Determining the roles of dendritic cells and ICAM-1 in the transpresentation of IL-15 to CD8 T cells

The maintenance and generation of memory CD8 T cells is dependent on the cytokine IL-15. IL-15 is delivered by a novel mechanism termed transpresentation: IL-15 is presented by a cell expressing IL-15Ralpha to the CD8 T cell which responds via IL-2Rbeta/gammac. The identity of what cells transpresent IL-15 to support the survival and homeostatic proliferation of memory CD8 T cells is unknown. Using a transgenic mouse model that limits IL-15 transpresentation to DCs, I have demonstrated that DCs transpresent IL-15 to CD8 T cells. DCs transpresent IL-15 to CD8 T cells during the contraction of an immune response and also drive homeostatic proliferation of memory CD8 T cells. Additionally, I identified a role for ICAM-1 in promoting homeostatic proliferation. Wt memory CD8 T cells displayed impaired homeostatic proliferation in ICAM-1-/- hosts but not in models of acute IL-15-driven proliferation. In this way, the role of ICAM-1 in IL-15 transpresentation resembles the role for ICAM-1 in antigenpresentation: where antigen or IL-15 is limited, adhesion molecules are important for generating maximal responses. In vitro cultures between CD8 T cells and bone marrowdifferentiated DCs (BMDC) activated with a TLR agonist established a model of proliferation and signaling in CD8 T cells that was dependent on IL-15 transpresentation and required ICAM-1 expression by BMDCs. Regarding the expression of IL-15, I demonstrated that in normal mice it is undetectable without stimulation but is elevated in lymphopenic mice, suggesting a role for T cells in regulating IL-15 expression. Overall, these studies have identified many novel aspects of the interaction between DCs and CD8 T cells that were previously unknown. The study of adhesion molecules in IL-15 transpresentation describes a novel role for these well-known adhesion molecules and it will be interesting for future studies to further characterize this relationship for other IL-15-dependent cell types.

Determining the roles of dendritic cells and ICAM-1 in the transpresentation of IL-15 to CD8 T cells

The maintenance and generation of memory CD8 T cells is dependent on the cytokine IL-15. IL-15 is delivered by a novel mechanism termed transpresentation: IL-15 is presented by a cell expressing IL-15Ralpha to the CD8 T cell which responds via IL-2Rbeta/gammac. The identity of what cells transpresent IL-15 to support the survival and homeostatic proliferation of memory CD8 T cells is unknown. Using a transgenic mouse model that limits IL-15 transpresentation to DCs, I have demonstrated that DCs transpresent IL-15 to CD8 T cells. DCs transpresent IL-15 to CD8 T cells during the contraction of an immune response and also drive homeostatic proliferation of memory CD8 T cells. Additionally, I identified a role for ICAM-1 in promoting homeostatic proliferation. Wt memory CD8 T cells displayed impaired homeostatic proliferation in ICAM-1-/- hosts but not in models of acute IL-15-driven proliferation. In this way, the role of ICAM-1 in IL-15 transpresentation resembles the role for ICAM-1 in antigenpresentation: where antigen or IL-15 is limited, adhesion molecules are important for generating maximal responses. In vitro cultures between CD8 T cells and bone marrowdifferentiated DCs (BMDC) activated with a TLR agonist established a model of proliferation and signaling in CD8 T cells that was dependent on IL-15 transpresentation and required ICAM-1 expression by BMDCs. Regarding the expression of IL-15, I demonstrated that in normal mice it is undetectable without stimulation but is elevated in lymphopenic mice, suggesting a role for T cells in regulating IL-15 expression. Overall, these studies have identified many novel aspects of the interaction between DCs and CD8 T cells that were previously unknown. The study of adhesion molecules in IL-15 transpresentation describes a novel role for these well-known adhesion molecules and it will be interesting for future studies to further characterize this relationship for other IL-15-dependent cell types.

