway diseases; however, the distinct function of individual Notch ligands remains elusive. We investigated whether Notch ligands, Jagged1 and DLL4, exert differential effects in OVA-induced allergic asthma. We found that whilst Jagged1 inhibition mitigated Th2-dominated airway inflammation, blockage of DLL4 aggravated the Th2-mediated asthma phenotypes. Additionally, Jagged1 signaling blockage enhanced IL-17 production and neutrophilic airway infiltration. In vitro, exogenous Jagged1 induced Th2-skewed responses, whereas augmented DLL4 signaling displayed a dual role by promoting expansion of both Tregs and Th17. In vivo, DLL4 blockage impaired Treg differentiation which plausibly resulted in exaggerated asthma phenotypes. On the contrary, administration of DLL4-expressing antigen-presenting cells promoted endogenous Treg expansion and ameliorated the allergic responses. Therefore, whilst Jagged1 induces Th2-skewed inflammation, DLL4 elicits an essential self-regulatory mechanism via Treg-mediated pathway that counterbalances Jagged1-induced Th2 responses and facilitates resolution of the airway inflammation to restore homeostasis. These findings uncover a disparate function of Jagged1 and DLL4 in allergic airway diseases, hinting feasibility of Notch ligand-specific targeting in therapy of allergic airway diseases.

Lymph node trafficking of regulatory T cells is prerequisite for immune suppression.

Regulatory T cells have a crucial role in health and disease because of their immune regulation function. However, the anatomic sites where regulatory T cells exert optimal immune regulation are open to debate. In our current study with the use of a shear-stress flow assay, we found that regulatory T cells exhibited significantly decreased adhesion to either activated endothelial monolayer or intercellular adhesion molecule 1 or E-selectin-coated surfaces compared with activated effector T cells. The less transmigration capacity of the regulatory T cells prompted our speculation of preferential lymph node localization for the regulatory T cells that endowed these cells with immune regulation function in the most efficient manner. To test this hypothesis, the role of lymph node localization in regulatory T cell-mediated immune suppression was evaluated with a footpad inflammation model. We found that adoptively transferred regulatory T cells inhibited the development of footpad inflammation. In addition, although blockage of CCR7 or CD62L had no effect on the immune suppressive function of the regulatory T cells per se, pretreatment of the regulatory T cells with either CCR7 or CD62L blocking antibodies prevented their recruitment into draining lymph nodes and concomitantly abrogated the immune suppressive effects of adoptively transferred regulatory T cells during footpad inflammation. Our data demonstrate the crucial role of lymph node localization in regulatory T cell-mediated immune suppression and suggest a probable hierarchy in the anatomic sites for optimal immune regulation. Elucidating the relationships between the transmigration characteristics of the regulatory T cells and their immune regulation function will provide insightful information for regulatory T cell-based cell therapy.

Feedback regulation of IFN-α/ß signaling by Axl receptor tyrosine kinase modulates HBV immunity.

Hepatitis B virus (HBV) is known to cause age-dependent infection outcomes wherein most infections during young age result in chronicity. The mechanism underlying the differential outcome remains elusive. By using hydrodynamic injection of the replication-competent pAAV-HBV, we established a mouse model in which HBV persistence was generated in 4-5 w/o C57BL/6 young mice, but not in adult mice over 10 w/o. HBV-tolerant young mice expressed higher interferon (IFN)-α/ß levels in hepatocytes and intrahepatic plasmacytoid DCs (pDCs) than adult mice after pAAV-HBV injection. Excessive IFN-α/ß expression in young mice was associated with induction of the Axl regulatory pathway and expansion of intrahepatic Treg cells. In line with these findings, augmented IFN-ß expression increased Axl expression in the liver and HBV persistence in adult mice, whereas IFN-α/ß signaling blockage decreased Axl expression and HBV persistence in young mice. Accordingly, Axl overexpression decreased HBV clearance of adult mice whereas Axl silencing enhanced HBV clearance of young mice. In vitro, IFN-ß priming of pDCs and Axl-overexpressing macrophages enhanced Treg-cell differentiation. These findings suggest that age-dependent HBV chronicity is attributed to IFN-ß-Axl immune regulation, which is selectively induced in young mice by excessive IFN-α/ß production at early stage of HBV infection.

Severe lymphopenia is associated with elevated plasma interleukin-15 levels and increased mortality during severe sepsis.

