Synergistic regulation mechanism of selectin and integrin on leukocyte adhesion under shear flow

In the process of inflammation, the hydrodynamic process of circulating leukocyte recruitment to the inflammatory site requires the rolling adhesion of leukocytes in blood vessels mediated by selectin and integrin molecules. Although a number of experiments have demonstrated that cooperative effects exist between selectins and integrins in leukocyte rolling adhesion under shear flow, the mechanisms underlying how the mechanics of selectins and integrins synergistically may govern the dynamics of cell rolling is not yet fully resolved. Here we present a mechanical model on selectin- and integrin- jointly mediated rolling adhesion of leukocyte in shear flow, by considering two pairs' binding/unbinding events as Markov processes and describing kinetics of leukocyte by the approach of continuum mechanics. Through examining the dynamics of leukocyte rolling as a function of relative fraction of selectin and integrin pairs, we show that, during recruitment, the elongation of intermittent weak selectin bonds consuming the kinetic energy of rolling leukocyte decelerates the rolling speed and enables the integrin pairs to form strong bonds, therefore achieving the arrestment of leukocyte (firm adhesion). The coexistence of selectins and integrins may also be required for effective phase transition from firm adhesion to rolling adhesion, due to dynamic competition in pairs' formation and elongation. These results are verified by the relevant Monte Carlo simulations and related to reported experimental observations.

Binding of Rap1 and Riam to Talin1 Fine-Tune β2 Integrin Activity During Leukocyte Trafficking

β2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent β2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the β2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that β2 integrin regulation primarily occurs via these two pathways.

Adhesion of monocytes and endothelial cells isolated from the human aorta suppresses by miRNA-PEI particles

Background Knowledge of stenosis in coronary arteries requires an understanding of the cellular and molecular processes that occur throughout the leukocyte rolling process. In this study, the roles of miR-125a-5p and miR-495-3p were investigated on the adhesion of endothelial cells (ECs) isolated from the human aorta. Methods Human primary endothelial cells were obtained from the aorta of people who had died of brain death. Whole blood was used to isolate the monocytes. The miR-125 and miR-495 were predicted and transfected into ECs using Poly Ethylene Imine (PEI). The expression levels of adhesion molecules and monocyte recruitment were identified by the RT-qPCR technique and Leukocyte-Endothelial Adhesion Assay kit, respectively. Results The ICAM-1, ICAM-2 and VCAM-1 expression levels decreased significantly in the miR-495/PEI-transfected ECs (P < 0.05) while in the miR-125/PEI-transfected ECs only the ICAM-2 and ITGB-2 expression levels decreased significantly (P < 0.05) as compared to the miR-synthetic/PEI-transfected ECs. Furthermore, the monocyte adhesion was decreased in the miR-125 and miR-mix/PEI-transfected ECs as compared to the miR-synthetic/PEI-transfected ECs (P = 0.01 and P = 0.04, respectively). Conclusion According to the findings, the efficient relations between miR-125 and adhesion molecules may be responsible for the inhibition of monocyte rolling.

P043 Succinate receptor (SUCNR1) mediates leukocyte-endothelial cell interactions induced by TNFa

Background The Krebs cycle metabolite succinate contributes to inflammatory conditions like arthritis and colitis by activating its receptor SUCNR1. We aimed to analyze whether the succinate-SUCNR1 pathway contributes to the leukocyte-endothelial cell interactions that initiate the inflammatory response. Methods We evaluated leukocyte rolling and adhesion by intravital microscopy in cremaster venules of wild-type (WT) and Sucrn1−/− mice treated, 4h before, with succinate (1 Mm, intraescrotally), combined or not with the common pro-inflammatory cytokine TNFα (500 ng/mice, i.p.), or with their vehicles. We analyzed the activity of the NF-κB signaling pathway in endothelial cells (HUVEC) treated (4h) with succinate (0.5–1 mM) ± TNFα (20ng/ml), by detecting this transcription factor, its inhibitor iκBα, and their phosphorylated forms, by western blot. Results are expressed as mean±SEM (N≥6) and analyzed by ANOVA + Newman-Keuls test. Results In WT mice, succinate tended to induce leukocyte rolling and adhesion, and amplified the effects of TNFα on rolling. SUCNR1-deficient mice failed to increase leukocyte rolling in response to these treatments while showed an attenuated response to the effect of TNFα on leukocyte adhesion (Figure 1). Succinate treatment intensified the activation of the NF-κB pathway in HUVEC treated with TNFα, as shown by the increased IκBα phosphorylation, which allows its degradation, the reduced total IκBα, and the increased NF-κB/IκBα ratio (Figure 2). Conclusion Our results suggest that the endogenous release of succinate and the consequent stimulation of the SUCNR1 mediates the leukocyte-endothelial cell interactions induced by TNFα, and this pro-inflammatory activity may be related with an increased activation of the NF-κB signaling pathway in endothelial cells. Thus, the accumulation of succinate observed in various inflammatory diseases probably contributes to the initiation of the inflammatory focus by promoting the accumulation of leukocytes. Bibliography