Cell surface levels of endothelial ICAM-1 influence the transcellular or paracellular T-cell diapedesis across the blood-brain barrier

The extravasation of CD4(+) effector/memory T cells (TEM cells) across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS). Endothelial ICAM-1 and ICAM-2 are essential for CD4(+) TEM cell crawling on the BBB prior to diapedesis. Here, we investigated the influence of cell surface levels of endothelial ICAM-1 in determining the cellular route of CD4(+) TEM -cell diapedesis across cytokine treated primary mouse BBB endothelial cells under physiological flow. Inflammatory conditions, inducing high levels of endothelial ICAM-1, promoted rapid initiation of transcellular diapedesis of CD4(+) T cells across the BBB, while intermediate levels of endothelial ICAM-1 favored paracellular CD4(+) T-cell diapedesis. Importantly, the route of T-cell diapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4(+) TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM-1 and ICAM-2 via an obviously alternatively regulated transcellular pathway. In vivo, this translated to the development of ameliorated EAE in ICAM-1(null) //ICAM-2(-/-) C57BL/6J mice. Taken together, our study demonstrates that cell surface levels of endothelial ICAM-1 rather than the inflammatory stimulus or BBB integrity influence the pathway of T-cell diapedesis across the BBB.

β2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

The physiological roles of ICAM-1 and ICAM-2 in neutrophil migration into tissues

PURPOSE OF REVIEW Neutrophil extravasation from the blood into tissues is initiated by tethering and rolling of neutrophils on endothelial cells, followed by neutrophil integrin activation and shear resistant arrest, crawling, diapedesis and breaching the endothelial basement membrane harbouring pericytes. Endothelial intercellular cell adhesion molecule (ICAM)-1 and ICAM-2, in conjunction with ICAM-1 on pericytes, critically contribute to each step. In addition, epithelial ICAM-1 is involved in neutrophil migration to peri-epithelial sites. The most recent findings on the role of ICAM-1 and ICAM-2 for neutrophil migration into tissues will be reviewed here. RECENT FINDINGS Signalling via endothelial ICAM-1 and ICAM-2 contributes to stiffness of the endothelial cells at sites of chronic inflammation and junctional maturation, respectively. Endothelial ICAM-2 contributes to neutrophil crawling and initiation of paracellular diapedesis, which then proceeds independent of ICAM-2. Substantial transcellular neutrophil diapedesis across the blood-brain barrier is strictly dependent on endothelial ICAM-1 and ICAM-2. Endothelial ICAM-1 or ICAM-2 is involved in neutrophil-mediated plasma leakage. ICAM-1 on pericytes assists the final step of neutrophil extravasation. Epithelial ICAM-1 rather indirectly promotes neutrophil migration to peri-epithelial sites. SUMMARY ICAM-1 and ICAM-2 are involved in each step of neutrophil extravasation, and have redundant but also distinct functions. Analysis of the role of endothelial ICAM-1 requires simultaneous consideration of ICAM-2.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-Å resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus–ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the α chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

Dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion

Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single “fully competent” LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an α-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/ΔD1–2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/ΔD1–2 and with an ≈2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/ΔD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/ΔD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, “fully competent” LFA-1-binding surface.

Vitamin C supplementation in normal subjects reduces constitutive ICAM-1 expression

Regulation of monocyte adhesion molecule gene expression is via redox sensitive transcription factors. We have investigated whether dietary antioxidant supplementation with vitamin C (250mg/day) can modulate monocyte ICAM-1 expression in healthy male subjects with low plasma vitamin C at baseline. In a randomised, double-blind, crossover study, monocyte ICAM-1 mRNA was analysed using quantitative reverse transcriptase PCR. Protein was determined by flow cytometry (monocytes) and ELISA (plasma). Monocyte numbers were unaltered by supplementation. Subjects with low plasma vitamin C (<50μM) prior to supplementation expressed higher levels of monocyte ICAM-1mRNA, and showed a significant (50%) reduction in ICAM-1mRNA expression after 6 weeks of 250mg/day vitamin C supplementation (p<0.05). This was paralleled by a reduction in sICAM-1 (p<0.05). For the first time, these results show that dietary vitamin C can modulate monocyte ICAM-1 gene expression in vivo, where regulation of gene expression represents a novel mechanism for benefit from dietary antioxidants. © 2003 Elsevier Inc. All rights reserved.

