Dendritic cells (DCs) form a key component of the immune response, as they are involved in the innate and adaptive immunity and in the process of tolerance. Under normal conditions, DCs are absent from the Central Nervous System (CNS), as the blood brain barrier (BBB) restricts their entry. However, DCs have recently been implicated in the pathogenesis of several CNS diseases. The molecular mechanisms that mediate DC trafficking across the BBB are poorly understood. The objectives of this study were to examine the role of endothelial cell adhesion molecules (eCAMs) and their ligands in the process of DC adhesion to the BBB endothelium, and to investigate the participation of DCs in human CNS diseases. To study DC adhesion, DCs were generated in vitro by culturing human blood monocytes in the presence of GM-CSF and IL- 4, and DC maturation was induced by adding inflammatory cytokines (TNF-α, IL-1β, IL-6) and PGE₂. Immature and mature DCs displayed differences in their expression of surface molecules, including eCAM ligands, by flow cytometry. Adhesion to the cerebral endothelium was investigated using an in vitro model of the BBB consisting of primary cultures of human brain microvessel endothelial cells (HBMEC). Immature or mature DCs were incubated with resting or TNF-α-activated HBMEC for up to one hour. Only a few DCs adhered to resting HBMEC, but adhesion was upregulated upon activating HBMEC (p<O.Ol). Moreover, immature DCs adhered to activated HBMEC to a greater extent compared to mature DCs (p<O.OOl). Blocking experiments indicated that the adhesion of both immature and mature DCs to HBMEC was dependent upon ICAM-1-CD18 or ICAM-2-CD18, ICAM-2-DC-SIGN, and PECAM-l PECAM-l interactions. In addition, VCAM-1-VLA-4 interactions mediated the adhesion of immature but not mature DCs to activated HBMEC. Using immunohistochemistry for DC markers, we also examined the presence of DCs in human inflammatory, infectious, and neurodegenerative diseases, stroke and tumours. The results indicate accumulation of DC SIGN—, fascin—, and MHC class Il—expressing DCs in the CNS under most pathological conditions. These findings provide further insight into the mechanisms of neuroinflammation, and highlight the role of DCs and the BBB endothelium in this process. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

There is a clear need for testing targeted drug carrier systems in a more realistic microenvironment where both biochemical interactions and shear forces are present. This is critical both for understanding of the molecular mechanisms involved in this process and during the drug discovery process. Current in vitro models of the leukocyte adhesion cascade cannot be used for real-time studies of the entire leukocyte adhesion cascade including rolling, adhesion and migration in a single assay. In this study, we have developed and validated a novel bioinspired microfluidic device (bMFD) and used it to test the hypothesis that blocking of specific steps in the adhesion/migration cascade significantly affects other steps of the cascade. The bMFD consists of an endothelialized microvascular network in communication with a tissue compartment via a 3 µm porous barrier. Human neutrophils in bMFD preferentially adhered to activated human endothelial cells near bifurcations with rolling and adhesion patterns in close agreement with in vivo observations. Treating endothelial cells with monoclonal antibodies to E-selectin or ICAM-1 or treating neutrophils with wortmannin reduced rolling, adhesion, and migration of neutrophils to 60%, 20% and 18% of their respective control values. Antibody blocking of specific steps in the adhesion/migration cascade (e.g. mAb to E-selectin) significantly downregulated other steps of the cascade (e.g. migration). This novel in vitro assay provides a realistic human cell based model for basic science studies, identification of new treatment targets, selection of pathways to target validation, and rapid screening of candidate agents. / Mechanical Engineering