Sepsis-related mortality has been found increased in RAG-1 knockout mice. However, in patients admitted to medical intensive care units, it is unknown whether severe lymphocyte depletion at admission is associated with increased interleukin (IL)-7 and IL-15 levels in circulation, and increased mortality. We prospectively enrolled 92 patients who were admitted to medical intensive care units for severe sepsis or septic shock. At admission, 24 patients (26.1%) had severe lymphopenia, defined as lymphocyte counts of less than 0.5 × 10(3)/µL. Severe lymphopenia was associated with significantly higher plasma levels of tumor necrosis factor α, IL-6, IL-8, and IL-10 and was also independently associated with 28-day mortality (adjusted hazard ratio, 3.532; 95% confidence interval, 1.482-8.416; P = 0.004). The levels of plasma IL-15, but not IL-7, were increased modestly in patients with severe lymphopenia compared with those without (median, 12.2 vs. 6.4 pg/mL; P = 0.005). The elevated plasma IL-15 levels were contrarily associated with significantly decreased B-cell lymphoma 2 mRNA expression in peripheral blood mononuclear cells. In conclusion, severe lymphopenia was associated with increased mortality in patients with severe sepsis. We found that patients with sepsis with severe lymphopenia had down-regulated B-cell lymphoma 2 mRNA expression in peripheral blood mononuclear cells, despite increased plasma IL-15 concentrations. Whether IL-7 and IL-15 are insufficient in patients with severe lymphopenia during severe sepsis warrants further investigations.

Prognostic significance of TWEAK expression in colorectal cancer and effect of its inhibition on invasion.

BACKGROUND: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been implicated in tumor development and progression. The aim of this study was to investigate the role of TWEAK in colorectal cancer (CRC) progression. METHODS: To investigate the involvement of TWEAK in the progression of human CRC, normal, and tumor specimens from 174 patients were analyzed immunohistochemically for the expression of TWEAK. TWEAK recombinant protein treatment, transfection of expression plasmids, and small interfering RNA to knockdown TWEAK expression were performed to test invasive ability with a Boyden chamber. The mRNA expression profile in recombinant TWEAK treatment was compared to a control group by microarray analysis. To identify downstream effectors, Raf kinase inhibitor (RKIP) and its correlation with TWEAK in vitro and in vivo were examined by quantitative real-time polymerase chain reaction and invasion assays. RESULTS: CRC patients whose tumors displayed high TWEAK expression had a statistically significantly higher overall survival and a disease-free advantage over those with a low TWEAK expression. In in vitro invasion assays, alterations in TWEAK expression in CRC cell lines inversely modulated their invasive ability. By means of integrated genomics, we identified RKIP as a downstream effector in TWEAK-mediated invasion inhibition. Knockout of RKIP expression in HCT116 cells by short hairpin RNA (shRKIP) resulted in increased invasiveness. Clinically, RKIP and TWEAK mRNA expression showed strong positive correlations in CRC patient samples. CONCLUSIONS: Our results implicate TWEAK as a key regulator of CRC invasion, and it appears to be a useful prognostic factor for patients with CRC.

Lipopolysaccharide-induced Notch signaling activation through JNK-dependent pathway regulates inflammatory response.

BACKGROUND: Notch and TLR pathways were found to act cooperatively to activate Notch target genes and to increase the production of TLR-induced cytokines in macrophages. However, the mechanism of LPS-induced Notch activation and its role in sepsis still remains unclear. METHODS: We analyzed the expression patterns of Notch components in a LPS-stimulated murine macrophage cell line using real-time PCR and western blotting. The role of DAPT, a gamma-secretase inhibitor that is known to be a potent Notch inhibitor, in LPS-induced cytokine release and experimental sepsis in mice was also explored. Student's t-test was used to analyze the difference between the two groups. RESULTS: We found that Notch signaling was activated after LPS stimulation. The expression of Jagged 1, a Notch ligand, induced by LPS occurred in a JNK-dependent manner. In addition, Notch target genes were upregulated by early Notch-independent activation followed by delayed Notch-dependent activation after LPS stimulation. Disruption of Notch signaling by DAPT attenuated the LPS-induced inflammatory responses, including vascular endothelial growth factor (VEGF) and high-mobility group box chromosomal protein 1 (HMGB1), both in vitro and in vivo and partially improved experimental sepsis survival. CONCLUSIONS: These findings support the existence of a synergistic effect of Notch signaling and the LPS pathway both in vitro and in vivo. Therefore, in the future Notch inhibitors may be utilized as adjunctive agents for the treatment of sepsis syndrome.

Notch signaling prevents mucous metaplasia in mouse conducting airways during postnatal development.

