Mast cell activation is characterized by upregulation of a functional anaphylatoxin C5a receptor

<p>Abstract</p> <p>Background</p> <p>Mast cells (MC) are key effector cells of allergic diseases and resistance to helminthic parasites and induce or amplify diverse innate and adaptive immune responses. The signals controlling MC mobilization during inflammation are not fully understood.</p> <p>Results</p> <p>Since anaphylatoxins are attractive candidates as MC chemoattractants, we investigated expression and function of anaphylatoxin receptors in murine MC. Precursor cell-derived MC cultured with IL-3 in the presence or absence of SCF did not express significant amounts of surface C5a receptor (C5aR) or C3a receptor (C3aR). MC required approximately 4 h of stimulation with Ag (DNP-albumin, following preincubation with IgE anti-DNP), ionomycin, or PMA to enable a strong chemotactic response towards C5a, paralleled by a distinct C5aR upregulation. Likewise, C5a induced intracellular calcium fluxes solely in activated MC. In contrast, C3a proved to be a weak MC chemotaxin and unable to increase intracellular calcium. Primary peritoneal MC did not express detectable amounts of anaphylatoxin receptors, however, similar to precursor cell-derived MC, stimulation with Ag or ionomycin for 4 h induced a prominent surface expression of C5aR whereas C3aR remained undetectable.</p> <p>Conclusion</p> <p>Collectively, our results suggest that Ag-dependent as well as -independent activation induces an inflammatory MC phenotype which is distinguished by neoexpression of a functional C5aR as a novel effector mechanism in MC-mediated pathogenesis.</p

Generation and characterization of monoclonal antibodies against the anaphylatoxin receptors