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Les neutrophiles jouent un rôle central dans la première ligne de défense mise en place contre les agents pathogènes. L’une de leur fonction essentielle est caractérisée par la capacité à rejoindre rapidement la circulation sanguine et de migrer vers les sites inflammatoires, où ils seront activés et exerceront des fonctions effectrices extrêmement puissantes. Ces processus doivent être finement contrôlés afin de moduler leur effet dévastateur dans l'organisme. L'hypothèse de travail de ce travail de thèse est que le CD31, un corécepteur ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif), exprimé de manière constitutive à la surface des neutrophiles, joue un rôle essentiel dans la régulation du recrutement et l'activation des neutrophiles au sein des sites inflammatoires. Nous avons pu mettre en évidence que le CD31 membranaire était clivé lors de l’activation neutrophilaire, et pouvait perdre une grande partie de son domaine extracellulaire, tout en conservant sa portion membranaire. Afin d’étudier le rôle du gain de fonction du CD31 neutrophilaire, nous avons utilisé un peptide synthétique homotypique ayant la capacité de se lier spécifiquement au domaine persistant du CD31 clivé, et permettant le maintien de sa fonction moléculaire. Parallèlement, des souris invalidées pour CD31 ont été utilisées dans divers modèles expérimentaux d'inflammation aiguë afin d’étudier le rôle de sa suppression. Dans leur ensemble, nos données suggèrent que le CD31 joue un rôle complexe et important dans la régulation du recrutement des neutrophiles au sein des sites d'inflammation aiguë. Nous avons pu montrer que des lymphocytes isolés à partir de souris invalidées pour CD31 adhèrent plus fortement aux cellules endothéliales activées, bien que leur progression hors du vaisseau est retardée. A l’inverse, l’ajout du peptide agoniste CD31 diminue leur adhérence, tout en favorisant leur détachement. La littérature rapporte que des neutrophiles isolés à partir de souris déficientes en CD31 sont incapables de migrer hors des vaisseaux, ce qui suggère un rôle important du CD31 dans les processus de transmigration. Cependant, les mécanismes sous-jacents restent inconnus. Nous avons pu montrer qu’au sein du front de migration neutrophilaire, le CD31 empêche l’ouverture des intégrines associées en clusters, par le biais du recrutement de phosphatase. Ce processus abouti à un diminution de l’adhérence neutrophilaires à l'endothélium. A l’inverse, la polarisation du CD31 à l'uropode semble être nécessaire à leur détachement. Cette dernière étape implique la fermeture des intégrines et semble être favorisée par la signalisation CD31. Dans la deuxième partie de ce travail de thèse, nos résultats suggèrent que le CD31 régule également l'activation des neutrophiles au sein des sites inflammatoires. Nous avons tenté de comprendre le rôle du CD31 dans le burst oxydatif et la dégranulation des neutrophiles. SHIP1, une phosphatase impliquée dans la signalisation de CD31, découple l'activation neutrophilaire induite par le récepteur Fc. En utilisant le modèle auto-immun de glomérulonéphrite, nous avons pu montrer que la signalisation du CD31 module les effets délétères causés par les protéases neutrophilaires. Dans ce modèle l’ajout du peptide agoniste CD31 prévient les lésions induites, alors que les souris déficientes en CD31 possèdent des neutrophiles plus activés et des lésions glomérulaires plus importantes. En conclusion, les résultats des expériences effectuées lors de ce travail de thèse suggèrent (1) que CD31 peut moduler le trafic neutrophilaire en contrôlant les intégrines et (2) que CD31 établit un seuil immunologique impliqué dans l'activation des neutrophiles, limitant leur activation excessive pendant la phase aiguë de l'inflammation / Neutrophils play a crucial role in the first line of defense against noxious agents. Central to their function, is their ability to rapidly exit the circulation and migrate to the site of inflammation where they get activated and exert extremely efficient effector functions. A tight regulation of these processes is mandatory to restrict their devastating effects in the organism. The working hypothesis of my thesis was that CD31, an ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) co-receptor constitutively expressed at the surface of neutrophils, plays a critical role in the regulation of the recruitment and activation of neutrophils at sites of inflammation. We found that, upon cell activation, the surface CD31 molecules underwent a cleavage and shedding of a large part of the extracellular portion, but leaving a portion that lingers at the cellular surface. A synthetic homotypic peptide that has the ability to specifically bind and maintain functional molecular clusters of CD31 was used to study the role of CD31 in neutrophils biology, by “gain-of-function” approach, whereas CD31 genetically invalidated mice were used as a “loss-of-function” approach both in vitro and in vivo, in experimental mouse models of acute