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Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis
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Immunology Letters xxx (2013) xxx–xxx
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis
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Kerstin Siegmund a,c,∗ , Woo-Yong Lee c , Vincent S. Tchang a , Michael Stiess a , Luigi Terracciano b , Paul Kubes c , Jean Pieters a,∗∗ a
Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland Molecular Pathology Division, Institute of Pathology, University Hospital, Schönbeinstrasse 40, CH-4003 Basel, Switzerland Q2 c The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, HRIC 4AA16, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1 Canada b
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Article history: Received 25 April 2013 Received in revised form 25 June 2013 Accepted 30 June 2013 Available online xxx
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Keywords: Coronin 1 Leukocytes Hepatitis Concanavalin A
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1. Introduction
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Coronin 1, a member of the evolutionary conserved coronin protein family, is highly expressed in all leukocytes. In mice and human, genetic inactivation of coronin 1 results in immuno-deficiencies that are linked to a strong reduction of naïve T cell numbers in peripheral organs, while memory/effector T cells, B cells, monocytes and neutrophils are less or not at all affected. Whether or not coronin 1 is important for leukocyte functions such as migration and phagocytosis has been a matter of debate. The current work addresses coronin 1-dependent leukocyte function by analyzing the response of coronin 1-deficient mice in a model of concanavalin A (Con A)-induced liver injury. Histological evaluation and determination of serum liver enzyme levels showed that coronin 1-deficient mice develop signs of acute hepatitis similar to Con A-treated wild type mice despite a reduced activation of T cells in the absence of coronin 1. Furthermore, analysis by intravital microscopy following Con A stimulation revealed that Gr-1+ neutrophils and CD4+ T cell adhesion in the post-sinusoidal venules increased in wild type as well as in coronin 1-deficient mice. These results suggest that coronin 1, in contrast to its important role in naïve T cell survival, is dispensable for leukocyte function under inflammatory conditions in vivo. © 2013 Published by Elsevier B.V.
Coronins, which comprise a family of WD-repeat proteins that are conserved from yeast to man, are involved in the regulation of a wide variety of cellular processes including pathogen survival, cell migration, and T cell homeostasis (reviewed in [1,2]). Coronin 1 (also known as P57 or TACO) is highly expressed in cells of the hematopoietic lineage and has been implicated in immune cell function of the innate as well as adaptive immune system [1,3–6]. Recent work revealed that coronin 1 is especially important for naïve T cell survival, activation and migration [7–10]. As such, coronin 1-deficient mice are characterized by strongly reduced naïve T cell numbers in the periphery, while in the thymus the numbers and frequencies of T cell subpopulations (double negative, double
∗ Corresponding author. Present address: Division of Translational Cell Genetics, Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Peter-Mayr-Str. 1a, A-6020 Innsbruck, Austria. Tel.: +43 512 9003 70555; fax: +43 512 9003 73518. ∗∗ Corresponding author. Tel.: +41 61 267 1494; fax: +41 61 267 2148. E-mail addresses: [email protected] (K. Siegmund), [email protected] (J. Pieters).
positive, CD4-single and CD8-single positive T cells) are similar to wild type mice [11]. This reduction of the peripheral naïve T cell pool, caused by the lack of coronin 1 expression, results in protection against the development of autoimmunity in a murine model of multiple sclerosis [12,13]. Furthermore, mutations of the coronin 1 gene have been linked with immunodeficiency in humans [14,15]. In contrast to the profound survival and activation defect of naïve T cells, other immune cells such as B cells, macrophages, dendritic cells and neutrophils seem to be only marginally affected by the lack of coronin 1 [16–20]. For example, using coronin 1deficient mice, it was demonstrated that coronin 1 is dispensable for macrophage chemotaxis and phagocytosis in vitro as well as dendritic cell function to process antigen and activate antigenspecific T cells [18,19,21]. While in human, coronin 1 expression has been related to reduced neutrophil apoptosis in cystic fibrosis patients [22], in mice lacking coronin 1 neutrophil functionality does not seem to be compromised with respect to development, survival, migration, spreading, phagocytosis and NADPH oxidase activity [16]. Despite the role of coronin 1 as an important regulator of naïve T cell viability and function, only little is known about its function in effector/memory T cells. In contrast to naïve T cell numbers, coronin
0165-2478/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.imlet.2013.06.005
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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1-deficient mice have almost equal numbers of T cells displaying an effector/memory phenotype compared to wild type mice in their peripheral lymphoid organs. Furthermore, coronin 1-deficient T cells seem to be fully competent to express the Th1, Th2 and Th17 effector cytokines, IFN-␥, IL-4 and IL-17A, respectively, upon polyclonal stimulation with antibodies recognizing CD3 and CD28 [12]. Additionally, in vitro differentiated coronin 1-deficient Th17 cell cultures contained significantly more IFN-␥ as well as IFN-␥/IL-17A co-producers than wild type cultures [13]. To investigate the role of coronin 1 for effector T cells and neutrophil function in vivo, we here analyzed coronin 1-deficient mice in an inflammation model of acute hepatitis induced by intravenous application of concanvalin A (Con A). In vitro studies have shown that Con A activates T cells to proliferate and produce pro-inflammatory cytokines such as interferon-␥ (INF-␥) by cross-linking glyco-proteins on the cell surface [23]. In vivo Con A treatment is commonly used to induce acute hepatitis in mice, which mimics human disease in many respects [24]. Within hours of intravenous administration of Con A the number of leukocytes such as CD4+ T cells and neutrophils in the liver [25,26] and liver enzymes as well as INF-␥ in the serum increase significantly [27]. CD4+ T cells have been described as the main effector cell in Con A-induced liver injury, but also neutrophils, Kupffer cells and natural killer T cells have been implicated to contribute essentially to the inflammatory process [26,28,29]. Here we show that both in the presence and absence of coronin 1, Con A treatment resulted in an increased adhesion of neutrophils as well as CD4+ T cells in the post-sinusoidal venules. Con Ainduced liver injury in the absence of coronin 1 was comparable to that observed in wild type mice even though coronin 1-deficient T cells showed impaired activation and INF-␥ expression upon Con A treatment. Together, these results suggest that in vivo, coronin 1 is dispensable for leukocyte-mediated liver damage in a Con Ainduced hepatitis model. 2. Materials and methods 2.1. Mice Coronin 1-deficient mice were generated in our laboratory as described [18] and backcrossed to C57BL/6 for eight generations. The corresponding littermates (knockout and wild type) were bred in-house as homozygous lines. For the experiments mice of age 8–16 weeks were used. All experiments were approved either by the Kantonales Veterinäramt Basel-Stadt or by the University of Calgary Animal Care Committee (Protocol #MO8131) and conform to the guidelines established by the Canadian Council for Animal Care. 2.2. Concanavalin A (Con A)-induced hepatic injury, in vivo leukocyte activation and ex vivo read-outs Liver injury was induced by intravenous administration (tail vein) of 13 mg/kg concanavalin A (Con A, Sigma–Aldrich, Oakville, ON, Canada) dissolved in PBS. For vehicle-treated control mice PBS was injected. Four or 12 h after treatment (single injection of Con A or PBS) the mice were sacrificed using high percentage CO2 . For determining the disease severity livers were analyzed by histology and liver enzyme levels in serum were determined. For histology the livers were excised, fixed in 10% formaldehyde and prepared for microscopic assessment after hematoxylin–eosin staining and Sirius Red staining. Slides observations and images acquisition were performed at Zeiss photomicroscope. To determine the degree of liver damage, the slides were scored by a pathologist in a “blinded fashion” for lobular inflammation, portal inflammation, confluent necrosis, endothelialitis and an
overall score was provided. Degree of inflammation: 0 = absent, 1 = minimal/slight, 2 = moderate, 3 = severe. Serum was obtained by centrifugation of coagulated blood, which has been obtained by heart puncture 12 h after Con A treatment. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured at the laboratory medicine of the University Hospital of Basel using commercially available diagnostic kits (Roche-Diagnostics, Mannheim, Germany). To assess the effect of Con A treatment on leukocytes in vivo, cells were isolated from blood, spleen and liver four or 12 h after intravenous Con A administration and analyzed by flow cytometry. For detection of intracellular INF-␥ spleen and liver cells were isolated 12 h after Con A treatment and incubated in vitro for additional 3 h in RPMI-1640 (Gibco, Zug, Switzerland) supplemented with MEM non-essential amino acids (Invitrogen, Zug, Switzerland), 1 mM sodium pyruvate (Sigma–Aldrich, Buchs, Switzerland), penicillin (100 units)-streptomycin (100 g/ml) (Sigma-Aldrich, Buchs, Switzerland), 2-mercaptoethanol (Invitrogen, Zug, Switzerland), 10% fetal bovine serum (PAA laboratories, Linz, Austria) in the presence of 10 g/ml brefeldin A (Sigma–Aldrich, Buchs, Switzerland). For determination of serum INF-␥, mice were sacrificed 4 h after Con A administration. Blood was taken by cardiac puncture and centrifuged after coagulation to obtain serum. The concentration of INF-␥ in serum was measured in Maxisorp plates (Nunc, Langenselbold, Germany) by ELISA (Biolegend, Fell, Germany). 2.3. Isolation of intrahepatic leukocytes Livers were perfused with PBS via the portal vein to remove blood-derived leukocytes. Then the livers were excised and minced with scissors in digestive medium composed of RPMI-1640 (Gibco, Zug, Switzerland) containing 0.05% collagenase D (Worthington, Lakewood, NJ, USA), 50,000 units/ml DNaseI (Roche, Basel, Switzerland) and 0.01% trypsin inhibitor (Worthington, Lakewood, NJ, USA). After 30 min of incubation at 37 ◦ C and 250 rpm, the digested tissue was passed through a 70 m cell strainer (Falcon, Allschwil, Switzerland) and rinsed with cold PBS. The cell pellet was washed twice with cold PBS (centrifugation 300 × g, 10 min, 4 ◦ C). Leukocytes were then isolated using a Percoll gradient (density 1.131 g/ml; GE healthcare, Glattbrug, Switzerland). The cell pellet was resuspended in 90% Percoll solution (diluted with HBSS, Gibco, Zug, Switzerland), overlaid with 37% and 30% Percoll solution and centrifuged at 800 × g for 20 min at room temperature. Leukocytes were washed in PBS and counted in trypan blue (Invitrogen, Zug, Switzerland) using a hemocytometer. 2.4. Surgery and spinning disk confocal intravital microscopy of the liver Mice were anesthetized by intraperitoneal injection of a mixture of 10 mg/kg xylazine and 200 mg/kg ketamine hydrochloride (both Bimeda-MTC, Cambridge, ON, Canada) prior to surgery. Body temperature was maintained at 37 ◦ C using an infrared heat lamp. The right vena jugularis was cannulated to allow for i.v. administration of antibodies and additional anesthetic as required. The following antibodies were administered: Alexa Fluor 647-conjugated antiCD4 (clone GK1.5; 1.6 g, eBioscience, San Diego, CA, USA) and phycoerythrin-conjugated anti-Gr-1 (clone RB6-8C5; 1.6 g, eBioscience, San Diego, CA, USA). The liver was prepared for in vivo microscopic observation, as previously described [30]. Briefly, the abdomen was opened with a midline incision, and the skin and abdominal wall were removed along the costal margin to the midaxillary line. The falciform ligament was then dissected away from the gallbladder and liver. Mice were placed in a right lateral position on a plexiglas microscope stage and the left lateral lobe of the liver was exposed. The liver surface was then covered with a
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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small piece of saline-soaked Kimwipes to hold the organ in position. All exposed tissues were moistened with saline-soaked gauze to prevent dehydration. Images were acquired with an Olympus IX81 inverted microscope (Olympus, Center Valley, PA) using a 10×/0.40 UPlanSApo objective. The microscope was equipped with a confocal light path (WaveFx; Quorum Technologies, Guelph, Ontario, Canada) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation, Tokyo, Japan). The liver and labeled cells were imaged using either a 488-, 561- or 635-nm laser excitation (Cobolt, Stockholm, Sweden)) and visualized with the appropriate long-pass filters (Semrock, Rochester, NY, USA). A 512 × 512 pixel back-thinned electron-multiplying charge-coupled device camera (C9100-13, Hamamatsu, Bridgewater, NJ, USA) was used for fluorescence detection. The confocal microscope was driven by Volocity acquisition software (Perkin Elmer, Waltham, MA, USA).