Goblet cell metaplasia and mucus overproduction contribute to the pathogenesis of chronic lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). Notch signaling regulates cell fate decisions and is crucial in controlling goblet cell differentiation in the gut epithelium. Little is known, however, about how endogenous Notch signaling influences the goblet cell differentiation program that takes place in the postnatal lung. Using a combination of genetic and in vitro approaches here we provide evidence of a novel role for Notch in restricting goblet cell differentiation in the airway epithelium during the postnatal period. Conditional inactivation of the essential Notch pathway component Pofut1 (protein O-fucosyltransferase1) in Tgfb3-Cre-expressing mice resulted in an aberrant postnatal airway phenotype characterized by marked goblet cell metaplasia, decreased Clara cell number and increase in ciliated cells. The presence of the same phenotype in mice in which the Notch transcriptional effector Rbpjk was deleted indicated the involvement of the canonical Notch pathway. Lineage study in vivo suggested that goblet cells originated from a subpopulation of Clara cells largely present in proximal airways in which Notch was disrupted. The phenotype was confirmed by a panel of goblet cell markers, showed no changes in cell proliferation or altered expression of proinflammatory cytokines and was associated with significant downregulation of the bHLH transcriptional repressor Hes5. Luciferase reporter analysis suggested that Notch directly repressed MUC5AC transcription in lung epithelial cells. The data suggested that during postnatal life Notch is required to prevent Clara cells from differentiating into goblet cells.

Hepatitis B virus surface antigen can activate dendritic cells and modulate T helper type immune response.

Hepatitis B virus surface antigen (HBsAg) is a major antigen of hepatitis B virus (HBV). Dendritic cells (DC) of HBV carriers have been reported to exhibit functional impairment. In this study, the role of HBsAg on mice bone marrow-derived dendritic cells and immune responses in vivo was studied. The immune modulatory function of HBsAg was explored by using mice bone marrow-derived dendritic cells in vitro and also by examining an ovalbumin (OVA) specific immune response in vivo. Treatment of dendritic cells with HBsAg resulted in enhanced cell surface expression of cluster of differentiation (CD) 80, CD83, CD86, and major histocompatibility complex (MHC) class II, and enhanced production of interleukin (IL)-12 p40 and IL-12 p70. Treatment of dendritic cells with HBsAg resulted in decreased T cell secretion of IL-5 by OVA stimulation. In addition, the results showed stronger OVA-specific immunoglobulin (Ig) M and weaker IgG responses in mice sera when they had been immunized with OVA and co-injected with HBsAg. It was also found that the mice exhibited significant enhancement of anti-OVA IgG2a antibody (Ab), as well as marked inhibition of IgG1 Ab production. In cellular immune responses, IL-5 production was significantly decreased and interferon (IFN)-γ increased in the group co-injected with HBsAg. On the other hand, the induction of lymphoproliferative response to OVA stimulation in spleen cells was decreased in the HBsAg co-injected group. These results demonstrate that HBsAg can affect the differentiation of T helper (Th) cells, which might provide a strategy for improving its prophylactic and therapeutic efficacy.

Cellular localization of the organic cation transporters, OCT1 and OCT2, in brain microvessel endothelial cells and its implication for MPTP transport across the blood-brain barrier and MPTP-induced dopaminergic toxicity in rodents.

The cellular localization of organic cation transporter (OCT) 1 and OCT2 in isolated brain microvessel endothelial cells from humans, rats, and mice and in cultured adult rat brain endothelial cells was examined by confocal microscopy and in isolated luminal and abluminal membrane fractions by Western blot analysis. Cellular uptake of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was measured with or without OCT1/OCT2 silencing. The interaction between MPTP and amantadine was studied by in vitro kinetic analysis and in vivo brain microdialysis. MPTP-induced dopaminergic toxicity was examined by measuring dopamine levels in the brain striatum and by positron emission tomography scanning. The results showed that both OCT1 and OCT2 were mainly expressed on the luminal side of brain microvessel endothelial cells and adult rat brain endothelial cells. Cellular uptake of MPTP was significantly (p < 0.05) decreased by about 53%, 60%, or 91% following silencing of OCT1, OCT2, or both, respectively. Amantadine competitively inhibited MPTP uptake in vitro and significantly (p < 0.05) reduced the area under the time-concentration curve for MPTP and MPP(+) in the brain extracellular fluid in rats and mice by 65-70% and 35-85%, respectively. MPTP-induced dopaminergic toxicity in mice was ameliorated by amantadine without stimulating dopamine turnover. In conclusion, OCT1 and OCT2 are important for MPTP transfer across the blood-brain barrier and amantadine reduces the blood-brain barrier transfer of MPTP and MPTP-induced dopaminergic toxicity in rodents.

Regulatory T cells negatively regulate neovasculature of airway remodeling via DLL4-Notch signaling.