inflammation. Altogether our data unveil a complex and important role for CD31 in the regulation of the recruitment of the neutrophils at the site of acute inflammation. We show that CD31-/- neutrophils established stronger adhesion on activated endothelial cells but their progression out of the vessel was delayed. At the opposite, the use of the CD31 agonist peptide prevented the attachment while it favored the detachment of the migrating neutrophils. It was previously observed that CD31-/- neutrophils are unable to migrate out of the vessels and it had been proposed that CD31 is necessary for the transmigration but the underlying mechanism had remained unknown. We show that, by co-clustering with the integrins at the migration front, the phosphatases recruited and activated by CD31 prevented their opening and hence inhibited the adhesion of neutrophils on the endothelium. At the opposite, CD31 polarization at the uropod appeared to be crucial for the detachment of the neutrophils. We found that this step, that relies upon the closure of the integrins, was favored by the CD31 signaling. Furthermore, our findings showed that CD31 not only regulates the recruitment but also the activation of neutrophils at sites of inflammation. Indeed, in the second part of my thesis, I extensively studied how the engagement of CD31 also regulates the oxidative burst and the degranulation of neutrophils. The phosphatase involved in the function of CD31 is reported to be the SHIP1 inositol phosphatase which uncouples the Fc-receptor dependent neutrophil activation. By using the autoimmune glomerulonephritis model that develops antibody-dependent acute inflammation, we showed that CD31 signaling is crucial for limiting the collateral damage caused by neutrophil-derived proteases: the CD31 agonist peptide prevented the damage whereas the induction of the disease in mice invalidated for the CD31 resulted in greater neutrophil activation and glomerular damage. In conclusion, the results of the experiments performed during my thesis work suggest that CD31 is an integrin regulator that controls neutrophil trafficking. In addition, CD31 sets an immunologic threshold for neutrophil activation that limits their excessive activation during the acute phase of inflammation

The Leukocyte Immunoglobulin-like Receptors (LILRs) are a family of receptors that is broadly expressed on all leukocytes and have the ability to regulate their function. A substantial amount of evidence suggests that LILRs may be involved in immune homeostasis but also immune dysregulation. We therefore studied the role of LILRs in relation to the autoimmune disease, rheumatoid arthritis (RA). RA is a chronic and systemic inflammatory disease involving inflammation of the joints affecting the synovial membrane, cartilage and bone. Much of the tissue damage is a result of an inappropriate immune response within the joint space caused by the unwarranted activation of leukocytes. Here were report that LILRA2 (an activating receptor) that has been previously shown to be highly expressed in the rheumatoid synovium, induces the production of pro-inflammatory cytokines TNF-α, IL-1, IL-6, IFN-γ and IL-10 in primary monocytes. These cytokines are known to have an important role in the pathogenesis of RA indicating a pathway by which LILRA2 exacerbates RA. Co-ligation of LILRB4 (an inhibitory receptor) with LILRA2 abolishes cytokine production suggesting that LILRB4 is able to suppress the function of LILRA2. Expression of both LILRA2 and LILRB4 are regulated by inflammatory cytokines and LPS, indicative of a feedback mechanism. There is also cross-talk between LILRs and TLR4 as co-stimulation with LPS and either LILRA2 or LILRB4 inhibits cytokine production. A differential expression of LILRs has also been identified on lymphocytes of patients with RA whereby an increase of LILRA1 (activating) and LILRB1 (inhibitory) expressing circulating lymphocytes is present in RA patients when compared to healthy control subjects. From these studies, we propose that LILRs have a functional role in RA by regulating local and systemic inflammation. The presence of LILRA2 in the RA joint is detrimental since its potent ability to induce inflammatory cytokines, particularly TNF-α, can initiate leukocyte recruitment and activation of proteases. Along with TLR4, LILRA2 and LILRB4 have the potential to moderate the innate immune system via regulation of cytokine production. Furthermore, suppression of LILRA2 function may serve as a therapeutic tool in many inflammatory diseases.