Dietary supplementation with vitamin C but not vitamin E reduces constitutive expression of ICAM-1 in peripheral blood monocytes of normal subjects with low plasma vitamin C levels

Monocytes play a central role in inflammatory responses through systemic antigen presentation and cytokine secretion. Regulation of monocyte adhesion molecule and inflammatory gene expression is via redox sensitive transcription factors. Therefore we have investigated the hypothesis that dietary antioxidant supplementation with vitamins C (250mg/d) or E (400iU/d) for six weeks can modulate monocyte ICAM-1 expression in healthy male subjects with low plasma vitamin C at baseline. In a randomised, double-blind, crossover study, ICAM-1 mRNA and protein was analysed using quantitative RTPCR with ELISA measurement of PCR products and by flow cytometry and ELISA respectively. Monocyte numbers were unaltered by supplementation. Subjects with low plasma vitamin C (<50uM) prior to supplementation expressed higher levels of monocyte ICAM-1 mRNA, and showed a significant (50%) reduction in ICAM-1 mRNA expression after 6 weeks of 250mg/d vitamin C supplementation compared to subjects with normal plasma vitamin C. This was paralleled by a reduction in plasma sICAM-1. Vitamin E supplementation had no effect on ICAM-1 expression. For the first time, these results show that dietary vitamin C can modulate monocyte ICAM-1 gene expression in vivo, where regulation of gene expression represents a novel mechanism for benefit from dietary antioxidants.

Implication de ICAM-1 et de ICAM soluble dans l'ostéoclastogénèse

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Implication de ICAM-1 et de ICAM soluble dans l'ostéoclastogénèse

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Increased Circulating Pedf And Low Sicam-1 Are Associated With Sickle Cell Retinopathy.

Sickle cell retinopathy (SCR) develops in up to 30% of sickle cell disease patients (SCD) during the second decade of life. Treatment for this affection remains palliative, so studies on its pathophysiology may contribute to the future development of novel therapies. SCR is more frequently observed in hemoglobin SC disease and derives from vaso-occlusion in the microvasculature of the retina leading to neovascularization and, eventually, to blindness. Circulating inflammatory cytokines, angiogenic factors, and their interaction may contribute to the pathophysiology of this complication. Angiopoietin (Ang)-1, Ang-2, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule (ICAM)-1, E-selectin, P-selectin, IL1-β, TNF-α, pigment epithelium derived factor (PEDF) and vascular endothelial growth factor plasmatic levels were determined in 37 SCD patients with retinopathy, 34 without retinopathy, and healthy controls. We observed that sICAM-1 is significantly decreased, whereas PEDF is elevated in HbSC patients with retinopathy (P=0.012 and P=0.031, respectively). Ang-1, Ang-2 and IL1-β levels were elevated in SCD patients (P=0.001, P<0.001 and P=0.001, respectively), compared to controls, and HbSS patients presented higher levels of Ang-2 compared to HbSC (P<0.001). Our study supports the possible influence of sICAM-1 and PEDF on the pathophysiology of retinal neovascularization in SCD patients.

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C578L/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. (C) 2010 Elsevier Inc. All rights reserved.

Expression of transcription factors NF-kappa B and AP-1 in nasal polyposis

Background The treatment and prognosis of nasal polyposis (NP) may be influenced by transcription factors, but their expression is poorly understood. Objective To determine the expression of transcription factors [(nuclear factor-kappa B) NF-kappa B and (activator protein) AP-1], cytokines [IL-1 beta, TNF-alpha and (granulocytes and macrophage colony-stimulating factor) GM-CSF], growth factor (b-FGF), chemokine (eotaxin-2) and adhesion molecule (ICAM-1) in NP in comparison with nasal mucosa controls. Methods Cross-sectional study. Twenty biopsies of nasal polyps were compared with eight middle turbinate biopsies. p65, c-Fos, IL-1 beta, TNF-alpha, ICAM-1, b-FGF, eotaxin-2 and GM-CSF were analysed through RQ-PCR, and p65 and c-Fos were also analysed through Western blotting. Results NF-kappa B expression was increased in patients with NP when compared with control mucosa (P < 0.05), whereas AP-1 expression did not differ significantly between groups. Expressions of IL-1 beta, eotaxin-2 and b-FGF were also increased in patients with NP compared with controls (P < 0.05). Conclusions The transcription factor NF-kappa B is more expressed in NP than in control mucosa. This is important in NP because NF-kappa B can induce the transcription of cytokines, chemokines and adhesion molecules, which play an important role in the inflammatory process. Moreover, transcription factors influence the response to corticosteroids, which are the basis of NP treatment. Transcription factor AP-1 does not seem to have a significant role in the pathological process.