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1.
bioRxiv ; 2024 May 20.
Article in English | MEDLINE | ID: mdl-38903096

ABSTRACT

The pair of transcription factors Bcl6-Blimp1 is well-known for follicular T helper (Tfh) cell fate determination, however, the mechanism(s) for Bcl6-independent regulation of CXCR5 during Tfh migration into germinal center (GC) is still unclear. In this study, we uncovered another pair of transcription factors, Bhlhe40-Pou2af1, that regulates CXCR5 expression. Pou2af1 was specifically expressed in Tfh cells whereas Bhlhe40 expression was found high in non-Tfh cells. Pou2af1 promoted Tfh formation and migration into GC by upregulating CXCR5 but not Bcl6, while Bhlhe40 repressed this process by inhibiting Pou2af1 expression. RNA-Seq analysis of antigen-specific Tfh cells generated in vivo confirmed the role of Bhlhe40-Pou2af1 axis in regulating optimal CXCR5 expression. Thus, the regulation of CXCR5 expression and migration of Tfh cells into GC involves a transcriptional regulatory circuit consisting of Bhlhe40 and Pou2af1, which operates independent of the Bcl6-Blimp1 circuit that determines the Tfh cell fate.

2.
Sci Signal ; 15(743): eabl9169, 2022 07 19.
Article in English | MEDLINE | ID: mdl-35857633

ABSTRACT

The integrin lymphocyte function-associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP3-binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.


Subject(s)
Lymphocyte Function-Associated Antigen-1 , Phosphatidylinositol 3-Kinases , Animals , Antigens, CD , Cell Adhesion/genetics , Cell Adhesion Molecules , Clustered Regularly Interspaced Short Palindromic Repeats , GTPase-Activating Proteins , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/genetics , Lymphocyte Function-Associated Antigen-1/metabolism , Mice , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , T-Lymphocytes/metabolism
3.
Neurosci Lett ; 760: 136042, 2021 08 24.
Article in English | MEDLINE | ID: mdl-34118306

ABSTRACT

The airways are constantly exposed to a multitude of inhaled particles and, as such, require a finely tuned discrimination between harmful or potentially threatening stimuli, and discrete responses to maintain homeostasis. Both the immune and nervous systems have the ability to sense environmental (and internal) signals, to integrate the obtained information and to initiate a protective reaction. Lung immunity and innervation are known to be individually involved in these processes, but it is becoming clear that they can also influence one another via a multitude of complex mechanisms. Here, we specifically describe how sensory innervation affects airways immunity with a focus on pathological conditions such as asthma or infections, describing cellular and molecular mechanisms, and highlighting potentially novel therapeutic targets.


Subject(s)
Asthma/immunology , Neuroimmunomodulation , Respiratory System/immunology , Respiratory Tract Infections/immunology , Sensory Receptor Cells/metabolism , Animals , Disease Models, Animal , Humans , Respiratory System/innervation
5.
Cell Immunol ; 350: 103865, 2020 04.
Article in English | MEDLINE | ID: mdl-30297084

ABSTRACT

The airway mucosa is the primary tissue site exposed to inhaled particulate matter, which includes pathogens and allergens. While most inhaled particles are eliminated from the airways via mucociliary clearance, some pathogens may penetrate the mucosal epithelial barrier and an effective activation of the mucosal immune system is required to prevent further pathogen spread. Similarly, inhaled environmental allergens may induce an aberrant activation of immune cells in the airway mucosa, causing allergic airway disease. During the last years, several investigators employed advanced microscopic imaging on both intravital and tissue explant preparations to observe the dynamic behavior of various immune cells within their complex tissue environment. In the respiratory tract, most imaging studies focused on immune responses of the alveolar compartment in the lung periphery. However, equally important immunological events occur more proximally in the mucosa of the conducting airways, both during infection and allergic responses, calling for a more detailed imaging analysis also at this site. In this review, I will outline the technical challenges of designing microscopic imaging experiments in the conducting airways and summarize our recent efforts in understanding airway mucosal immune cell dynamics in steady-state conditions, during infection and allergy.