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2.5. In vitro activation of lymphocytes
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To analyze in vitro activation of T cells single cell suspensions of spleen and peripheral and mesenteric lymph nodes were prepared using metal sieves or 70 m cell strainers (Falcon). Erythrocytes lysis was obtained by 2 min incubation in hypotonic buffer (ACK buffer, 0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) followed by a wash step. Isolated cells were then resuspended in RPMI-1640 supplemented with MEM non-essential amino acids (Invitrogen, Zug, Switzerland), 1 mM sodium pyruvate (Sigma–Aldrich, Buchs, Switzerland), penicillin (100 units)–streptomycin (100 g/ml) (Sigma–Aldrich, Buchs, Switzerland), 2-mercaptoethanol (Invitrogen, Zug, Switzerland), 10% fetal bovine serum (PAA laboratories, Linz, Austria). 2.5 × 106 cells were stimulated in a 24-well plate with 1 g/ml Con A or 500 ng/ml ionomycin and 20 ng/ml phorbol–myristate–acetate (all from Sigma–Aldrich, Buchs, Switzerland) for 4 h. Cell activation was assessed by analyzing CD69 and CD62L expression of CD4+ and CD8+ cells by flow cytometry.
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2.6. Flow cytometry
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The following antibodies were used for staining cells for flow cytometric analyses: CD16/32 (clone 93, Biolegend, Fell, Germany), anti-CD4 Pacific blue (RM4-5, Invitrogen, Zug, Switzerland), CD69PE-Cy7 (clone H1.2F3 BD Pharmingen), INF-␥ PE Cy7 (clone XMG1.2, Biolegend, Fell, Germany), CD8a APC Cy7 (clone 53–6.7, Biolegend, Fell, Germany), NK1.1 Alexa fluor 647 (clone PK136, Biolegend, Fell, Germany), CD3e PE (clone 145-2C11, Biolegend, Fell, Germany), CD62L PerCP (MEL14, Biolegend, Fell, Germany). For surface staining the cells were incubated with the labeled antibodies for 15 min on ice in the dark and then washed once with PBS/0.5% BSA. For intracellular staining, cells were first stained for surface antigens as described and then fixed with 2% paraformaldehyde for 20 min at room temperature. After a washing step with PBS/0.5%BSA the cells were permeabilized using 0.5% saponin (Sigma–Aldrich, Buchs, Switzerland) in PBS/0.5% BSA and stained in this buffer for 15 min with anti-INF-␥ antibody at room temperature. This was followed by one washing step with 0.5% saponin/PBS/0.5%BSA and one washing step with PBS/0.5% BSA. Incubation with FcR block (anti-CD16/32 antibody, Biolegend, Fell, Germany) was included. The samples were measured on a FACS Canto II (BD Biosciences) and the data were analyzed with the FlowJo software (Tree Star, Ashland, OR, USA).
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2.7. Statistical analyses
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The data are depicted in the graphs as values obtained of all individual mice analyzed and the mean (±SD). FACS dot plots and
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microscopic pictures show representative results. The numbers of mice (n) that have been analyzed are listed in each figure legend. Statistically significance was analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparison test (for comparing multiple samples: wtPBS with wtCon A , koPBS with koCon A , wtPBS with koPBS and wtCon A with koCon A ), Mann–Whitney U-test (for comparing only two samples) using GraphPad Prism software (GraphPad, San Diego, CA, USA). In the figures only significant differences are depicted. ***p value <0.001, **p value <0.01, *p value <0.05, ns p value >0.05.
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3. Results
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3.1. Con A-induced liver injury
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Coronin 1 is essential for the activation of naïve T lymphocytes and has been implicated in immune activation in the context of autoimmune diseases such as lupus and experimental autoimmune encephalomyelitis [9,12,13]. However, the impact of coronin 1 on immune functions in the context of acute inflammation has thus far not been addressed. To analyze a role for coronin 1 in acute inflammation, both wild type and coronin 1-deficient animals were injected intravenously with the lectin concanavalin A (Con A), which is a broadly used model for acute hepatitis and leads to acute liver injury after a few hours [24]. To assess the overall consequences of coronin 1 deficiency on the extent of liver injury following Con A injection, histology was performed. Histological evaluation of liver parenchyma revealed that both wild type as well as coronin 1-deficient mice developed clear-cut histological signs of Con A-induced hepatitis characterized by large confluent necrosis, intra-lobular mononuclear inflammation and focal endothelialitis. The overall pathological evaluation revealed a similar inflammation score for wild type and coronin 1-deficient mice (Fig. 1). Furthermore, increased AST and ALT serum levels, which correlate with hepatocyte death, were not significantly different between both mouse strains (Fig. 1).
3.2. Leukocyte recruitment to the liver of Con A-treated mice in the presence and absence of coronin 1 analyzed by intravital microscopy Inflammation is characterized by infiltration of a variety of immune cells in the affected organ. The role for coronin 1 in leukocyte migration remains however unclear; based on in vitro migration analysis these processes have been suggested to both be dependent as well as independent on coronin 1 [7,11]. Whether or not there is a role for coronin 1 in migration under inflammatory conditions in vivo is not known. To investigate CD4+ T cell and neutrophil migration during inflammation in the presence and absence of coronin 1, intravital microscopy of the liver was performed using wild type and coronin 1-deficient mice upon Con A treatment. To visualize CD4+ T cells and neutrophils in vivo, fluorescently labeled anti-CD4 and anti-Gr-1 antibodies were injected into the vena jugularis (see also Suppl. Movies 1–8). As expected, given the overall reduction of T cell numbers in the absence of coronin 1 [7,8,10], intravital microscopy of the livers of PBS- or Con A-treated mice showed that the overall number of CD4+ cells in coronin 1-deficient animals was significantly lower than in wild type mice (Fig. 2A). Importantly, as shown in Fig. 2A, injection of Con A had no significant effect in either wild type or coronin 1-deficient animals on the total number of CD4+ T cells per field of view (FOV, PBS vs. Con A) obtained by intravital microscopy of the liver 1.5–4 h after treatment. Nonetheless, the number of CD4+ cells that adhered in the
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 1. Liver injury of wild type and coronin 1-deficient mice induced by Con A injection. Mice were treated once either with Con A (13 mg/kg) or PBS by i.v. injection and the analysis was performed at 12 h after administration. (A) Sections of formalin-fixed, hematoxylin–eosin stained and Sirius Red stained livers of PBS and Con A-treated mice. Magnification is indicated in the figure, the box marks the region that was further magnified. Arrows point to large confluent necrosis. (B) The graph depicts the disease score in Con A-treated mice, which represents the degree of inflammation/score 0–3: 0 = absent, 1 = minimal/slight, 2 = moderate, 3 = severe (n = 4). Serum ALT and AST activities were determined 12 h after treatment using diagnostic kits (nwt = 22, nko = 20). ns, not significant.
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post-sinusoidal venules did increase after Con A treatment in both mouse strains (Fig. 2A). Analysis of Gr-1+ cells by intravital microscopy revealed comparable numbers in the livers of PBS-treated coronin 1-deficient and wild type mice, which strongly increased after Con A treatment in the whole field of view as well as the post-sinusoidal venules. The Gr-1+ cell counts per FOV and post-sinusoidal venules was not significantly different between mice of both genotypes (Fig. 2B). 3.3. Immune responses to Con A treatment in the presence and absence of coronin 1
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To further analyze the immune response to Con A treatment, leukocyte counts in blood, spleen and liver at 4 h after treatment with either PBS or Con A were determined using a hemocytometer. Con A treatment resulted in increased numbers of leukocytes predominantly in the spleen (Fig. 3A). Con A-treated coronin 1deficient mice had significantly reduced blood leukocyte counts compared to Con A-treated wild type mice. In contrast, leukocyte numbers in liver and spleen were comparable between wild type and coronin 1-deficient mice whether or not treated with Con A (Fig. 3A). CD4+ and CD8+ T cells in the liver were quantified using flow cytometry. PBS- as well as Con A-treated coronin 1-deficient mice possessed lower T cells counts (CD3+ NK1.1− ) in their livers than wild type mice (Fig. 3B). Four hours after Con A treatment no considerable change in intrahepatic T cells counts was observed. However, it resulted in an increase in CD4+ T cell frequency solely in wild type mice (Fig. 3C).