During inflammation, leukocytes modulate α4β1(VLA-4) integrin avidity in order to rapidly stabilize nascent adhesive contacts to VCAM-1-expressing endothelial cells and resist detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization under conditions of fluid flow remains poorly understood. We modeled leukocyte (U937 cell, mouse lymphocyte and human monocyte) arrest and adhesion stabilization through the use of a parallel plate flow chamber and visualized cells by phase contrast or fluorescent confocal microscopy. Live cell imaging with Lifeact-transfected U937 cells revealed that mechanical forces imparted by fluid flow induced formation of upstream tension-bearing anchors attached to the VCAM-1-coated surface. Scanning electron microscopy confirmed that flow-induced mechanical force culminates in the formation of structures that anchor monocyte adhesion. These structures are critical for adhesion stabilization, since disruption of actin polymerization dramatically inhibited VLA-4-dependent resistance to detachment, but did not affect VLA-4 expression, affinity modulation, and clustering or constitutive linkage to F-actin. Transfection of dominant-negative constructs and inhibition of kinase function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. Rap1 was shown to be critical for resistance to flow-induced detachment and accumulated in its GTP form at the sites of anchor formation. A key mediator of force-induced Rac activation and actin polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension-bearing anchors and blockade of PI3K or deficiency of PI3Kγ isoform reproduced the adhesion defect produced by inhibition of actin polymerization. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces; these included activation of Rap1, phosphoinositide 3-kinase γ-isoform and Rac, but not Cdc42.

During pregnancy, the woman carries a fetus partly foreign to her immune system, because of the expression of paternal antigens. Despite this, the fetus is normally tolerated and not rejected, as is often the case with organs in allogeneic transplantations. Systemic changes in maternal blood occur during pregnancy but, perhaps of greater importance, are changes in tissues locally in the uterus. The pregnant uterine endometrium, the decidua, is infiltrated by large numbers of leukocytes, mainly natural killer (NK) cells but also macrophages and T lymphocytes. Further, various cytokines are known to be secreted at the fetomaternal interface. However, the functions of these cells and the cytokine networks are not fully understood. The aim of this thesis was to investigate the local immune balance in normal human pregnancy decidua, both in the early phase of pregnancy and at parturition. First trimester decidual mononuclear cells, NK cells and macrophages were all shown to secrete IFN-γ, IL-4 and IL-10, as detected by ELISPOT. The secretion was not mirrored in blood from the same subjects. A significantly larger number of decidual macrophages secreted IL-10 than did their blood counterparts, indicating potential regulatory functions of this cell type. Further examination of early pregnancy decidual macrophages by microarray revealed 120 genes being differentially regulated at the transcriptional level in decidual compared to blood monocytes/macrophages. Several genes were associated with alternative activation/M2 polarization of macrophages, including CCL-18, CD209, IGF-1, MRC-1 and FN-1. Genes connected to immune regulation and tissue remodelling were common, in line with the potential functions for this cell type in utero. In addition, some molecules not previously connected to decidual macrophages, such as TREM-2, A2M and PGDS, were found to be upregulated, gaining new insights into the regulatory functions of decidual macrophages. Term decidual mononuclear cells spontaneously secrete IFN-γ, TNF, IL-4, IL-10, and TGF-β. No differences were seen between tissues obtained before and after the onset of labour, indicating that decidual mononuclear cells are not the main cell population responsible for plausible cytokine regulation in the process of labour induction. Placental and fetal membranes as well as cells in the maternal systemic circulation may instead contribute to a possible shift in immune balance prior to pregnancy termination. In conclusion, decidual leukocytes, including NK cells and macrophages, are potential producers of both Th1-like/pro-inflammatory and Th2-like/anti-inflammatory cytokines in early pregnancy as well as at parturition. Decidual macrophages are of a specialized phenotype with effector functions contributing to a proper invasion of the placenta and to immunological protection of the semi-allogeneic fetus. This thesis adds new knowledge on local immune balance during normal human pregnancy, however, the clinical significance of the presented data needs to be clarified.