Subject(s)
Intravital Microscopy/methods , Respiratory Mucosa/diagnostic imaging , Respiratory Mucosa/immunology , Allergens/immunology , Humans , Hypersensitivity/diagnostic imaging , Hypersensitivity/immunology , Immune System/immunology , Inflammation/pathology , Lung/immunology , Microscopy, Fluorescence, Multiphoton/methods , Mucous Membrane/diagnostic imaging , Mucous Membrane/immunology
6.
J Neuroinflammation ; 15(1): 128, 2018 May 01.
Article in English | MEDLINE | ID: mdl-29716612

ABSTRACT

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial cell molecule and primary amine oxidase that mediates leukocyte entry to sites of inflammation. However, there is limited knowledge of the inflammation-related expression of VAP-1 in the central nervous system (CNS). Therefore, we investigated the expression of VAP-1 within the CNS vasculature in two focal rat models of experimental autoimmune encephalomyelitis (EAE) mimicking multiple sclerosis (MS). METHODS: EAE was induced either with Bacillus Calmette-Guérin, resulting in a delayed-type hypersensitivity-like pathogenesis (fDTH-EAE), or with myelin oligodendrocyte glycoprotein (fMOG-EAE). A subgroup of fMOG-EAE rats were treated daily with a selective VAP-1 inhibitor (LJP1586; 5 mg/kg). On 3 and 14 days after lesion activation, rat brains were assessed using magnetic resonance imaging (MRI), and ex vivo autoradiography was conducted to evaluate the binding of Gallium-68-labelled VAP-1 ligand. Histology and immunohistochemistry (OX-42, VAP-1, intercellular adhesion protein-1 [ICAM-1], P-selectin) supported the ex vivo autoradiography. RESULTS: EAE lesions showed MRI-detectable signal changes and binding of the VAP-1-targeting radiotracer in both rat models. Some of the VAP-1 positive vessels showed morphological features typical for high endothelial-like venules at sites of inflammation. Inhibition of VAP-1 activity with small molecule inhibitor, LJP1586, decreased lymphocyte density in the acute inflammatory phase of fMOG-EAE lesions (day 3, P = 0.026 vs. untreated), but not in the remission phase (day 14, P = 0.70 vs. untreated), and had no effect on the amount of OX-42-positive cells in either phase. LJP1586 treatment reduced VAP-1 and ICAM-1 expression in the acute inflammatory phase, whereas P-selectin remained not detectable at all studied stages of the disease. CONCLUSIONS: Our results revealed that VAP-1 is expressed and functionally active in vasculature within the induced focal EAE lesions during the acute phase of inflammation and remains expressed after the acute inflammation has subsided. The study indicates that VAP-1 is actively involved in the development of inflammatory CNS lesions. During this process, the endothelial cell lesion-related vasculature seem to undergo a structural transformation from regular flat-walled endothelium to HEV-like endothelium.


Subject(s)
Amine Oxidase (Copper-Containing)/biosynthesis , Cell Adhesion Molecules/biosynthesis , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Animals , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Inflammation/metabolism , Inflammation/pathology , Male , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Rats , Rats, Inbred Lew
7.
Sci Rep ; 7(1): 694, 2017 04 06.
Article in English | MEDLINE | ID: mdl-28386104

ABSTRACT

The mucosal layer of conducting airways is the primary tissue exposed to inhaled microorganisms, allergens and pollutants. We developed an in vivo two-photon microscopic approach that allows performing dynamic imaging studies in the mouse trachea, which is a commonly used in vivo model of human small-diameter bronchi. By providing stabilized access to the tracheal mucosa without intubation, our setup uniquely allows dynamic in vivo imaging of mucociliary clearance and steady-state immune cell behavior within the complex airway mucosal tissue.


Subject(s)
Intravital Microscopy , Microscopy, Fluorescence, Multiphoton , Respiratory Mucosa/cytology , Respiratory Mucosa/diagnostic imaging , Trachea/cytology , Trachea/diagnostic imaging , Animals , Cilia , Humans , Image Processing, Computer-Assisted , Intravital Microscopy/instrumentation , Intravital Microscopy/methods , Mice , Microscopy, Fluorescence, Multiphoton/instrumentation , Microscopy, Fluorescence, Multiphoton/methods , Mucociliary Clearance , X-Ray Microtomography
8.
FASEB J ; 31(6): 2477-2491, 2017 06.
Article in English | MEDLINE | ID: mdl-28251930

ABSTRACT

In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface-expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4+ lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3-knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.


Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Cell Adhesion Molecules/metabolism , Pulmonary Fibrosis/enzymology , Amine Oxidase (Copper-Containing)/genetics , Animals , Antibiotics, Antineoplastic/toxicity , Bleomycin/toxicity , Carboxylic Acids , Cell Adhesion Molecules/genetics , Gene Expression Regulation, Enzymologic/physiology , Humans , Lung/enzymology , Lung/pathology , Lymphocytes/physiology , Mice , Mice, Knockout , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/metabolism , Pyrrolidines
9.
Contrast Media Mol Imaging ; 2017: 7645070, 2017.
Article in English | MEDLINE | ID: mdl-29463960

ABSTRACT

Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a ligand of inflammation-inducible vascular adhesion protein-1 (VAP-1). We compared 68Ga-DOTA- and 18F-fluorodeoxyribose- (FDR-) labeled Siglec-9 motif peptides for PET imaging of inflammation. Methods. Firstly, we examined 68Ga-DOTA-Siglec-9 and 18F-FDR-Siglec-9 in rats with skin/muscle inflammation. We then studied 18F-FDR-Siglec-9 for the detection of inflamed atherosclerotic plaques in mice and compared it with previous 68Ga-DOTA-Siglec-9 results. Lastly, we estimated human radiation dosimetry from the rat data. Results. In rats, 68Ga-DOTA-Siglec-9 (SUV, 0.88 ± 0.087) and 18F-FDR-Siglec-9 (SUV, 0.77 ± 0.22) showed comparable (P = 0.29) imaging of inflammation. In atherosclerotic mice, 18F-FDR-Siglec-9 detected inflamed plaques with a target-to-background ratio (1.6 ± 0.078) similar to previously tested 68Ga-DOTA-Siglec-9 (P = 0.35). Human effective dose estimates for 68Ga-DOTA-Siglec-9 and 18F-FDR-Siglec-9 were 0.024 and 0.022 mSv/MBq, respectively. Conclusion. Both tracers are suitable for PET imaging of inflammation. The easier production and lower cost of 68Ga-DOTA-Siglec-9 present advantages over 18F-FDR-Siglec-9, indicating it as a primary choice for clinical studies.


Subject(s)
Dermatitis/diagnostic imaging , Fluorodeoxyglucose F18/pharmacology , Gallium Radioisotopes/pharmacology , Myositis/diagnostic imaging , Organometallic Compounds/pharmacology , Positron-Emission Tomography/methods , Sialic Acid Binding Immunoglobulin-like Lectins/pharmacology , Animals , Inflammation/diagnostic imaging , Myositis/diagnosis , Radiometry , Rats , Rats, Sprague-Dawley
10.
J Immunol ; 198(2): 895-907, 2017 01 15.
Article in English | MEDLINE | ID: mdl-27903737

ABSTRACT

Allergic asthma develops in the mucosal tissue of small bronchi. At these sites, local cytokine production by Th2/Th17 cells is believed to be critical for the development of tissue eosinophilia/neutrophilia. Using the mouse trachea as a relevant model of human small airways, we performed advanced in vivo dynamic and in situ static imaging to visualize individual cytokine-producing T cells in the airway mucosa and to define their immediate cellular environment. Upon allergen sensitization, newly recruited CD4+ T cells formed discrete Ag-driven clusters with dendritic cells (DCs). Within T cell-DC clusters, a small fraction of CD4+ T cells produced IL-13 or IL-17 following prolonged Ag-specific interactions with DCs. As a result of local Th2 cytokine signaling, eosinophils were recruited into these clusters. Neutrophils also infiltrated these clusters in a T cell-dependent manner, but their mucosal distribution was more diffuse. Our findings reveal the focal nature of allergen-driven responses in the airways and define multiple steps with potential for interference with the progression of asthmatic pathology.