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3.4. In vivo activation of T cells by concanavalin A
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Since CD4+ T cells have been described as major players of liver injury after Con A administration [24], we addressed whether coronin 1-deficient T cells were able to respond to Con A treatment. To analyze T cell activation following Con A stimulation in vivo, upregulation of the early activation marker CD69 was analyzed on CD4+ and CD8+ T cells isolated from the livers of wild type and
coronin 1-deficient mice 4 h after Con A or PBS treatment. Con A treatment increased CD69 expression on T cells of both mouse strains. A comparison between Con A-treated wild type and coronin 1-deficient mice revealed that CD69 expression on CD4+ as well as CD8+ T cells was higher in wild type than in coronin 1-deficient mice as analyzed by FACS (Fig. 4A). A similar increase in CD69expression was observed for CD4+ and CD8+ T cells isolated from the spleen of Con A-treated animals (data not shown). When analyzing INF-␥ expression of intrahepatic T lymphocytes as a read-out for cell activation, no significant difference in frequency and numbers of INF-␥+ CD8+ T cells was observed between Con A-treated wild type and Con A-treated coronin 1-deficient mice; even though there was a trend to lower expression in the absence of coronin 1. In contrast, CD4+ INF-␥-expressing cells in the liver were reduced in numbers in coronin 1-deficient mice (Fig. 4B). Unlike the data obtained for the livers, almost no INF-␥ expression above background was observed when T cells were isolated from the spleen after Con A treatment in both wild type and coronin 1-deficient mice (data not shown). In addition to intracellular INF-␥ expression by T cells, the INF␥ concentration in the serum of Con A-treated mice was analyzed by ELISA. While in the serum of PBS-treated mice no INF-␥ above detection limit could be measured, the concentration of INF-␥ was strongly increased in the serum of wild type but not coronin 1deficient mice 4 h after Con A treatment (Fig. 4C). 3.5. In vitro activation of T cells by concanavalin A In addition to the analysis ex vivo, we addressed whether coronin 1-deficient T cells can be activated by Con A in vitro. To this end, spleen and lymph node cells were stimulated with Con A and as a read-out for T cell activation CD69 and CD62L expression was analyzed by flow cytometry (Fig. 5). In both wild type and coronin 1-deficient T cells, increased CD69 and decreased CD62L expression was observed. As observed ex vivo, coronin 1-deficient lymphocytes showed a slight decreased response to Con A treatment with regard to CD69 up-regulation, but the difference between wild type
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 2. Gr-1+ and CD4+ T cell recruitment to the livers of wild type and coronin 1-deficient mice in response to Con A treatment. Mice were treated with Con A (single intravenous administration) 60–90 min before anesthesia, cannulation of the vena jugularis and surgical exposure of the livers to perform intravital microscopy analyses using an inverted spinning disk confocal microscope. To visualize CD4 (A) and Gr-1 (B) expressing cells, fluorochrome-labeled antibodies recognizing these antigens were applied via the vein catheter. The mice were imaged between 1.5 and 4 h after Con A treatment. Still images, showing CD4 (blue), Gr-1 (red) and background fluorescence and in case of coronin 1-deficient mice GFP (green) from one experiment, are depicted. The lines outline the location of the post-sinusoidal venule. For quantification the number of crawling or adherent Gr-1+ and CD4+ cells per field of view (FOV, 512 m × 512 m) using a 10× objective and in post-sinusoidal venules (calculated per 500 m length) were counted. The scale bar represents 100 m, 2–6 FOV per mouse were analyzed. n(wtPBS ) = 2, n(wtcon A ) = 4, n(coro1−/− PBS ) = 2, n(coro1−/− con A ) = 6).
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 3. Changes in leukocyte numbers after Con A treatment of wild type and coronin 1-deficient mice. (A) Leukocyte counts in blood, spleen and liver of PBS or Con A-treated mice were determined 4 h after treatment using trypan blue and a hemocytometer. (B) and (C) Leukocytes in the liver of PBS and Con A-treated mice were stained with antiGr-1, anti-CD3, anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Doublettes and dead cells were excluded by FSC-A and FSC-H gating and propidium iodide staining, respectively. Representative FACS dot blots are shown. Numbers of Gr-1high granulocytes (high in sideward scatter) and CD3+ T cells (NK1.1− ) and the frequency of CD4+ and CD8+ T cells in the livers are depicted. n = 6 (Con A), nwt/ko = 8/9 (PBS).
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and coronin 1-deficient mice was less pronounced in vitro (Fig. 5). Coronin 1-deficient CD4+ cells exhibited already without any stimulation lower CD62L expression, which is in accordance with the reduced number of naïve T cells in coronin 1-deficient mice. However, the degree of CD62L down-regulation after Con A treatment was similar between coronin 1-deficient and wild type CD4+ cells, while coronin 1-deficient CD8+ cells seemed to be impaired (Fig. 5). Taken together these results show that while activation of T cells was impaired in coronin 1-deficient mice, CD4+ T cells and neutrophils were similarly recruited to the liver in wild type as well as coronin 1-deficient mice upon Con A treatment causing liver injury in both mice strains.
effector T cells to peripheral tissues under inflammatory conditions. In this study, we analyzed the role of coronin 1 for leukocyte recruitment to the liver using the well-established Con A-induced hepatitis model [24,26]. Furthermore, it was addressed whether the absence of coronin 1 affects the extend of acute liver damage, which can be assessed by increased liver enzyme levels in blood as early as 4 h upon intravenous administration of Con A [24]. We found that in both wild type as well as coronin 1-deficient animals Con A-induced hepatitis resulted in similar levels of liver injury, as well as a comparable recruitment of leukocytes to the inflamed liver. Together these results suggest that coronin 1 is dispensable for leukocyte recruitment under inflammatory conditions in vivo.
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4. Discussion
4.1. Coronin 1, neutrophils and Con A-induced hepatitis
Acute inflammation is characterized by tissue infiltration of activated leukocytes, which is a tightly controlled process involving several steps such as rolling and adhesion preceding cell extravasation [31–35]. Thus far it was unknown whether coronin 1 is involved in recruitment of leukocytes such as neutrophils and
Neutrophils, that are the first cells to arrive at sides of inflammation, have been reported to be key initiators of lymphocyte recruitment to the liver after Con A treatment [26] and furthermore to be necessary for INF-␥ production by T cells [36]. Depletion of neutrophils before Con A treatment reduced liver injury, T cell
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Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 4. In vivo cell activation after intravenous administration of Con A. (A) CD69 expression on hepatic T cells (gated for viable CD3+ NK1.1− cells) of PBS and Con A-treated (4 h) wild type and coronin 1-deficient mice was analyzed by flow cytometry. Representative FACS dot blots gated for CD4+ T cells are shown. CD69 expression was quantified by determining the mean fluorescent intensity (MFI) of CD69 staining on CD4+ and CD8+ T cells. (B) INF-␥ expression of hepatic T cells was analyzed 12 h after PBS or Con A-treatment by intracellular staining and flow cytometry. Directly after isolation from the livers leukocytes were incubated for 3 h in the presence of brefeldin A to trap the produced INF-␥ in the cells. Thereafter surface staining with antibodies recognizing NK1.1, CD3, CD4 and CD8 was performed and the cells were subsequently fixed with 2% PFA. The fixed cells were then permeabilized using 0.05% saponin in PBS, stained for intracellular INF-␥ and analyzed by flow cytometry. Representative FACS dot blots, gated CD4+ and CD8+ T cells (NK1.1− CD3+ ), are shown. For quantification the frequency and cell counts of INF-␥-positive hepatic CD4+ and CD8+ T cells were determined. Each symbol represents data obtained from an individual animal. n = 6. (C) INF-␥ concentration in the serum of wild type or coronin 1-deficient mice treated with either PBS or Con A was analyzed by ELISA at 4 h after intravenous injection. n = 6; nd = not detectable.
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adhesion in the post-sinusoidal venules and cytokine production by T cells [36]. Even though neutrophils express high levels of coronin 1, a role for coronin 1 in neutrophils, if any, remains unknown. Previous work using coronin 1-deficient mice suggests that both neutrophil phagocytosis and chemotaxis are independent of coronin 1 [11,16]. Furthermore, the here observed recruitment of coronin 1-deficient neutrophils to the liver upon Con A treatment is consistent with the previously reported increase of granulocytes in blood upon immunization with incomplete Freud adjuvant, suggesting a normal mobilization from the bone marrow in the absence of coronin 1 [12]. Together these data suggest that coronin 1 is dispensable for these neutrophil functions in vivo.
