ABSTRACT
Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients.
Subject(s)
Antigens, CD/genetics , Cadherins/genetics , Dermatitis, Contact/pathology , Hemorrhage/physiopathology , Neutrophils/physiology , Thrombocytopenia/physiopathology , Transendothelial and Transepithelial Migration/physiology , Vasculitis/pathology , Analysis of Variance , Animals , Dermatitis, Contact/etiology , Dermatitis, Contact/immunology , Flow Cytometry , Hemorrhage/etiology , Hemorrhage/prevention & control , Histological Techniques , Humans , Mice , Mice, Inbred C57BL , Mutation, Missense/genetics , Transendothelial and Transepithelial Migration/drug effects , Ultraviolet Rays , Vasculitis/etiology , Vasculitis/immunologyABSTRACT
Matrix metalloproteinases (MMPs) play a critical role in various pathological conditions including cutaneous inflammation. Thus far, serial assessment of MMP activity in ongoing inflammation is hampered due to technical limitations. Here, we present an innovative method for longitudinal detection of MMP activity by in vivo imaging. First, we analysed skin sections from patients suffering from leucocytoclastic vasculitis (LcV) and detected a significant MMP signal via immunofluorescence staining. Then, we mimicked LcV in mice in a well-studied model of immune complex-mediated vasculitis (ICV). This acute inflammatory process was serially visualized in vivo using the fluorescence-labelled MMP tracer Cy5.5-AF443. The deposition of fluorescence-labelled immune complexes and MMP tracer distribution was visualized repeatedly and non-invasively by fluorescence reflectance imaging. In correlation with the presence of MMP-2 and MMP-9 in immunofluorescence stainings, Cy5.5-AF443 accumulated in ICV spots in the skin of C57BL/6 mice. This tracer accumulation could also be observed in mice equipped with a dorsal skinfold chamber, where microscopic observations revealed an increased recruitment of fluorescence-labelled leucocytes during ICV. The specificity of the MMP tracer was supported by (i) analysis of mice deficient in functional ß2 -integrins (CD18(-/-) ) and (ii) subsequent MMP immunofluorescence staining. These findings let us conclude that MMP accumulation in the acute phase of ICV depends on ß2 -mediated leucocyte recruitment. In summary, we show that MMPs are involved in ICV as determined by Cy5.5-AF443, a new optical marker to longitudinally and non-invasively follow MMP activity in acute skin inflammation in vivo.
Subject(s)
Matrix Metalloproteinases/metabolism , Molecular Imaging/methods , Neutrophils/enzymology , Vasculitis, Leukocytoclastic, Cutaneous/pathology , Animals , Arthus Reaction , CD18 Antigens/metabolism , Carbocyanines/chemistry , Female , Humans , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Microcirculation , Microscopy, Fluorescence , Skin/pathology , Vasculitis, Leukocytoclastic, Cutaneous/enzymologyABSTRACT
IC-mediated vasculitis (ICV) can be life threatening. The cellular and immune mechanisms controlling ICV are poorly understood. Therefore, we investigated the role of α-1-antitrypsin (α1AT) and IFN-γ in reducing the severity of ICV in a mouse model in vivo. To induce ICV, mice were challenged with the reverse passive Arthus reaction (RPA), the prototypic in vivo model for leukocytoclastic vasculitis (LcV), and the modulation of vascular permeability, edema formation, and leukocyte recruitment was studied. To further analyze the dynamics of RPA, we applied intravital microscopy in the dorsal skinfold chamber. α1AT continuously led to reduced leukocyte recruitment. α1AT interfered with neutrophil recruitment through a KC-dependent mechanism and reduced KC-elicited neutrophil activation. In contrast to α1AT, IFN-γ-reduced leukocyte recruitment during RPA was clearly independent of KC. We also revealed that the recruitment of neutrophils during RPA was a prerequisite for full KC expression. Thus, therapeutic administration of α1AT and IFN-γ might be beneficial for limiting the duration and severity of ICV.