The role of neutrophils in the pathogenesis of Salmonella typhimurium-induced ruminant and human enteritis and diarrhea remains incompletely understood. To address this question, the in vivo bovine ligated ileal loop model of non-typhoidal salmonellosis was used in calves with the naturallyoccurring Bovine Leukocyte Adhesion Deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from BLAD calves were compared to those from genetically normal calves negative for the BLAD mutation. Morphologic studies showed that the absence of significant tissue influx of neutrophils in intestine infected by S. typhimurium resulted in less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion compared to regular calves. Study of gene expression profile of cytokines by quantitative Real-Time PCR (qRTPCR) revealed that the massive tissue influx of neutrophils during acute infection is mainly driven by the CXC chemokine GRO- α especially in the last stages of acute infection and to a lesser extent, IL-8. In contrast, the pro-inflammatory cytokines IL-1 β and TNF- α were not significantly correlated with the presence or absence of tissue neutrophils. The precise in situ localization of gene expression of these major cytokines and chemokines was investigated by qRTCPR from specific groups of intestinal cells captured by Laser Capture Microdissection in S. typhimuriuminfected ileal loops from BLAD animals. Our data confirmed that gene expression of IL-8, GRO- α, and IL-1 β was predominantly localized to enterocytes of crypts with less expression in enterocytes of villi tips and cells that form the domed villi were not an important source of TNF- α gene expression. Microarray technology was used to determine the global transcriptional profile of bovine intestinal loops inoculated with S. typhimurium. The host samples were hybridized on a 13K bovine-specific oligoarray and microarray data was analyzed using a suite of gene expression analysis and modeling tools. Analysis of our data revealed that the tissue influx of neutrophils in ileal loops greatly influenced the host gene expression. Major differences in gene expression in relevant fields of Salmonella research including inflammation and immune response, Toll-like receptor signaling, cytokine profiles, apoptosis, and intracellular defense against infection are discussed.

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein, is involved in neutrophil recruitment into peritoneum. Because mechanisms of excessive migration of activated neutrophils into inflamed lungs, credited with tissue damage, are not fully understood, we explored the hitherto unknown expression and role of LSP1 in neutrophil migration in a mouse model of acute lung inflammation. We induced acute lung inflammation through intranasal E. coli lipopolysacharide (LPS) (80μg) in wild-type 129/SvJ (WT) and LSP1 deficient (LSP1-/-) mice. WT (n=10) and LSP1-/- (n=11) mice showed significant neutrophilia and more neutrophils in bronchoalveolar lavage (BAL) at 9 hour post-LPS challenge compared to respective saline-treated controls (WT=7; LSP1-/-=10). LPS treatment induced more BAL neutrophils (P<0.001), myeloperoxidase concentrations and Gr-1+ neutrophils in lung tissues in WT mice compared to LSP1-/- mice. Lung myeloperoxidase and Gr-1+ (P<0.05) were higher in LPS-treated WT compared to the LSP1-/- mice. Lung tissue and BAL fluid KC, MCP-1, MIP-1α and MIP-1β concentration and vascular permeability were not different between LPS-treated WT and LSP1-/- mice but TNF-α concentration was higher in LPS-treated WT mice. Hematoxylin and eosin staining showed more septal congestion in LPS-treated WT mice compared to LSP1-/- mice. LSP1 expression was increased in lungs from LPS-treated mice compared to saline control. The autopsied lungs from septic humans, compared to their respective controls, showed increased expression of LSP1. These data show that LSP1 expression is modulated in acute lung inflammation and that LSP1 deficiency reduces neutrophil migration into acute lung inflammation.