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An essential role for CD4+ T cells in the induction of full-blown liver inflammation in Con A-induced hepatitis has been demonstrated using mice lacking CD4+ T cells, which were fully protected against Con A-induced hepatitis [24]. Interestingly, even though coronin 1-deficient mice possess reduced overall T cell numbers as well as impaired Con A-induced activation of intrahepatic T cells ex vivo, acute liver injury develops in the absence of coronin 1. Since the response to intravenous Con A is fast and occurs within a few hours, it is conceivable that effector/memory T cells and not naïve T cells are the major responsible subset, consistent with the effector/memory T cell compartment being less affected
by the absence of coronin 1 [7–10]. In contrast to the development of acute Con A-induced liver inflammation described here, coronin 1-deficient mice have been shown to be resistant to experimental autoimmune encephalomyelitis (EAE), which develops over several days and depends on the antigen-specific clonal expansion of naïve CD4+ T cells [12,13]. This resistance to EAE is in accordance with the strong reduction of naïve T cells in coronin 1-deficient mice [12]. However, coronin 1-deficient mice develop more severe chronic EAE when re-challenged with the same antigen, activating a memory response, suggesting that effector/memory T cells are fully functional in the absence of coronin 1 once they have developed from the naïve T cell pool [13]. The importance of naïve and effector/memory CD4+ cell subsets in EAE and Con A-induced hepatitis, respectively, is likely to explain the different outcome of these diseases in coronin 1-deficient mice, whose major defect lies in the naïve T cell compartment. Also, the differences in the pathophysiology of acute inflammation vs. auto-immune disease, the latter being mainly a chronic inflammatory response may explain the different outcomes observed in these CD4+ T cell-dependent disease models. Serum INF-␥-levels after in vivo Con A treatment were strongly reduced in coronin 1-deficient mice, arguing for a defective activation of leukocytes in the absence of coronin 1 expression. However, the differences in expression of activation markers were less pronounced between wild type and coronin 1-deficient mice, suggesting that cell types other than T cells may contribute to elevated serum INF-␥. This idea is supported by the finding that in contrast
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Fig. 5. Activation of T cells in response to Con A treatment in vitro. CD69 and CD62L expression on CD4+ and CD8+ T cells were analyzed by flow cytometry. Spleen and lymph node cells were isolated and stimulated in vitro for 4 h either with PMA/ionomycin or with 1 g/ml Con A or left un-stimulated. Thereafter the cells were stained for CD4, CD8, CD69 and CD62L and analyzed by flow cytometry. Representative FACS dot blots showing CD69 and CD62L expression on CD4+ T cells are depicted. CD69 and CD62L expression were quantified by determining the mean fluorescent intensity (MFI) for both stains on CD4+ and CD8+ T cells. n = minimum of 5 individual mice.
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to in vivo treatment, Con A induced a similar CD69 expression of wild type and coronin 1-deficient CD4+ T cell when treated in vitro. Furthermore, coronin 1-deficient T cells are fully competent to express Th1, Th2 and Th17 cytokines after polyclonal stimulation with antibodies against CD3 and CD28 in vitro arguing against a general defect in cytokine expression [12]. Interestingly, in this context, the largest difference in CD4+ T cell numbers between wild type and coronin 1-deficient mice was not observed in lymphoid tissue but in blood [12]. As a result, this impacts on the size of the T cell pool available for recruitment to peripheral sites upon inflammatory stimuli. Also, while after Con A treatment reduced numbers of adherent CD4+ cells were observed in the post-sinusoidal venules of coronin 1-deficient mice compared to wild type mice, the fold increase in adherent CD4+ cells induced by treatment was comparable between both mice strains. This finding suggests that CD4+ cell adhesion in the inflamed liver is independent of coronin 1 expression. In conclusion, the results presented here show that neutrophil as well as CD4+ T cell recruitment upon an inflammatory stimulus
and liver inflammation does occur also in the absence of coronin 1 expression. Acknowledgements This work was supported by the Canton of Basel, the Swiss National Science Foundation [to J.P.], EMBO [ALTF 1151-2011 to M.S. and ASTF No. 415-2010 to K.S] and the Canadian Institutes of Health Research [to P.K.]. We thank Fausta Chiaverio from the laboratory medicine of the University Hospital of Basel for coordinating the AST/ALT measurements in murine blood samples. We thank the animal care unit at the Biozentrum, University of Basel, and at the University of Calgary for help with all mice related issues, especially Carla Badick in the laboratory of Paul Kubes for the coordination of the mouse import to Calgary. We thank The Live Cell Imaging Facility at the University of Calgary funded by the Canada Foundation for Innovation for support concerning all microscopy related issues.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imlet. 2013.06.005. References
[1] Pieters J, Mueller P, Jayachandran R. On guard: coronin proteins in innate and 495 adaptive immunity. Nat Rev Immunol 2013 [in press]. 496 [2] Chan KT, Creed SJ, Bear JE. Unraveling the enigma: progress towards under497 standing the coronin family of actin regulators. Trends Cell Biol 2011;21: 498 481–8. 499 [3] Ferrari G, Langen H, Naito M, Pieters J. A coat protein on phagosomes involved 500 in the intracellular survival of mycobacteria. Cell 1999;97:435–47. 501 [4] Gatfield J, Pieters J. Essential role for cholesterol in entry of mycobacteria into 502 macrophages. Science 2000;288:1647–50. 503 [5] Pieters J. Mycobacterium tuberculosis and the macrophage: maintaining a bal504 ance. Cell Host Microbe 2008;3:399–407. 505 [6] Pieters J. Coronin 1 in innate immunity. Subcell Biochem 2008;48:116–23. 506 [7] Foger N, Rangell L, Danilenko DM, Chan AC. Requirement for coronin 1 507 in T lymphocyte trafficking and cellular homeostasis. Science 2006;313: 508 839–42. 509 [8] Mueller P, Massner J, Jayachandran R, Combaluzier B, Albrecht I, Gatfield J, 510 et al. Regulation of T cell survival through coronin-1-mediated generation of 511 inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor trig512 gering. Nat Immunol 2008;9:424–31. 513 [9] Haraldsson MK, Louis-Dit-Sully CA, Lawson BR, Sternik G, Santiago-Raber 514 ML, Gascoigne NR, et al. The lupus-related Lmb3 locus contains a disease515 suppressing Coronin-1A gene mutation. Immunity 2008;28:40–51. 516 [10] Shiow LR, Roadcap DW, Paris K, Watson SR, Grigorova IL, Lebet T, et al. 517 The actin regulator coronin 1A is mutant in a thymic egress-deficient mouse 518 strain and in a patient with severe combined immunodeficiency. Nat Immunol 519 2008;9:1307–15. 520 [11] Mueller P, Liu X, Pieters J. Migration and homeostasis of naive T cells depends 521 on coronin 1-mediated prosurvival signals and not on coronin 1-dependent 522 filamentous actin modulation. J Immunol 2011;186:4039–50. 523 [12] Siegmund K, Zeis T, Kunz G, Rolink T, Schaeren-Wiemers N, Pieters J. Coronin 1524 mediated naive T cell survival is essential for the development of autoimmune 525 encephalomyelitis. J Immunol 2011;186:3452–61. 526 [13] Kaminski S, Hermann-Kleiter N, Meisel M, Thuille N, Cronin S, Hara 527 H, et al. Coronin 1A is an essential regulator of the TGFbeta recep528 tor/SMAD3 signaling pathway in Th17 CD4(+) T cells. J Autoimmun 2011;37: 529 198–208. 530 [14] Shiow LR, Paris K, Akana MC, Cyster JG, Sorensen RU, Puck JM. Severe combined 531 immunodeficiency (SCID) and attention deficit hyperactivity disorder (ADHD) 532 associated with a coronin-1A mutation and a chromosome 16p11.2 deletion. 533 Clin Immunol 2008. 534 [15] Moshous D, Martin E, Carpentier W, Lim A, Callebaut I, Canioni D, et al. 535 Whole-exome sequencing identifies Coronin-1A deficiency in 3 siblings with 536 Q4 immunodeficiency and EBV-associated B-cell lymphoproliferation. J Allergy 537 Clin Immunol 2013. 538 [16] Combaluzier B, Pieters J. Chemotaxis and phagocytosis in neutrophils is independent of coronin 1. J Immunol 2009;182:2745–52.
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[17] Combaluzier B, Mueller P, Massner J, Finke D, Pieters J. Coronin 1 is essential for IgM-mediated Ca2+ mobilization in B cells but dispensable for the generation of immune responses in vivo. J Immunol 2009;182:1954–61. [18] Jayachandran R, Sundaramurthy V, Combaluzier B, Mueller P, Korf H, Huygen K, et al. Survival of mycobacteria in macrophages is mediated by coronin 1dependent activation of calcineurin. Cell 2007;130:37–50. [19] Westritschnig K, Bosedasgupta S, Tchang V, Siegmund K, Pieters J. Antigen processing and presentation by dendritic cells is independent of coronin 1. Mol Immunol 2013;53:379–86. [20] Tchang V, Mekker A, Siegmund K, Karrer U, Pieters J. Diverging role for coronin 1 in antiviral CD4+ and CD8+ T cell responses. Mol Immunol 2013 [in press]. [21] Jayachandran R, Gatfield J, Massner J, Albrecht I, Zanolari B, Pieters J. RNA interference in J774 macrophages reveals a role for Coronin 1 in mycobacterial trafficking but not in actin-dependent processes. Mol Biol Cell 2008;19:1241–51. [22] Moriceau S, Kantari C, Mocek J, Davezac N, Gabillet J, Guerrera IC, et al. Coronin1 is associated with neutrophil survival and is cleaved during apoptosis: potential implication in neutrophils from cystic fibrosis patients. J Immunol 2009;182:7254–63. [23] Wimer BM, Mann PL. Mitogen information summaries. Cancer Biother Radiopharm 2002;17:569–97. [24] Tiegs G, Hentschel J, Wendel AA. T cell-dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest 1992;90:196–203. [25] Miyazawa Y, Tsutsui H, Mizuhara H, Fujiwara H, Kaneda K. Involvement of intrasinusoidal hemostasis in the development of concanavalin A-induced hepatic injury in mice. Hepatology 1998;27:497–506. [26] Bonder CS, Ajuebor MN, Zbytnuik LD, Kubes P, Swain MG. Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis. J Immunol 2004;172:45–53. [27] Kusters S, Gantner F, Kunstle G, Tiegs G. Interferon gamma plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A. Gastroenterology 1996;111:462–71. [28] Schumann J, Wolf D, Pahl A, Brune K, Papadopoulos T, van Rooijen N, et al. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am J Pathol 2000;157:1671–83. [29] Takeda K, Hayakawa Y, Van Kaer L, Matsuda H, Yagita H, Okumura K. Critical contribution of liver natural killer T cells to a murine model of hepatitis. Proc Natl Acad Sci USA 2000;97:5498–503. [30] Wong J, Johnston B, Lee SS, Bullard DC, Smith CW, Beaudet AL, et al. A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature. J Clin Invest 1997;99:2782–90. [31] Imhof BA, Aurrand-Lions M. Adhesion mechanisms regulating the migration of monocytes. Nat Rev Immunol 2004;4:432–44. [32] Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 1995;57:827–72. [33] Catalina MD, Estess P, Siegelman MH. Selective requirements for leukocyte adhesion molecules in models of acute and chronic cutaneous inflammation: participation of E- and P- but not L-selectin. Blood 1999;93:580–9. [34] Mohamadzadeh M, DeGrendele H, Arizpe H, Estess P, Siegelman M. Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion. J Clin Invest 1998;101:97–108. [35] Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 2007;7:678–89. [36] Hatada S, Ohta T, Shiratsuchi Y, Hatano M, Kobayashi Y. A novel accessory role of neutrophils in concanavalin A-induced hepatitis. Cell Immunol 2005;233:23–9.