Anaphylatoxine sind potente Entzündungsmediatoren, die ihre biologische Wirkung durch eine hochaffine Bindung an spezifische, zelluläre Rezeptoren entfalten. Aufgrund der widersprüchlichen Angaben in der Literatur über die Expression der Anaphylatoxin-Rezeptoren sollten in der vorliegenden Arbeit neue, hochspezifische monoklonale Antikörper gegen den humanen C5aR und erstmals solche Reagenzien gegen den Maus-C3aR hergestellt werden. Mit Hilfe dieser neu generierten monoklonalen Antikörper sollte die Expression sowohl des C5a- als auch des C3a-Rezeptors im normalen und pathologisch veränderten Nierengewebe untersucht werden. Zur Herstellung monoklonaler Antikörper gegen den humanen C5aR wurden RBL-2H3-Transfektanten eingesetzt, die den Rezeptor auf der Oberfläche exprimieren. Nach Bestimmung der Rezeptorspezifität wurden acht monoklonale Antikörper identifiziert, die den C5aR immunhistochemisch erkannten. Anhand ihrer Reaktivität gegen zwei unterschiedliche Peptide wurden sie in drei Gruppen eingeteilt, die an verschiedene Epitope der N-terminalen Domäne des C5aR binden. Monoklonale Antikörper aller drei Gruppen ließen eine deutliche C5aR-Expression in Gefrierschnitten der menschlichen Niere erkennen, die allerdings auf Zellen des mononukleär-phagozytären Systems beschränkt blieb. Mono- sowie polyklonale Antikörper, die durch Immunisierung mit C5aR-Peptiden generiert worden waren, hatten in der Vergangenheit eine C5aR-Expression auch in tubulären Zellen der menschlichen Niere nachgewiesen. Dieser Befund konnte in der vorliegenden Arbeit mittels der neu hergestellten monoklonalen Antikörper nicht bestätigt werden. Eine immunzytochemische Untersuchung sowie die RT-PCR-Analyse zum Nachweis von C5aR-mRNA in kultivierten proximal tubulären Zellen der Niere erbrachten ebenfalls keinen Hinweis auf eine C5aR-Expression. Für die Generierung monoklonaler Antikörper gegen den murinen C3aR wurden zunächst stabil transfizierte RBL-2H3 Zellen, die den murinen C3aR exprimieren, hergestellt. Mit diesen wurden Lou/C-Ratten immunisiert. Insgesamt konnten sechs monoklonale Antikörper generiert und charakterisiert werden, die zum Nachweis des C3aR sowohl auf Zelloberflächen als auch in den Gefrierschnitten eingesetzt wurden. Mittels Kreuz-Kompetitions-Experimenten konnten mindestens zwei Gruppen von monoklonalen Antikörpern definiert werden, die unterschiedliche Epitope erkennen. Die durchflusszytometrische Untersuchung muriner Zellen mit den neu generierten monoklonalen Antikörpern wies nach, dass der C3aR auf Peritoneal-Makrophagen und auf Makrophagen, die mittels M-CSF aus Knochenmarksvorläuferzellen differenziert wurden, exprimiert wird. Auf neutrophilen Granulozyten konnte keine C3aR-Expression nachgewiesen werden. Dendritische Zellen aus murinem Knochenmark zeigten eine schwache C3aR-Expression. Die Untersuchung von Milzzellen und Thymozyten der Maus belegten, dass der C3aR weder auf T-Lymphozyten noch auf B-Lymphozyten exprimiert wird. Die immunhistochemische Untersuchung von Gefrierschnitten der Niere LPS-behandelter oder an Lupus-Nephritis erkrankter Tiere (MRL/lpr-Mäuse) zeigte, dass beide Anaphylatoxin-Rezeptoren ausschließlich in residenten und infiltrierenden Makrophagen exprimiert werden. Es konnte keine Expression von Anaphylatoxin-Rezeptoren in proximal tubulären Zellen normaler oder entzündlich veränderter Nieren nachgewiesen werden. Die Ergebnisse der vorliegenden Arbeit ziehen eine Beteiligung nicht-myeloischer Zellen an Anaphylatoxin-vermittelten Entzündungsreaktionen in der Niere in Zweifel. Ferner haben unsere Ergebnisse eine über Anaphylatoxin-Rezeptoren hinausgehende Bedeutung, da sie die generelle Notwendigkeit einer kritischen Beurteilung immunhistochemisch erzielter Ergebnisse hervorheben.Anaphylatoxins are potent pro-inflammatory mediators that induce their biological effects through high affinity binding to specific cellular receptors. Due to controversial evidence in the literature on the expression of anaphylatoxin receptors, it was the purpose of the present study to raise novel highly specific monoclonal antibodies against the human C5aR and, for the first time, against the mouse C3aR. These reagents should be applied to investigate, expression of both the C5a and the C3a receptor in normal and diseased kidney tissues. For the generation of monoclonal antibodies against the human C5aR, RBL-2H3 transfectants expressing the receptor on the cell surface were employed. After determining their receptor specificity eight monoclonal antibodies were identified, which recognized the C5aR in immunohistochemistry. On the basis of their reactivity against two different peptides they were arranged in three groups, which bind to distinct epitopes of the N-terminal domain of C5aR. Monoclonal antibodies from these three groups detected a prominent C5aR expression in frozen sections of the human kidney, which was restricted to cells of the mononuclear phagocytic system. Mono- and polyclonal antibodies which had been raised against C5aR peptides, had previously detected C5aR expression also in tubular cells of the human kidney. This finding could not be confirmed using our newly established monoclonal antibodies. Immunocytochemistry along with the RT-PCR analysis of C5aR mRNA in cultivated proximal tubular cells of the kidney also failed to detect C5aR expression. For generating monoclonal antibodies against the murine C3aR stably transfected RBL-2H3 cells that express the murine C3aR were produced, which then served to immunize Lou/C-rats. A total of six monoclonal antibodies could be generated and characterized, which served to detect the C3aR on cell surface as well as in frozen sections. By cross competition experiments at least two groups of monoclonal antibodies were defined, that recognize different epitopes. Flow cytometric investigation of murine cells using the newly generated monoclonal antibodies demonstrated that C3aR is expressed on peritoneal macrophages and on macrophages, which had been differentiated from bone marrow cells by M-CSF. C3aR expression could not be found on neutrophil granulocytes. Dendritic cells from murine bone marrow displayed a weak C3aR signal. Investigation of murine spleen cells and thymocytes demonstrated that C3aR is neither expressed on T lymphocytes nor on B lymphocytes. The immunohistochemical investigation of frozen kidney sections from mice treated with LPS or suffering from lupus-type nephritis (MRL/lpr mice) demonstrated that both anaphylatoxin receptors are expressed exclusively in resident and infiltrated macrophages. No C5a and C3a receptors were detectable in proximal tubular cells of the normal or inflamed kidney. The results of the present study question a role for non-myeloid cells in anaphylatoxin-mediated inflammatory reactions in the kidney. Moreover, the implications of our study may go beyond the narrow scope of anaphylatoxin receptors as they emphasize the need for a critical assessment of immunohistochemically acquired data in general

