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      The differential role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in the adherence, migration and podosome formation of human macrophages and dendritic cells under inflammatory conditions

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          Abstract

          CR3 and CR4, the leukocyte specific β 2-integrins, involved in cellular adherence, migration and phagocytosis, are often assumed to have similar functions. Previously however, we proved that under physiological conditions CR4 is dominant in the adhesion to fibrinogen of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Here, using inflammatory conditions, we provide further evidence that the expression and function of CR3 and CR4 are not identical in these cell types. We found that LPS treatment changes their expression differently on MDMs and MDDCs, suggesting a cell type specific regulation. Using mAb24, specific for the high affinity conformation of CD18, we proved that the activation and recycling of β 2-integrins is significantly enhanced upon LPS treatment. Adherence to fibrinogen was assessed by two fundamentally different approaches: a classical adhesion assay and a computer-controlled micropipette, capable of measuring adhesion strength. While both receptors participated in adhesion, we demonstrated that CR4 exerts a dominant role in the strong attachment of MDDCs. Studying the formation of podosomes we found that MDMs retain podosome formation after LPS activation, whereas MDDCs lose this ability, resulting in a significantly reduced adhesion force and an altered cellular distribution of CR3 and CR4. Our results suggest that inflammatory conditions reshape differentially the expression and role of CR3 and CR4 in macrophages and dendritic cells.

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          Most cited references64

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          Ligand binding to integrins.

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            Selective Recruitment of Immature and Mature Dendritic Cells by Distinct Chemokines Expressed in Different Anatomic Sites

            DCs (dendritic cells) function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led us to investigate the chemokine responsiveness of DCs during their development and maturation. DCs were differentiated either from CD34+ hematopoietic progenitor cells (HPCs) cultured with granulocyte/macrophage colony–stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-α or from monocytes cultured with GM-CSF plus interleukin 4. Immature DCs derived from CD34+ HPCs migrate most vigorously in response to macrophage inflammatory protein (MIP)-3α, but also to MIP-1α and RANTES (regulated on activation, normal T cell expressed and secreted). Upon maturation, induced by either TNF-α, lipopolysaccharide, or CD40L, DCs lose their response to these three chemokines when they acquire a sustained responsiveness to a single other chemokine, MIP-3β. CC chemokine receptor (CCR)6 and CCR7 are the only known receptors for MIP-3α and MIP-3β, respectively. The observation that CCR6 mRNA expression decreases progressively as DCs mature, whereas CCR7 mRNA expression is sharply upregulated, provides a likely explanation for the changes in chemokine responsiveness. Similarly, MIP-3β responsiveness and CCR7 expression are induced upon maturation of monocyte- derived DCs. Furthermore, the chemotactic response to MIP-3β is also acquired by CD11c+ DCs isolated from blood after spontaneous maturation. Finally, detection by in situ hybridization of MIP-3α mRNA only within inflamed epithelial crypts of tonsils, and of MIP-3β mRNA specifically in T cell–rich areas, suggests a role for MIP-3α/CCR6 in recruitment of immature DCs at site of injury and for MIP-3β/CCR7 in accumulation of antigen-loaded mature DCs in T cell–rich areas.
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              Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation

              Dendritic cells (DC) migrate into inflamed peripheral tissues where they capture antigens and, following maturation, to lymph nodes where they stimulate T cells. To gain insight into this process we compared chemokine receptor expression in immature and mature DC. Immature DC expressed CCR1, CCR2, CCR5 and CXCR1 and responded to their respective ligands, which are chemokines produced at inflammatory sites. Following stimulation with LPS or TNF-alpha maturing DC expressed high levels of CCR7 mRNA and acquired responsiveness to the CCR7 ligand EBI1 ligand chemokine (ELC), a chemokine produced in lymphoid organs. Maturation also resulted in up-regulation of CXCR4 and down-regulation of CXCR1 mRNA, while CCR1 and CCR5 mRNA were only marginally affected for up to 40 h. However, CCR1 and CCR5 were lost from the cell surface within 3 h, due to receptor down-regulation mediated by chemokines produced by maturing DC. A complete down-regulation of CCR1 and CCR5 mRNA was observed only after stimulation with CD40 ligand of DC induced to mature by LPS treatment. These different patterns of chemokine receptors are consistent with "inflammatory" and "primary response" phases of DC function.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draft
                Role: Data curationRole: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: Writing – review & editing
                Role: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                4 May 2020
                2020
                : 15
                : 5
                : e0232432
                Affiliations
                [1 ] MTA-ELTE Immunology Research Group, Eötvös Loránd University, Budapest, Hungary
                [2 ] Department of Biological Physics, Eötvös Loránd University, Budapest, Hungary
                [3 ] Nanobiosensorics “Lendület” Group, Institute of Technical Physics and Material Sciences, Centre for Energy Research, Hungarian Academy of Sciences, Budapest, Hungary
                [4 ] Department of Immunology, Eötvös Loránd University, Budapest, Hungary
                [5 ] CellSorter Company for Innovations, Budapest, Hungary
                Thomas Jefferson University, UNITED STATES
                Author notes

                Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: BS and BF are employees of CellSorter Company for Innovations. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

                Author information
                http://orcid.org/0000-0003-3499-8422
                Article
                PONE-D-20-03587
                10.1371/journal.pone.0232432
                7197861
                32365067
                b77bd2d7-b0c3-41f9-9042-a287a85e8d54
                © 2020 Lukácsi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 February 2020
                : 14 April 2020
                Page count
                Figures: 6, Tables: 0, Pages: 21
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003825, Magyar Tudományos Akadémia;
                Award Recipient :
                Funded by: Hungarian National Science Fund (OTKA)
                Award ID: K112011
                Award Recipient :
                This study was funded by the Hungarian National Science Fund (OTKA) [grant K112011 for AE] and by the Hungarian Academy of Sciences for AE and ZB. CellSorter Company for Innovations ( https://www.singlecellpicker.com/) provided its technology, including hardware and software, and funding in the form of salaries for authors BS and BF. The specific roles of these authors are articulated in the ‘author contributions’ section.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Biology and Life Sciences
                Cell Biology
                Cellular Types
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