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Contents lists available at SciVerse ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis
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Kerstin Siegmund a,c,∗ , Woo-Yong Lee c , Vincent S. Tchang a , Michael Stiess a , Luigi Terracciano b , Paul Kubes c , Jean Pieters a,∗∗ a
Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland Molecular Pathology Division, Institute of Pathology, University Hospital, Schönbeinstrasse 40, CH-4003 Basel, Switzerland Q2 c The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, HRIC 4AA16, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1 Canada b
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Article history: Received 25 April 2013 Received in revised form 25 June 2013 Accepted 30 June 2013 Available online xxx
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Keywords: Coronin 1 Leukocytes Hepatitis Concanavalin A
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1. Introduction
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Coronin 1, a member of the evolutionary conserved coronin protein family, is highly expressed in all leukocytes. In mice and human, genetic inactivation of coronin 1 results in immuno-deficiencies that are linked to a strong reduction of naïve T cell numbers in peripheral organs, while memory/effector T cells, B cells, monocytes and neutrophils are less or not at all affected. Whether or not coronin 1 is important for leukocyte functions such as migration and phagocytosis has been a matter of debate. The current work addresses coronin 1-dependent leukocyte function by analyzing the response of coronin 1-deficient mice in a model of concanavalin A (Con A)-induced liver injury. Histological evaluation and determination of serum liver enzyme levels showed that coronin 1-deficient mice develop signs of acute hepatitis similar to Con A-treated wild type mice despite a reduced activation of T cells in the absence of coronin 1. Furthermore, analysis by intravital microscopy following Con A stimulation revealed that Gr-1+ neutrophils and CD4+ T cell adhesion in the post-sinusoidal venules increased in wild type as well as in coronin 1-deficient mice. These results suggest that coronin 1, in contrast to its important role in naïve T cell survival, is dispensable for leukocyte function under inflammatory conditions in vivo. © 2013 Published by Elsevier B.V.
Coronins, which comprise a family of WD-repeat proteins that are conserved from yeast to man, are involved in the regulation of a wide variety of cellular processes including pathogen survival, cell migration, and T cell homeostasis (reviewed in [1,2]). Coronin 1 (also known as P57 or TACO) is highly expressed in cells of the hematopoietic lineage and has been implicated in immune cell function of the innate as well as adaptive immune system [1,3–6]. Recent work revealed that coronin 1 is especially important for naïve T cell survival, activation and migration [7–10]. As such, coronin 1-deficient mice are characterized by strongly reduced naïve T cell numbers in the periphery, while in the thymus the numbers and frequencies of T cell subpopulations (double negative, double
∗ Corresponding author. Present address: Division of Translational Cell Genetics, Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Peter-Mayr-Str. 1a, A-6020 Innsbruck, Austria. Tel.: +43 512 9003 70555; fax: +43 512 9003 73518. ∗∗ Corresponding author. Tel.: +41 61 267 1494; fax: +41 61 267 2148. E-mail addresses: [email protected] (K. Siegmund), [email protected] (J. Pieters).
positive, CD4-single and CD8-single positive T cells) are similar to wild type mice [11]. This reduction of the peripheral naïve T cell pool, caused by the lack of coronin 1 expression, results in protection against the development of autoimmunity in a murine model of multiple sclerosis [12,13]. Furthermore, mutations of the coronin 1 gene have been linked with immunodeficiency in humans [14,15]. In contrast to the profound survival and activation defect of naïve T cells, other immune cells such as B cells, macrophages, dendritic cells and neutrophils seem to be only marginally affected by the lack of coronin 1 [16–20]. For example, using coronin 1deficient mice, it was demonstrated that coronin 1 is dispensable for macrophage chemotaxis and phagocytosis in vitro as well as dendritic cell function to process antigen and activate antigenspecific T cells [18,19,21]. While in human, coronin 1 expression has been related to reduced neutrophil apoptosis in cystic fibrosis patients [22], in mice lacking coronin 1 neutrophil functionality does not seem to be compromised with respect to development, survival, migration, spreading, phagocytosis and NADPH oxidase activity [16]. Despite the role of coronin 1 as an important regulator of naïve T cell viability and function, only little is known about its function in effector/memory T cells. In contrast to naïve T cell numbers, coronin
0165-2478/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.imlet.2013.06.005
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1-deficient mice have almost equal numbers of T cells displaying an effector/memory phenotype compared to wild type mice in their peripheral lymphoid organs. Furthermore, coronin 1-deficient T cells seem to be fully competent to express the Th1, Th2 and Th17 effector cytokines, IFN-␥, IL-4 and IL-17A, respectively, upon polyclonal stimulation with antibodies recognizing CD3 and CD28 [12]. Additionally, in vitro differentiated coronin 1-deficient Th17 cell cultures contained significantly more IFN-␥ as well as IFN-␥/IL-17A co-producers than wild type cultures [13]. To investigate the role of coronin 1 for effector T cells and neutrophil function in vivo, we here analyzed coronin 1-deficient mice in an inflammation model of acute hepatitis induced by intravenous application of concanvalin A (Con A). In vitro studies have shown that Con A activates T cells to proliferate and produce pro-inflammatory cytokines such as interferon-␥ (INF-␥) by cross-linking glyco-proteins on the cell surface [23]. In vivo Con A treatment is commonly used to induce acute hepatitis in mice, which mimics human disease in many respects [24]. Within hours of intravenous administration of Con A the number of leukocytes such as CD4+ T cells and neutrophils in the liver [25,26] and liver enzymes as well as INF-␥ in the serum increase significantly [27]. CD4+ T cells have been described as the main effector cell in Con A-induced liver injury, but also neutrophils, Kupffer cells and natural killer T cells have been implicated to contribute essentially to the inflammatory process [26,28,29]. Here we show that both in the presence and absence of coronin 1, Con A treatment resulted in an increased adhesion of neutrophils as well as CD4+ T cells in the post-sinusoidal venules. Con Ainduced liver injury in the absence of coronin 1 was comparable to that observed in wild type mice even though coronin 1-deficient T cells showed impaired activation and INF-␥ expression upon Con A treatment. Together, these results suggest that in vivo, coronin 1 is dispensable for leukocyte-mediated liver damage in a Con Ainduced hepatitis model. 2. Materials and methods 2.1. Mice Coronin 1-deficient mice were generated in our laboratory as described [18] and backcrossed to C57BL/6 for eight generations. The corresponding littermates (knockout and wild type) were bred in-house as homozygous lines. For the experiments mice of age 8–16 weeks were used. All experiments were approved either by the Kantonales Veterinäramt Basel-Stadt or by the University of Calgary Animal Care Committee (Protocol #MO8131) and conform to the guidelines established by the Canadian Council for Animal Care. 2.2. Concanavalin A (Con A)-induced hepatic injury, in vivo leukocyte activation and ex vivo read-outs Liver injury was induced by intravenous administration (tail vein) of 13 mg/kg concanavalin A (Con A, Sigma–Aldrich, Oakville, ON, Canada) dissolved in PBS. For vehicle-treated control mice PBS was injected. Four or 12 h after treatment (single injection of Con A or PBS) the mice were sacrificed using high percentage CO2 . For determining the disease severity livers were analyzed by histology and liver enzyme levels in serum were determined. For histology the livers were excised, fixed in 10% formaldehyde and prepared for microscopic assessment after hematoxylin–eosin staining and Sirius Red staining. Slides observations and images acquisition were performed at Zeiss photomicroscope. To determine the degree of liver damage, the slides were scored by a pathologist in a “blinded fashion” for lobular inflammation, portal inflammation, confluent necrosis, endothelialitis and an
overall score was provided. Degree of inflammation: 0 = absent, 1 = minimal/slight, 2 = moderate, 3 = severe. Serum was obtained by centrifugation of coagulated blood, which has been obtained by heart puncture 12 h after Con A treatment. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured at the laboratory medicine of the University Hospital of Basel using commercially available diagnostic kits (Roche-Diagnostics, Mannheim, Germany). To assess the effect of Con A treatment on leukocytes in vivo, cells were isolated from blood, spleen and liver four or 12 h after intravenous Con A administration and analyzed by flow cytometry. For detection of intracellular INF-␥ spleen and liver cells were isolated 12 h after Con A treatment and incubated in vitro for additional 3 h in RPMI-1640 (Gibco, Zug, Switzerland) supplemented with MEM non-essential amino acids (Invitrogen, Zug, Switzerland), 1 mM sodium pyruvate (Sigma–Aldrich, Buchs, Switzerland), penicillin (100 units)-streptomycin (100 g/ml) (Sigma-Aldrich, Buchs, Switzerland), 2-mercaptoethanol (Invitrogen, Zug, Switzerland), 10% fetal bovine serum (PAA laboratories, Linz, Austria) in the presence of 10 g/ml brefeldin A (Sigma–Aldrich, Buchs, Switzerland). For determination of serum INF-␥, mice were sacrificed 4 h after Con A administration. Blood was taken by cardiac puncture and centrifuged after coagulation to obtain serum. The concentration of INF-␥ in serum was measured in Maxisorp plates (Nunc, Langenselbold, Germany) by ELISA (Biolegend, Fell, Germany). 