Subject(s)
Asthma/immunology , CD4-Positive T-Lymphocytes/immunology , Chemotaxis, Leukocyte/immunology , Cytokines/biosynthesis , Dendritic Cells/immunology , Adoptive Transfer , Animals , Asthma/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cytokines/immunology , Disease Models, Animal , Female , Flow Cytometry , Hypersensitivity/immunology , Immunity, Mucosal/immunology , Immunohistochemistry , Male , Mice , Microscopy, Confocal , Respiratory Mucosa/immunology
11.
Sci Rep ; 6: 35089, 2016 10 12.
Article in English | MEDLINE | ID: mdl-27731409

ABSTRACT

Given the important role of inflammation and the potential association of the leukocyte trafficking-associated adhesion molecule vascular adhesion protein 1 (VAP-1) with atherosclerosis, this study examined whether functional VAP-1 is expressed in atherosclerotic lesions and, if so, whether it could be targeted by positron emission tomography (PET). First, immunohistochemistry revealed that VAP-1 localized to endothelial cells of intra-plaque neovessels in human carotid endarterectomy samples from patients with recent ischemic symptoms. In low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR-/-ApoB100/100), VAP-1 was expressed on endothelial cells lining inflamed atherosclerotic lesions; normal vessel walls in aortas of C57BL/6N control mice were VAP-1-negative. Second, we discovered that the focal uptake of VAP-1 targeting sialic acid-binding immunoglobulin-like lectin 9 based PET tracer [68Ga]DOTA-Siglec-9 in atherosclerotic plaques was associated with the density of activated macrophages (r = 0.58, P = 0.022). As a final point, we found that the inhibition of VAP-1 activity with small molecule LJP1586 decreased the density of macrophages in inflamed atherosclerotic plaques in mice. Our results suggest for the first time VAP-1 as a potential imaging target for inflamed atherosclerotic plaques, and corroborate VAP-1 inhibition as a therapeutic approach in the treatment of atherosclerosis.


Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Cell Adhesion Molecules/metabolism , Plaque, Atherosclerotic/metabolism , Adult , Animals , Antigens, CD/metabolism , Apolipoprotein B-100/metabolism , Carotid Stenosis/diagnostic imaging , Carotid Stenosis/metabolism , Carotid Stenosis/pathology , Female , Gallium Radioisotopes , Heterocyclic Compounds, 1-Ring , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Plaque, Atherosclerotic/diagnostic imaging , Plaque, Atherosclerotic/pathology , Positron Emission Tomography Computed Tomography , Radioligand Assay , Receptors, LDL/deficiency , Receptors, LDL/genetics , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism
12.
Eur J Immunol ; 44(11): 3232-9, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25116373

ABSTRACT

Amine oxidase, copper containing 3 (AOC3, also known as vascular adhesion protein-1 (VAP-1)) is an endothelial adhesion molecule that contributes to the extravasation of neutrophils, macrophages, and lymphocytes to sites of inflammation. However, the role of AOC3/VAP-1 in allergic responses remains unknown. Here, we studied eosinophil and CD4+ T-cell recruitment to the airways using AOC3/VAP-1-deficient mice. In an OVA-triggered asthma model, AOC3/VAP-1 slightly contributed to the accumulation of leukocytes in lungs in an age-dependent manner. We then established a new model to kinetically measure recruitment of OVA-specific CD4+ T cells to different airway immune compartments during the priming and effector phases of an adaptive immune response. The results showed that in the absence of AOC3/VAP-1, recruitment of antigen-specific CD4+ T cells to draining bronchial lymph nodes is reduced by 89% on day 3 after tracheal allergen exposure, but this difference was not observed on day 6. The dispersal of effector cells to lung and tracheal mucosa is AOC3/VAP-1 independent. Thus, in allergic airway reactions, AOC3/VAP-1 transiently contributes to the antigen-specific, CD4+ T-cell traffic to secondary lymphatic tissues, but not to airway mucosa or lung parenchyma. Our results suggest a largely redundant function for AOC3/VAP-1 in allergic inflammatory responses of the airways.