Microcirculatory aspects of neurogenic bladder syndrome

The microcirculatory aspects of inflammatory disorders are of importance in the pathology of the urinary system. Our first aim was to perform a comparative analysis of the microcirculatory responses of the urinary bladder in infectious and non-infectious inflammatory animal models with direct or indirect endothelial damage. To this end, we compared the local microcirculatory consequences of experimental interstitial (IC) cystitis and hemorrhagic cystitis (HC) with those of bladder ischemia/reperfusion (IR). We found that not only IR, where direct endothelial damage is present, but also HC and IC, where microcirculatory inflammatory reactions are secondary after urothelial and interstitial damage, are associated with manifest polymorphonuclear leukocyte (PMN)–endothelial cell interactions. This finding confirms the common role of PMN-mediated microcirculatory reactions in the pathogenesis of bladder diseases. The overexpression of transient receptor potential vanilloid type 1 (TRPV1) has been demonstrated in IC cases as well as in cases of neurogenic bladder. In addition, TRPV1 agonists have also previously been used during pharmacological management of these diseases. Therefore, our next aim was to examine the microcirculatory effects of local capsaicin treatment (the archetypical TRPV1 agonist). In our study, capsaicin induced rapid increases in PMN leukocyte rolling and adhesion and in adhesion molecule expression in the postcapillary venules of the urinary bladder, which was prevented by neonatal sensory chemodenervation with capsaicin and competitive TRPV1 antagonism. The effect of specific receptor antagonist therapy showed that TRPV1-induced calcitonin gene-related peptide (CGRP) release initiates the PMN–endothelial cell interaction by promoting leukocyte rolling, but adhesion is influenced by both CGRP and substance P. Our final goal is to implement medical research in clinical practice. Depending on the underlying cause, neurogenic bladder can manifest in both overactive and underactive forms. Diagnosis and treatment of both manifestations of neurogenic bladder remain a challenge in urology practice because disease severity cannot easily be assessed and the most common symptoms are non-specific. For this reason, our last aim was to provide an algorithm that aims to facilitate rapid and efficient diagnosis and assessment of disease severity. This may also aid in decision making on the potential pharmacological and invasive therapeutic approaches to protecting the upper urinary tract by maintaining low pressure values in the bladder.

L-Arginine Modulates Neonatal Leukocyte Recruitment in a Gestational Age-Dependent Manner

(1) Background: L-arginine is a complex modulator of immune functions, and its levels are known to decrease under septic conditions. L-arginine may suppress leukocyte recruitment in vivo; however, little is known about the gestational age-specific effects of L-arginine on leukocyte recruitment in preterm infants. We now asked whether L-arginine alters leukocyte recruitment in preterm and term neonates. (2) Methods: Leukocytes were isolated from preterm (28 + 0 to 32 + 6 weeks of gestation) and term (>37 weeks of gestation) newborns as well as from healthy adults. After incubation with 10 µg/mL L-arginine, we assessed leukocyte rolling and adhesion in dynamic microflow chamber experiments and leukocyte transmigration in fluorescence assays. In addition, we measured the expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) in neutrophils by flow cytometry. (3) Results: Leukocyte rolling, adhesion, and transmigration increased with gestational age. Leukocyte rolling, adhesion, and transmigration were decreased by L-arginine in term-born infants and adults. Preterm leukocytes showed no change in recruitment upon L-arginine exposure. Leukocyte adhesion after L-arginine exposure reached similar levels among all groups. In line, the expression of iNOS and Arg-1 was similar in all three age groups. (4) Conclusion: L-arginine dampens the ex vivo recruitment capacity of leukocytes from term-born infants, whereas no effect was seen in premature infants. As levels of iNOS and Arg-1 in neutrophils remain ontogenetically unchanged, the anti-inflammatory effect of L-arginine on the leukocyte recruitment cascade needs further investigation. These results add to the controversial debate of L-arginine supplementation in premature infants in sepsis.

Empagliflozin Ameliorates Leukocyte – Endothelium Cell Interactions and Inflammation in Type 2 Diabetic Patients

Background: SGLT2 inhibitors (iSGLT2) such as empagliflozin can reduce cardiovascular risk in patients with type 2 diabetes, but the underlying molecular mechanisms are yet to be determined. In the present study we evaluate the effects of empagliflozin on anthropometric and endocrine parameters, leukocyte-endothelium interactions, adhesion molecules and NFkB-p65 transcription factor expression. Methods: Eighteen patients with type 2 diabetes were recruited for the study. Patients received 10 mg/day of empagliflozin according to standard clinical protocols and were followed-up during a 24-week period. Anthropometric and analytical measurements were performed at baseline, 12-weeks and 24-weeks. Interactions between polymorphonuclear leukocytes and human umbilical vein endothelial cells (HUVECs), serum levels of adhesion molecules (P-selectin, VCAM-1 and ICAM-1) and NFkB-p65 protein levels were measured. Results: We observed a decrease in body weight, BMI and HbA1C levels from 12 weeks of treatment, which had become more pronounced at 24 weeks and was accompanied by a significant reduction in waist circumference, glucose, and hs-CRP levels. Leukocyte-endothelium interactions were reduced due to an enhancement of leukocyte rolling velocity from 12 weeks onwards, together with a significant decrease in leukocyte rolling flux and adhesion at 24 weeks. Accordingly, a significant decrease in ICAM-1 levels and NFkB-p65 expression were observed. Conclusions: Empagliflozin reduced leukocyte-endothelium interactions, adhesion molecules and NFkB-p65 expression in type 2 diabetic patients after 24 weeks of treatment.