2.3. Isolation of intrahepatic leukocytes Livers were perfused with PBS via the portal vein to remove blood-derived leukocytes. Then the livers were excised and minced with scissors in digestive medium composed of RPMI-1640 (Gibco, Zug, Switzerland) containing 0.05% collagenase D (Worthington, Lakewood, NJ, USA), 50,000 units/ml DNaseI (Roche, Basel, Switzerland) and 0.01% trypsin inhibitor (Worthington, Lakewood, NJ, USA). After 30 min of incubation at 37 ◦ C and 250 rpm, the digested tissue was passed through a 70 m cell strainer (Falcon, Allschwil, Switzerland) and rinsed with cold PBS. The cell pellet was washed twice with cold PBS (centrifugation 300 × g, 10 min, 4 ◦ C). Leukocytes were then isolated using a Percoll gradient (density 1.131 g/ml; GE healthcare, Glattbrug, Switzerland). The cell pellet was resuspended in 90% Percoll solution (diluted with HBSS, Gibco, Zug, Switzerland), overlaid with 37% and 30% Percoll solution and centrifuged at 800 × g for 20 min at room temperature. Leukocytes were washed in PBS and counted in trypan blue (Invitrogen, Zug, Switzerland) using a hemocytometer. 2.4. Surgery and spinning disk confocal intravital microscopy of the liver Mice were anesthetized by intraperitoneal injection of a mixture of 10 mg/kg xylazine and 200 mg/kg ketamine hydrochloride (both Bimeda-MTC, Cambridge, ON, Canada) prior to surgery. Body temperature was maintained at 37 ◦ C using an infrared heat lamp. The right vena jugularis was cannulated to allow for i.v. administration of antibodies and additional anesthetic as required. The following antibodies were administered: Alexa Fluor 647-conjugated antiCD4 (clone GK1.5; 1.6 g, eBioscience, San Diego, CA, USA) and phycoerythrin-conjugated anti-Gr-1 (clone RB6-8C5; 1.6 g, eBioscience, San Diego, CA, USA). The liver was prepared for in vivo microscopic observation, as previously described [30]. Briefly, the abdomen was opened with a midline incision, and the skin and abdominal wall were removed along the costal margin to the midaxillary line. The falciform ligament was then dissected away from the gallbladder and liver. Mice were placed in a right lateral position on a plexiglas microscope stage and the left lateral lobe of the liver was exposed. The liver surface was then covered with a
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small piece of saline-soaked Kimwipes to hold the organ in position. All exposed tissues were moistened with saline-soaked gauze to prevent dehydration. Images were acquired with an Olympus IX81 inverted microscope (Olympus, Center Valley, PA) using a 10×/0.40 UPlanSApo objective. The microscope was equipped with a confocal light path (WaveFx; Quorum Technologies, Guelph, Ontario, Canada) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation, Tokyo, Japan). The liver and labeled cells were imaged using either a 488-, 561- or 635-nm laser excitation (Cobolt, Stockholm, Sweden)) and visualized with the appropriate long-pass filters (Semrock, Rochester, NY, USA). A 512 × 512 pixel back-thinned electron-multiplying charge-coupled device camera (C9100-13, Hamamatsu, Bridgewater, NJ, USA) was used for fluorescence detection. The confocal microscope was driven by Volocity acquisition software (Perkin Elmer, Waltham, MA, USA).
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To analyze in vitro activation of T cells single cell suspensions of spleen and peripheral and mesenteric lymph nodes were prepared using metal sieves or 70 m cell strainers (Falcon). Erythrocytes lysis was obtained by 2 min incubation in hypotonic buffer (ACK buffer, 0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) followed by a wash step. Isolated cells were then resuspended in RPMI-1640 supplemented with MEM non-essential amino acids (Invitrogen, Zug, Switzerland), 1 mM sodium pyruvate (Sigma–Aldrich, Buchs, Switzerland), penicillin (100 units)–streptomycin (100 g/ml) (Sigma–Aldrich, Buchs, Switzerland), 2-mercaptoethanol (Invitrogen, Zug, Switzerland), 10% fetal bovine serum (PAA laboratories, Linz, Austria). 2.5 × 106 cells were stimulated in a 24-well plate with 1 g/ml Con A or 500 ng/ml ionomycin and 20 ng/ml phorbol–myristate–acetate (all from Sigma–Aldrich, Buchs, Switzerland) for 4 h. Cell activation was assessed by analyzing CD69 and CD62L expression of CD4+ and CD8+ cells by flow cytometry.
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The following antibodies were used for staining cells for flow cytometric analyses: CD16/32 (clone 93, Biolegend, Fell, Germany), anti-CD4 Pacific blue (RM4-5, Invitrogen, Zug, Switzerland), CD69PE-Cy7 (clone H1.2F3 BD Pharmingen), INF-␥ PE Cy7 (clone XMG1.2, Biolegend, Fell, Germany), CD8a APC Cy7 (clone 53–6.7, Biolegend, Fell, Germany), NK1.1 Alexa fluor 647 (clone PK136, Biolegend, Fell, Germany), CD3e PE (clone 145-2C11, Biolegend, Fell, Germany), CD62L PerCP (MEL14, Biolegend, Fell, Germany). For surface staining the cells were incubated with the labeled antibodies for 15 min on ice in the dark and then washed once with PBS/0.5% BSA. For intracellular staining, cells were first stained for surface antigens as described and then fixed with 2% paraformaldehyde for 20 min at room temperature. After a washing step with PBS/0.5%BSA the cells were permeabilized using 0.5% saponin (Sigma–Aldrich, Buchs, Switzerland) in PBS/0.5% BSA and stained in this buffer for 15 min with anti-INF-␥ antibody at room temperature. This was followed by one washing step with 0.5% saponin/PBS/0.5%BSA and one washing step with PBS/0.5% BSA. Incubation with FcR block (anti-CD16/32 antibody, Biolegend, Fell, Germany) was included. The samples were measured on a FACS Canto II (BD Biosciences) and the data were analyzed with the FlowJo software (Tree Star, Ashland, OR, USA).
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2.7. Statistical analyses
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The data are depicted in the graphs as values obtained of all individual mice analyzed and the mean (±SD). FACS dot plots and
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microscopic pictures show representative results. The numbers of mice (n) that have been analyzed are listed in each figure legend. Statistically significance was analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparison test (for comparing multiple samples: wtPBS with wtCon A , koPBS with koCon A , wtPBS with koPBS and wtCon A with koCon A ), Mann–Whitney U-test (for comparing only two samples) using GraphPad Prism software (GraphPad, San Diego, CA, USA). In the figures only significant differences are depicted. ***p value <0.001, **p value <0.01, *p value <0.05, ns p value >0.05.
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3. Results
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3.1. Con A-induced liver injury
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Coronin 1 is essential for the activation of naïve T lymphocytes and has been implicated in immune activation in the context of autoimmune diseases such as lupus and experimental autoimmune encephalomyelitis [9,12,13]. However, the impact of coronin 1 on immune functions in the context of acute inflammation has thus far not been addressed. To analyze a role for coronin 1 in acute inflammation, both wild type and coronin 1-deficient animals were injected intravenously with the lectin concanavalin A (Con A), which is a broadly used model for acute hepatitis and leads to acute liver injury after a few hours [24]. To assess the overall consequences of coronin 1 deficiency on the extent of liver injury following Con A injection, histology was performed. Histological evaluation of liver parenchyma revealed that both wild type as well as coronin 1-deficient mice developed clear-cut histological signs of Con A-induced hepatitis characterized by large confluent necrosis, intra-lobular mononuclear inflammation and focal endothelialitis. The overall pathological evaluation revealed a similar inflammation score for wild type and coronin 1-deficient mice (Fig. 1). Furthermore, increased AST and ALT serum levels, which correlate with hepatocyte death, were not significantly different between both mouse strains (Fig. 1).
3.2. Leukocyte recruitment to the liver of Con A-treated mice in the presence and absence of coronin 1 analyzed by intravital microscopy Inflammation is characterized by infiltration of a variety of immune cells in the affected organ. The role for coronin 1 in leukocyte migration remains however unclear; based on in vitro migration analysis these processes have been suggested to both be dependent as well as independent on coronin 1 [7,11]. Whether or not there is a role for coronin 1 in migration under inflammatory conditions in vivo is not known. To investigate CD4+ T cell and neutrophil migration during inflammation in the presence and absence of coronin 1, intravital microscopy of the liver was performed using wild type and coronin 1-deficient mice upon Con A treatment. To visualize CD4+ T cells and neutrophils in vivo, fluorescently labeled anti-CD4 and anti-Gr-1 antibodies were injected into the vena jugularis (see also Suppl. Movies 1–8). As expected, given the overall reduction of T cell numbers in the absence of coronin 1 [7,8,10], intravital microscopy of the livers of PBS- or Con A-treated mice showed that the overall number of CD4+ cells in coronin 1-deficient animals was significantly lower than in wild type mice (Fig. 2A). Importantly, as shown in Fig. 2A, injection of Con A had no significant effect in either wild type or coronin 1-deficient animals on the total number of CD4+ T cells per field of view (FOV, PBS vs. Con A) obtained by intravital microscopy of the liver 1.5–4 h after treatment. Nonetheless, the number of CD4+ cells that adhered in the
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 1. Liver injury of wild type and coronin 1-deficient mice induced by Con A injection. Mice were treated once either with Con A (13 mg/kg) or PBS by i.v. injection and the analysis was performed at 12 h after administration. (A) Sections of formalin-fixed, hematoxylin–eosin stained and Sirius Red stained livers of PBS and Con A-treated mice. Magnification is indicated in the figure, the box marks the region that was further magnified. Arrows point to large confluent necrosis. (B) The graph depicts the disease score in Con A-treated mice, which represents the degree of inflammation/score 0–3: 0 = absent, 1 = minimal/slight, 2 = moderate, 3 = severe (n = 4). Serum ALT and AST activities were determined 12 h after treatment using diagnostic kits (nwt = 22, nko = 20). ns, not significant.