Subject(s)
Amine Oxidase (Copper-Containing)/immunology , Asthma/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Adhesion Molecules/immunology , Lung/immunology , Trachea/immunology , Adaptive Immunity , Adoptive Transfer , Amine Oxidase (Copper-Containing)/biosynthesis , Amine Oxidase (Copper-Containing)/genetics , Animals , CD4-Positive T-Lymphocytes/transplantation , Cell Adhesion/immunology , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Eosinophils/immunology , Humans , Inflammation/immunology , Leukocytes/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology
13.
J Immunol ; 190(3): 897-903, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23267021

ABSTRACT

Aeroallergen provocation induces the rapid accumulation of CD11c(+)MHC class II (MHC II)(+) dendritic cells (DCs) in the lungs, which is driven by an increased recruitment of blood-derived DC precursors. Recent data show, however, that well-differentiated DCs proliferate in situ in various tissues. This may also contribute to their allergen-induced expansion; therefore, we studied DC proliferation in the airways of mice in the steady state and after local aeroallergen provocation. Confocal whole-mount microscopy was used to visualize proliferating DCs in different microanatomical compartments of the lung. We demonstrate that in the steady state, CD11c(+)MHC II(+) DCs proliferate in both the epithelial and subepithelial layers of the airway mucosa as well as in the lung parenchyma. A 1-h pulse of the nucleotide 5-ethynyl-2'-deoxyuridine was sufficient to label 5% of DCs in both layers of the airway mucosa. On the level of whole-lung tissue, 3-5% of both CD11b(+) and CD11b(-) DC populations and 0.3% of CD11c(+)MHC II(low) lung macrophages incorporated 5-ethynyl-2'-deoxyuridine. Aeroallergen provocation caused a 3-fold increase in the frequency of locally proliferating DCs in the airway mucosa. This increase in mucosal DC proliferation was later followed by an elevation in the number of DCs. The recruitment of monocyte-derived inflammatory DCs contributed to the increasing number of DCs in the lung parenchyma, but not in the airway mucosa. We conclude that local proliferation significantly contributes to airway DC homeostasis in the steady state and that it is the major mechanism underlying the expansion of the mucosal epithelial/subepithelial DC network in allergic inflammation.


Subject(s)
Allergens/immunology , Dendritic Cells/immunology , Immunity, Mucosal , Ovalbumin/toxicity , Respiratory System/immunology , Adoptive Transfer , Aerosols , Animals , Bronchi/immunology , Bronchi/pathology , Cell Division , Cell Lineage , Crosses, Genetic , DNA Replication , Dendritic Cells/pathology , Epithelium/immunology , Epithelium/pathology , Immunization , Inflammation , Lung/immunology , Lung/pathology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/immunology , Monocytes/transplantation , Mucous Membrane/immunology , Mucous Membrane/pathology , Organ Specificity , Ovalbumin/administration & dosage , Ovalbumin/immunology , Receptor, Macrophage Colony-Stimulating Factor/analysis , Respiratory System/pathology
14.
PLoS One ; 7(9): e45951, 2012.
Article in English | MEDLINE | ID: mdl-23049899

ABSTRACT

The airway mucosal epithelium is permanently exposed to airborne particles. A network of immune cells patrols at this interface to the environment. The interplay of immune cells is orchestrated by different mediators. In the current study we investigated the impact of neuronal signals on key functions of dendritic cells (DC). Using two-photon microscopic time-lapse analysis of living lung sections from CD11c-EYFP transgenic mice we studied the influence of neuropeptides on airway DC motility. Additionally, using a confocal microscopic approach, the phagocytotic capacity of CD11c(+) cells after neuropeptide stimulation was determined. Electrical field stimulation (EFS) leads to an unspecific release of neuropeptides from nerves. After EFS and treatment with the neuropeptides vasoactive intestinal peptide (VIP) or calcitonin gene-related peptide (CGRP), airway DC in living lung slices showed an altered motility. Furthermore, the EFS-mediated effect could partially be blocked by pre-treatment with the receptor antagonist CGRP(8-37). Additionally, the phagocytotic capacity of bone marrow-derived and whole lung CD11c(+) cells could be inhibited by neuropeptides CGRP, VIP, and Substance P. We then cross-linked these data with the in vivo situation by analyzing DC motility in two different OVA asthma models. Both in the acute and prolonged OVA asthma model altered neuropeptide amounts and DC motility in the airways could be measured. In summary, our data suggest that neuropeptides modulate key features motility and phagocytosis of mouse airway DC. Therefore altered neuropeptide levels in airways during allergic inflammation have impact on regulation of airway immune mechanisms and therefore might contribute to the pathophysiology of asthma.