Analysis of the CCR7 expression on murine bone marrow-derived and spleen dendritic cells

About 40% of bone marrow-derived dendritic cells (BM-DCs) generated from stem cells of C57BL/6 (B6.WT) mice differentiate in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) without further stimuli to mature DCs. These cells are characterized by high levels of major histocompatibility complex class II, CD40, and CD86 on their surface. Recent studies have revealed that tumor necrosis factor (TNF) is crucial for maturation of BM-DCs. However, once matured, the phenotype of mature TNF-negative C57BL/6 (B6.TNF–/–) and B6.WT BM-DCs is comparable. Both expressed high levels of CD40 and CD86 and were positive for mRNA of the chemokine receptor (CCR)7. To extend our studies, we generated a monoclonal antibody (mAb) specific for mouse CCR7. This mAb allowed us to analyze the surface expression of CCR7 during maturation of B6.WT and B6.TNF–/– BM-DCs in the presence of GM-CSF and stimulated with TNF or lipopolysaccharide (LPS) and to compare it with the CCR7 expression on ex vivo-isolated splenic DCs with or without additional stimulation. Our results showed that CCR7 expression on murine BM-DCs is an indication of cell maturity. Incubation with LPS induced the maturation of all BM-DCs in culture but increased the number of mature CCR7+ splenic DCs only marginally

CCR7 Governs Skin Dendritic Cell Migration under Inflammatory and Steady-State Conditions

AbstractThe CC chemokine receptor CCR7 has been identified as a key regulator of homeostatic B and T cell trafficking to secondary lymphoid organs. Data presented here demonstrate that CCR7 is also an essential mediator for entry of both dermal and epidermal dendritic cells (DC) into the lymphatic vessels within the dermis while this receptor is dispensable for the mobilization of Langerhans cells from the epidermis to the dermis. Moreover, a distinct population of CD11c+MHCIIhigh DC showing low expression of the costimulatory molecules CD40, CD80, and CD86 in wild-type animals was virtually absent in skin-draining lymph nodes of CCR7-deficient mice under steady-state conditions. We provide evidence that these cells represent a semimature population of DC that is capable of initiating T cell proliferation under conditions known to induce tolerance. Thus, our data identify CCR7 as a key regulator that governs trafficking of skin DC under both inflammatory and steady-state conditions

TNF controls the infiltration of dendritic cells into the site of Leishmania major infection

TNF-negative C57BL/6 (B6.TNF−/−) mice are highly susceptible to Leishmania (L.) major infection and succumb rapidly to fatal leishmaniasis. A T helper type 1 (Th1) cell-mediated immune response is central for protective anti-leishmanial immunity. Therefore, the observed susceptibility of B6.TNF−/− mice to L. major parasites could be caused by a deficiency in mounting a Th1 response. Analysis of infected footpads revealed, that B6.TNF−/− mice exhibited a substantially diminished formation of DCs at the site of infection. Furthermore, Th1 cytokines such as IFN-γ were reduced in footpads of infected B6.TNF−/− mice. Cutaneous reconstitution of B6.TNF−/− mice with either bone marrow derived DCs (BM-DCs) or recombinant TNF simultaneous to infection resulted in an increased expression of cytokines such as IFN-γ and in an enhanced presence of Leishmania-antigen in skin draining lymph nodes. In addition, the individual time of survival was doubled. In conclusion we demonstrate that the expression of dermal TNF is necessary to provide an environment that initiates a local inflammatory response, but is not sufficient to induce protective immunity