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post-sinusoidal venules did increase after Con A treatment in both mouse strains (Fig. 2A). Analysis of Gr-1+ cells by intravital microscopy revealed comparable numbers in the livers of PBS-treated coronin 1-deficient and wild type mice, which strongly increased after Con A treatment in the whole field of view as well as the post-sinusoidal venules. The Gr-1+ cell counts per FOV and post-sinusoidal venules was not significantly different between mice of both genotypes (Fig. 2B). 3.3. Immune responses to Con A treatment in the presence and absence of coronin 1
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To further analyze the immune response to Con A treatment, leukocyte counts in blood, spleen and liver at 4 h after treatment with either PBS or Con A were determined using a hemocytometer. Con A treatment resulted in increased numbers of leukocytes predominantly in the spleen (Fig. 3A). Con A-treated coronin 1deficient mice had significantly reduced blood leukocyte counts compared to Con A-treated wild type mice. In contrast, leukocyte numbers in liver and spleen were comparable between wild type and coronin 1-deficient mice whether or not treated with Con A (Fig. 3A). CD4+ and CD8+ T cells in the liver were quantified using flow cytometry. PBS- as well as Con A-treated coronin 1-deficient mice possessed lower T cells counts (CD3+ NK1.1− ) in their livers than wild type mice (Fig. 3B). Four hours after Con A treatment no considerable change in intrahepatic T cells counts was observed. However, it resulted in an increase in CD4+ T cell frequency solely in wild type mice (Fig. 3C).
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3.4. In vivo activation of T cells by concanavalin A
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Since CD4+ T cells have been described as major players of liver injury after Con A administration [24], we addressed whether coronin 1-deficient T cells were able to respond to Con A treatment. To analyze T cell activation following Con A stimulation in vivo, upregulation of the early activation marker CD69 was analyzed on CD4+ and CD8+ T cells isolated from the livers of wild type and
coronin 1-deficient mice 4 h after Con A or PBS treatment. Con A treatment increased CD69 expression on T cells of both mouse strains. A comparison between Con A-treated wild type and coronin 1-deficient mice revealed that CD69 expression on CD4+ as well as CD8+ T cells was higher in wild type than in coronin 1-deficient mice as analyzed by FACS (Fig. 4A). A similar increase in CD69expression was observed for CD4+ and CD8+ T cells isolated from the spleen of Con A-treated animals (data not shown). When analyzing INF-␥ expression of intrahepatic T lymphocytes as a read-out for cell activation, no significant difference in frequency and numbers of INF-␥+ CD8+ T cells was observed between Con A-treated wild type and Con A-treated coronin 1-deficient mice; even though there was a trend to lower expression in the absence of coronin 1. In contrast, CD4+ INF-␥-expressing cells in the liver were reduced in numbers in coronin 1-deficient mice (Fig. 4B). Unlike the data obtained for the livers, almost no INF-␥ expression above background was observed when T cells were isolated from the spleen after Con A treatment in both wild type and coronin 1-deficient mice (data not shown). In addition to intracellular INF-␥ expression by T cells, the INF␥ concentration in the serum of Con A-treated mice was analyzed by ELISA. While in the serum of PBS-treated mice no INF-␥ above detection limit could be measured, the concentration of INF-␥ was strongly increased in the serum of wild type but not coronin 1deficient mice 4 h after Con A treatment (Fig. 4C). 3.5. In vitro activation of T cells by concanavalin A In addition to the analysis ex vivo, we addressed whether coronin 1-deficient T cells can be activated by Con A in vitro. To this end, spleen and lymph node cells were stimulated with Con A and as a read-out for T cell activation CD69 and CD62L expression was analyzed by flow cytometry (Fig. 5). In both wild type and coronin 1-deficient T cells, increased CD69 and decreased CD62L expression was observed. As observed ex vivo, coronin 1-deficient lymphocytes showed a slight decreased response to Con A treatment with regard to CD69 up-regulation, but the difference between wild type
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 2. Gr-1+ and CD4+ T cell recruitment to the livers of wild type and coronin 1-deficient mice in response to Con A treatment. Mice were treated with Con A (single intravenous administration) 60–90 min before anesthesia, cannulation of the vena jugularis and surgical exposure of the livers to perform intravital microscopy analyses using an inverted spinning disk confocal microscope. To visualize CD4 (A) and Gr-1 (B) expressing cells, fluorochrome-labeled antibodies recognizing these antigens were applied via the vein catheter. The mice were imaged between 1.5 and 4 h after Con A treatment. Still images, showing CD4 (blue), Gr-1 (red) and background fluorescence and in case of coronin 1-deficient mice GFP (green) from one experiment, are depicted. The lines outline the location of the post-sinusoidal venule. For quantification the number of crawling or adherent Gr-1+ and CD4+ cells per field of view (FOV, 512 m × 512 m) using a 10× objective and in post-sinusoidal venules (calculated per 500 m length) were counted. The scale bar represents 100 m, 2–6 FOV per mouse were analyzed. n(wtPBS ) = 2, n(wtcon A ) = 4, n(coro1−/− PBS ) = 2, n(coro1−/− con A ) = 6).
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 3. Changes in leukocyte numbers after Con A treatment of wild type and coronin 1-deficient mice. (A) Leukocyte counts in blood, spleen and liver of PBS or Con A-treated mice were determined 4 h after treatment using trypan blue and a hemocytometer. (B) and (C) Leukocytes in the liver of PBS and Con A-treated mice were stained with antiGr-1, anti-CD3, anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Doublettes and dead cells were excluded by FSC-A and FSC-H gating and propidium iodide staining, respectively. Representative FACS dot blots are shown. Numbers of Gr-1high granulocytes (high in sideward scatter) and CD3+ T cells (NK1.1− ) and the frequency of CD4+ and CD8+ T cells in the livers are depicted. n = 6 (Con A), nwt/ko = 8/9 (PBS).
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and coronin 1-deficient mice was less pronounced in vitro (Fig. 5). Coronin 1-deficient CD4+ cells exhibited already without any stimulation lower CD62L expression, which is in accordance with the reduced number of naïve T cells in coronin 1-deficient mice. However, the degree of CD62L down-regulation after Con A treatment was similar between coronin 1-deficient and wild type CD4+ cells, while coronin 1-deficient CD8+ cells seemed to be impaired (Fig. 5). Taken together these results show that while activation of T cells was impaired in coronin 1-deficient mice, CD4+ T cells and neutrophils were similarly recruited to the liver in wild type as well as coronin 1-deficient mice upon Con A treatment causing liver injury in both mice strains.
effector T cells to peripheral tissues under inflammatory conditions. In this study, we analyzed the role of coronin 1 for leukocyte recruitment to the liver using the well-established Con A-induced hepatitis model [24,26]. Furthermore, it was addressed whether the absence of coronin 1 affects the extend of acute liver damage, which can be assessed by increased liver enzyme levels in blood as early as 4 h upon intravenous administration of Con A [24]. We found that in both wild type as well as coronin 1-deficient animals Con A-induced hepatitis resulted in similar levels of liver injury, as well as a comparable recruitment of leukocytes to the inflamed liver. Together these results suggest that coronin 1 is dispensable for leukocyte recruitment under inflammatory conditions in vivo.
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4. Discussion
4.1. Coronin 1, neutrophils and Con A-induced hepatitis
Acute inflammation is characterized by tissue infiltration of activated leukocytes, which is a tightly controlled process involving several steps such as rolling and adhesion preceding cell extravasation [31–35]. Thus far it was unknown whether coronin 1 is involved in recruitment of leukocytes such as neutrophils and
Neutrophils, that are the first cells to arrive at sides of inflammation, have been reported to be key initiators of lymphocyte recruitment to the liver after Con A treatment [26] and furthermore to be necessary for INF-␥ production by T cells [36]. Depletion of neutrophils before Con A treatment reduced liver injury, T cell
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Fig. 4. In vivo cell activation after intravenous administration of Con A. (A) CD69 expression on hepatic T cells (gated for viable CD3+ NK1.1− cells) of PBS and Con A-treated (4 h) wild type and coronin 1-deficient mice was analyzed by flow cytometry. Representative FACS dot blots gated for CD4+ T cells are shown. CD69 expression was quantified by determining the mean fluorescent intensity (MFI) of CD69 staining on CD4+ and CD8+ T cells. (B) INF-␥ expression of hepatic T cells was analyzed 12 h after PBS or Con A-treatment by intracellular staining and flow cytometry. Directly after isolation from the livers leukocytes were incubated for 3 h in the presence of brefeldin A to trap the produced INF-␥ in the cells. Thereafter surface staining with antibodies recognizing NK1.1, CD3, CD4 and CD8 was performed and the cells were subsequently fixed with 2% PFA. The fixed cells were then permeabilized using 0.05% saponin in PBS, stained for intracellular INF-␥ and analyzed by flow cytometry. Representative FACS dot blots, gated CD4+ and CD8+ T cells (NK1.1− CD3+ ), are shown. For quantification the frequency and cell counts of INF-␥-positive hepatic CD4+ and CD8+ T cells were determined. Each symbol represents data obtained from an individual animal. n = 6. (C) INF-␥ concentration in the serum of wild type or coronin 1-deficient mice treated with either PBS or Con A was analyzed by ELISA at 4 h after intravenous injection. n = 6; nd = not detectable.
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adhesion in the post-sinusoidal venules and cytokine production by T cells [36]. Even though neutrophils express high levels of coronin 1, a role for coronin 1 in neutrophils, if any, remains unknown. Previous work using coronin 1-deficient mice suggests that both neutrophil phagocytosis and chemotaxis are independent of coronin 1 [11,16]. Furthermore, the here observed recruitment of coronin 1-deficient neutrophils to the liver upon Con A treatment is consistent with the previously reported increase of granulocytes in blood upon immunization with incomplete Freud adjuvant, suggesting a normal mobilization from the bone marrow in the absence of coronin 1 [12]. Together these data suggest that coronin 1 is dispensable for these neutrophil functions in vivo.