Subject(s)
Dendritic Cells/cytology , Mucous Membrane/cytology , Neuropeptides/pharmacology , Animals , Asthma/metabolism , Bone Marrow Cells/cytology , Bronchi/metabolism , CD11c Antigen/biosynthesis , Hypersensitivity/metabolism , Immune System , Inflammation/metabolism , Lung/metabolism , Mice , Mice, Transgenic , Microscopy/methods , Microscopy, Confocal/methods , Neurons/metabolism , Neuropeptides/chemistry , Phagocytosis , Substance P/metabolism
15.
Blood ; 118(13): 3725-33, 2011 Sep 29.
Article in English | MEDLINE | ID: mdl-21821708

ABSTRACT

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.


Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Antigens, CD/physiology , Cell Adhesion Molecules/metabolism , Inflammation/diagnostic imaging , Lectins/physiology , Neoplasms/diagnostic imaging , Positron-Emission Tomography/methods , Amine Oxidase (Copper-Containing)/chemistry , Animals , Antigens, CD/chemistry , Antigens, CD/metabolism , CHO Cells , Cell Adhesion Molecules/chemistry , Cricetinae , Cricetulus , Humans , Lectins/chemistry , Lectins/metabolism , Ligands , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Binding , Protein Interaction Domains and Motifs/physiology , Radioactive Tracers , Rats , Rats, Sprague-Dawley , Sialic Acid Binding Immunoglobulin-like Lectins
16.
Am J Pathol ; 179(2): 603-9, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21708113

ABSTRACT

Airway mucosal dendritic cells (DCs), located beneath the epithelium of the conducting airways, are believed to be specialized for immunosurveillance via sampling of antigens from the airway luminal surface. However, the dynamics of airway DC activity has not yet been visualized. We used two-photon microscopy to illuminate the endogenous mucosal DC network in the airways of mice. To characterize DC behavior, we used lung section preparations and an intravital microscopic approach. DCs displayed a heterogeneous movement pattern according to their localization within the airway mucosa: sessile intraepithelial DCs with a dendritiform shape exhibited active probing movements and occasionally formed transepithelial extensions into the airway lumen. In contrast, DCs within the deeper layers of the mucosal tissue migrated fast in an amoeboid manner, without probing movements, and slowed down after aeroallergen challenge. Strikingly, neither of these two mucosal DC populations ingested fluorescently labeled antigens after antigen administration to the airways in the steady state, in contrast to alveolar macrophage/DC populations in the lung periphery. Our results provide a first description of the dynamic behavior of airway mucosal DCs, with their exact role in antigen sampling remaining unclear.


Subject(s)
Dendritic Cells/cytology , Microscopy/methods , Allergens/chemistry , Animals , Bacterial Proteins/metabolism , CD11c Antigen/biosynthesis , Escherichia coli/metabolism , Histocompatibility Antigens Class II/metabolism , Image Processing, Computer-Assisted , Luminescent Proteins/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mucous Membrane/pathology , Photons , Respiratory System/immunology
17.
Proc Natl Acad Sci U S A ; 108(23): 9478-83, 2011 Jun 07.
Article in English | MEDLINE | ID: mdl-21606356

ABSTRACT

In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C(ß2)) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.


Subject(s)
Chemoreceptor Cells/metabolism , Receptors, Nicotinic/metabolism , Respiration , Trachea/physiology , Animals , Choline O-Acetyltransferase/genetics , Choline O-Acetyltransferase/metabolism , Female , Flow Cytometry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/genetics , Heterotrimeric GTP-Binding Proteins/metabolism , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Microfilament Proteins/metabolism , Microscopy, Confocal , Microscopy, Electron , Microvilli/metabolism , Microvilli/ultrastructure , Phospholipase C beta/genetics , Phospholipase C beta/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Taste , Trachea/cytology , Trachea/metabolism , Vesicular Acetylcholine Transport Proteins/metabolism
18.
J Gen Virol ; 92(Pt 6): 1279-1291, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21325477

ABSTRACT

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1(+) monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b(+) and CX3CR1(+) subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.