Regulatory T cells (Treg) have been shown to prevent the development of allergic asthma; however, the role of Treg in asthma with established airway remodeling is unknown. To address this, we exploited an OVA-induced chronic asthma mouse model wherein Treg were adoptively transferred to the mice at chronic stage of the model. We found that among the structural alterations of airway remodeling, Treg selectively reduced the vessel numbers in both peritracheal and peribronchial regions and the lung parenchyma. Extracellular matrix deposition, mucus metaplasia, muscular hyperplasia, and vasodilation, as were also induced by chronic allergen challenge, were not affected by Treg. TUNEL staining of the lung sections revealed an increased endothelial cell (EC) apoptosis in mice receiving Treg transfers compared with their asthmatic counterparts. By using Matrigel angiogenesis assays, we showed that Treg inhibited EC angiogenesis both in vitro and in vivo. Treg preferentially expressed Notch ligand DLL4, and an anti-DLL4 blocking Ab abrogated the inhibitory effect of Treg on EC tube formation. In vivo, decreased airway and lung vessel numbers as well as ameliorated airway hyperresponsiveness after Treg transfers were reverted when Treg-derived DLL4 signal was blocked by the anti-DLL4 Ab. Our findings demonstrate a novel function of Treg whereby Treg down-regulate remodeling angiogenesis via proapoptotic DLL4-Notch signaling, and suggest a therapeutic potential of Treg in alleviating airway hyperresponsiveness of chronic asthma.

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA(1/3), COX-2, and NF-kappaB activation- and EGFR transactivation-dependent mechanisms.

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.

ICAM-2 mediates neutrophil transmigration in vivo: evidence for stimulus specificity and a role in PECAM-1-independent transmigration.

ICAM-2 has been implicated in leukocyte transmigration in vitro, but there is little in vivo evidence to support this. To address this, neutrophil migration was investigated in ICAM-2-deficient mice (KO) and in wild-type (WT) mice treated with an anti-ICAM-2 blocking monoclonal antibody (mAb) (3C4). In a peritonitis model, IL-1beta-induced accumulation of neutrophils was significantly reduced in mice treated with 3C4 (51% inhibition) and in KO mice (41% inhibition). In contrast, TNF-alpha- or thioglycolate-induced responses were not suppressed in KO mice. Analysis of IL-1beta-induced leukocyte responses in cremasteric venules of KO animals by intravital microscopy indicated a defect in transmigration (44% inhibition) but not rolling or adhesion. As found before, TNF-alpha-induced leukocyte transmigration was unaltered in the KO mice. WT mice treated with the anti-ICAM-2 mAb also exhibited a selective reduction in leukocyte transmigration in response to IL-1beta while an anti-ICAM-1 mAb inhibited both leukocyte adhesion and transmigration. Interestingly, mAb 3C4 significantly suppressed IL-1beta-induced neutrophil transmigration in PE-CAM-1 KO animals in the peritonitis model but not in the cremaster muscle. The findings provide direct evidence for the involvement of ICAM-2 in neutrophil transmigration in vivo, though this role appears to be stimulus specific. Furthermore, ICAM-2 appears capable of mediating PECAM-1-independent leukocyte transmigration.

Endothelial intercellular adhesion molecule (ICAM)-2 regulates angiogenesis.

Endothelial junctions maintain endothelial integrity and vascular homeostasis. They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis. Junctional adhesion molecules such as vascular endothelial (VE)-cadherin and CD31/platelet endothelial cell adhesion molecule (PECAM) mediate contact between adjacent endothelial cells and regulate leukocyte transmigration and angiogenesis. The leukocyte adhesion molecule intercellular adhesion molecule 2 (ICAM-2) is expressed at the endothelial junctions. In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2-deficient mice and ICAM-2-deficient endothelial cells, we show that the lack of ICAM-2 expression results in impaired angiogenesis both in vitro and in vivo. We show that ICAM-2 supports homophilic interaction, and that this may be involved in tube formation. ICAM-2-deficient cells show defective in vitro migration, as well as increased apoptosis in response to serum deprivation, anti-Fas antibody, or staurosporine. ICAM-2 signaling in human umbilical vein endothelial cells (HUVECs) was found to activate the small guanosine triphosphatase (GTPase) Rac, which is required for endothelial tube formation and migration. These data indicate that ICAM-2 may regulate angiogenesis via several mechanisms including survival, cell migration, and Rac activation. Our findings identify a novel pathway regulating angiogenesis through ICAM-2 and a novel mechanism for Rac activation during angiogenesis.

PECAM-1 (CD31) homophilic interaction up-regulates alpha6beta1 on transmigrated neutrophils in vivo and plays a functional role in the ability of alpha6 integrins to mediate leukocyte migration through the perivascular basement membrane.

Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of alpha(6) integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1-mediated increased expression of alpha(6)beta(1) on transmigrating neutrophils in vivo. An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS((R)) analysis of neutrophils demonstrated increased expression of alpha(6)beta(1) on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of alpha(6) as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice. The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of alpha(6)beta(1) on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1-mediated neutrophil migration through the PBM.