Subject(s)
Cytomegalovirus Infections/virology , Muromegalovirus/physiology , Single-Cell Analysis/methods , Virus Activation , Virus Latency , Animals , Cytomegalovirus/genetics , Cytomegalovirus/physiology , Disease Models, Animal , Female , Gene Expression , Genes, Reporter , Herpesviridae Infections/virology , Host-Pathogen Interactions , Humans , Luciferases/genetics , Luciferases/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lung/cytology , Lung/virology , Male , Mice , Mice, Inbred BALB C , Muromegalovirus/genetics , Muromegalovirus/isolation & purification , Red Fluorescent Protein
19.
Pharmacol Ther ; 122(2): 203-14, 2009 May.
Article in English | MEDLINE | ID: mdl-19292991

ABSTRACT

Despite recent advances in the development of anti-asthmatic medication, asthma continues to be a major health problem worldwide. The symptoms of asthmatic patients include wheezing, chest tightness, cough and shortness of breath, which, together with airway hyperresponiveness, previously have been attributed to a dysfunction of airway nerves. However, research in the last two decades identified Th2-sensitization and the subsequent allergic reaction to innocuous environmental antigens as a basic immunological mechanism leading to chronic airway inflammation. Recent evidence suggests that the development of allergic asthma is influenced by events and circumstances in early childhood and even in utero. Allergen, ozone or stress exposure, as well as RSV infection in early life could be able to induce irreversible changes in the developing epithelial-mesenchymal trophic unit of the airways. The co-existence of chronic inflammation and neural dysfunction have recently drawn attention to the involvement of interaction pathways between the nervous and the immune system in the airways. Intensive basic research has accumulated morphological as well as functional evidence for the interaction between nerves and immune cells. Neuropeptides and neurotrophins have come into focus of attention as the key mediators of neuro-immune interactions, which lead to the development of several pharmacological compounds specifically targeting these molecules. This review will integrate our current knowledge on the involvement of neuro-immune pathways in asthma on the cellular and molecular level. It will summarize the results of pharmacological studies addressing the potential of neuropeptides and neurotrophins as novel therapeutic targets in asthma.


Subject(s)
Asthma/immunology , Neuroimmunomodulation , Anti-Allergic Agents/pharmacology , Anti-Asthmatic Agents/pharmacology , Asthma/drug therapy , Asthma/etiology , Dendritic Cells/cytology , Dendritic Cells/immunology , Humans , Hypersensitivity/drug therapy , Hypersensitivity/immunology , Pneumonia/immunology , Receptor Cross-Talk , Respiratory System/innervation , Stress, Psychological , T-Lymphocytes/cytology , T-Lymphocytes/immunology
20.
Am J Pathol ; 174(3): 808-17, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19179611

ABSTRACT

Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway inflammation, we applied whole-mount immunohistology and confocal microscopy to visualize dendritic cells and T cells together with nerves, epithelium, and smooth muscle in three dimensions. Proliferating T cells were identified by the detection of the incorporation of the nucleotide analogue 5-ethynyl-2'-deoxyuridine into the DNA. We developed a novel quantification method that enabled the accurate determination of cell-cell contacts in a semi-automated fashion. Dendritic cell-T cell interactions occurred beneath the smooth muscle layer, but not in the epithelium. Approximately 10% of the dendritic cells were contacted by nerves, and up to 4% of T cells formed clusters with these dendritic cells. T cells that were clustered with nerve-contacting dendritic cells proliferated only in the airways of mice with allergic inflammation but not in the airways of negative controls. Taken together, these results suggest that during the secondary immune response, sensory nerves influence dendritic cell-driven T cell activation in the airway mucosa.


Subject(s)
Dendritic Cells/pathology , Hypersensitivity/pathology , Inflammation/pathology , Respiratory System/pathology , T-Lymphocytes/pathology , Animals , CD11c Antigen/genetics , Cell Division , Dendritic Cells/immunology , Dendritic Cells/ultrastructure , Disease Models, Animal , Hypersensitivity/immunology , Inflammation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Nerve Fibers/pathology , Neurons/immunology , Neurons/pathology , Ovalbumin , Respiratory System/immunology , T-Lymphocytes/immunology , T-Lymphocytes/ultrastructure
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