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An essential role for CD4+ T cells in the induction of full-blown liver inflammation in Con A-induced hepatitis has been demonstrated using mice lacking CD4+ T cells, which were fully protected against Con A-induced hepatitis [24]. Interestingly, even though coronin 1-deficient mice possess reduced overall T cell numbers as well as impaired Con A-induced activation of intrahepatic T cells ex vivo, acute liver injury develops in the absence of coronin 1. Since the response to intravenous Con A is fast and occurs within a few hours, it is conceivable that effector/memory T cells and not naïve T cells are the major responsible subset, consistent with the effector/memory T cell compartment being less affected
by the absence of coronin 1 [7–10]. In contrast to the development of acute Con A-induced liver inflammation described here, coronin 1-deficient mice have been shown to be resistant to experimental autoimmune encephalomyelitis (EAE), which develops over several days and depends on the antigen-specific clonal expansion of naïve CD4+ T cells [12,13]. This resistance to EAE is in accordance with the strong reduction of naïve T cells in coronin 1-deficient mice [12]. However, coronin 1-deficient mice develop more severe chronic EAE when re-challenged with the same antigen, activating a memory response, suggesting that effector/memory T cells are fully functional in the absence of coronin 1 once they have developed from the naïve T cell pool [13]. The importance of naïve and effector/memory CD4+ cell subsets in EAE and Con A-induced hepatitis, respectively, is likely to explain the different outcome of these diseases in coronin 1-deficient mice, whose major defect lies in the naïve T cell compartment. Also, the differences in the pathophysiology of acute inflammation vs. auto-immune disease, the latter being mainly a chronic inflammatory response may explain the different outcomes observed in these CD4+ T cell-dependent disease models. Serum INF-␥-levels after in vivo Con A treatment were strongly reduced in coronin 1-deficient mice, arguing for a defective activation of leukocytes in the absence of coronin 1 expression. However, the differences in expression of activation markers were less pronounced between wild type and coronin 1-deficient mice, suggesting that cell types other than T cells may contribute to elevated serum INF-␥. This idea is supported by the finding that in contrast
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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Fig. 5. Activation of T cells in response to Con A treatment in vitro. CD69 and CD62L expression on CD4+ and CD8+ T cells were analyzed by flow cytometry. Spleen and lymph node cells were isolated and stimulated in vitro for 4 h either with PMA/ionomycin or with 1 g/ml Con A or left un-stimulated. Thereafter the cells were stained for CD4, CD8, CD69 and CD62L and analyzed by flow cytometry. Representative FACS dot blots showing CD69 and CD62L expression on CD4+ T cells are depicted. CD69 and CD62L expression were quantified by determining the mean fluorescent intensity (MFI) for both stains on CD4+ and CD8+ T cells. n = minimum of 5 individual mice.
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to in vivo treatment, Con A induced a similar CD69 expression of wild type and coronin 1-deficient CD4+ T cell when treated in vitro. Furthermore, coronin 1-deficient T cells are fully competent to express Th1, Th2 and Th17 cytokines after polyclonal stimulation with antibodies against CD3 and CD28 in vitro arguing against a general defect in cytokine expression [12]. Interestingly, in this context, the largest difference in CD4+ T cell numbers between wild type and coronin 1-deficient mice was not observed in lymphoid tissue but in blood [12]. As a result, this impacts on the size of the T cell pool available for recruitment to peripheral sites upon inflammatory stimuli. Also, while after Con A treatment reduced numbers of adherent CD4+ cells were observed in the post-sinusoidal venules of coronin 1-deficient mice compared to wild type mice, the fold increase in adherent CD4+ cells induced by treatment was comparable between both mice strains. This finding suggests that CD4+ cell adhesion in the inflamed liver is independent of coronin 1 expression. In conclusion, the results presented here show that neutrophil as well as CD4+ T cell recruitment upon an inflammatory stimulus
and liver inflammation does occur also in the absence of coronin 1 expression. Acknowledgements This work was supported by the Canton of Basel, the Swiss National Science Foundation [to J.P.], EMBO [ALTF 1151-2011 to M.S. and ASTF No. 415-2010 to K.S] and the Canadian Institutes of Health Research [to P.K.]. We thank Fausta Chiaverio from the laboratory medicine of the University Hospital of Basel for coordinating the AST/ALT measurements in murine blood samples. We thank the animal care unit at the Biozentrum, University of Basel, and at the University of Calgary for help with all mice related issues, especially Carla Badick in the laboratory of Paul Kubes for the coordination of the mouse import to Calgary. We thank The Live Cell Imaging Facility at the University of Calgary funded by the Canada Foundation for Innovation for support concerning all microscopy related issues.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imlet. 2013.06.005. References
[1] Pieters J, Mueller P, Jayachandran R. On guard: coronin proteins in innate and 495 adaptive immunity. Nat Rev Immunol 2013 [in press]. 496 [2] Chan KT, Creed SJ, Bear JE. Unraveling the enigma: progress towards under497 standing the coronin family of actin regulators. Trends Cell Biol 2011;21: 498 481–8. 499 [3] Ferrari G, Langen H, Naito M, Pieters J. A coat protein on phagosomes involved 500 in the intracellular survival of mycobacteria. Cell 1999;97:435–47. 501 [4] Gatfield J, Pieters J. Essential role for cholesterol in entry of mycobacteria into 502 macrophages. Science 2000;288:1647–50. 503 [5] Pieters J. Mycobacterium tuberculosis and the macrophage: maintaining a bal504 ance. Cell Host Microbe 2008;3:399–407. 505 [6] Pieters J. Coronin 1 in innate immunity. Subcell Biochem 2008;48:116–23. 506 [7] Foger N, Rangell L, Danilenko DM, Chan AC. Requirement for coronin 1 507 in T lymphocyte trafficking and cellular homeostasis. Science 2006;313: 508 839–42. 509 [8] Mueller P, Massner J, Jayachandran R, Combaluzier B, Albrecht I, Gatfield J, 510 et al. Regulation of T cell survival through coronin-1-mediated generation of 511 inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor trig512 gering. Nat Immunol 2008;9:424–31. 513 [9] Haraldsson MK, Louis-Dit-Sully CA, Lawson BR, Sternik G, Santiago-Raber 514 ML, Gascoigne NR, et al. The lupus-related Lmb3 locus contains a disease515 suppressing Coronin-1A gene mutation. Immunity 2008;28:40–51. 516 [10] Shiow LR, Roadcap DW, Paris K, Watson SR, Grigorova IL, Lebet T, et al. 517 The actin regulator coronin 1A is mutant in a thymic egress-deficient mouse 518 strain and in a patient with severe combined immunodeficiency. Nat Immunol 519 2008;9:1307–15. 520 [11] Mueller P, Liu X, Pieters J. Migration and homeostasis of naive T cells depends 521 on coronin 1-mediated prosurvival signals and not on coronin 1-dependent 522 filamentous actin modulation. J Immunol 2011;186:4039–50. 523 [12] Siegmund K, Zeis T, Kunz G, Rolink T, Schaeren-Wiemers N, Pieters J. Coronin 1524 mediated naive T cell survival is essential for the development of autoimmune 525 encephalomyelitis. J Immunol 2011;186:3452–61. 526 [13] Kaminski S, Hermann-Kleiter N, Meisel M, Thuille N, Cronin S, Hara 527 H, et al. Coronin 1A is an essential regulator of the TGFbeta recep528 tor/SMAD3 signaling pathway in Th17 CD4(+) T cells. J Autoimmun 2011;37: 529 198–208. 530 [14] Shiow LR, Paris K, Akana MC, Cyster JG, Sorensen RU, Puck JM. Severe combined 531 immunodeficiency (SCID) and attention deficit hyperactivity disorder (ADHD) 532 associated with a coronin-1A mutation and a chromosome 16p11.2 deletion. 533 Clin Immunol 2008. 534 [15] Moshous D, Martin E, Carpentier W, Lim A, Callebaut I, Canioni D, et al. 535 Whole-exome sequencing identifies Coronin-1A deficiency in 3 siblings with 536 Q4 immunodeficiency and EBV-associated B-cell lymphoproliferation. J Allergy 537 Clin Immunol 2013. 538 [16] Combaluzier B, Pieters J. Chemotaxis and phagocytosis in neutrophils is independent of coronin 1. J Immunol 2009;182:2745–52.
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[17] Combaluzier B, Mueller P, Massner J, Finke D, Pieters J. Coronin 1 is essential for IgM-mediated Ca2+ mobilization in B cells but dispensable for the generation of immune responses in vivo. J Immunol 2009;182:1954–61. [18] Jayachandran R, Sundaramurthy V, Combaluzier B, Mueller P, Korf H, Huygen K, et al. Survival of mycobacteria in macrophages is mediated by coronin 1dependent activation of calcineurin. Cell 2007;130:37–50. [19] Westritschnig K, Bosedasgupta S, Tchang V, Siegmund K, Pieters J. Antigen processing and presentation by dendritic cells is independent of coronin 1. Mol Immunol 2013;53:379–86. [20] Tchang V, Mekker A, Siegmund K, Karrer U, Pieters J. Diverging role for coronin 1 in antiviral CD4+ and CD8+ T cell responses. Mol Immunol 2013 [in press]. [21] Jayachandran R, Gatfield J, Massner J, Albrecht I, Zanolari B, Pieters J. RNA interference in J774 macrophages reveals a role for Coronin 1 in mycobacterial trafficking but not in actin-dependent processes. Mol Biol Cell 2008;19:1241–51. [22] Moriceau S, Kantari C, Mocek J, Davezac N, Gabillet J, Guerrera IC, et al. Coronin1 is associated with neutrophil survival and is cleaved during apoptosis: potential implication in neutrophils from cystic fibrosis patients. J Immunol 2009;182:7254–63. [23] Wimer BM, Mann PL. Mitogen information summaries. Cancer Biother Radiopharm 2002;17:569–97. [24] Tiegs G, Hentschel J, Wendel AA. T cell-dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest 1992;90:196–203. [25] Miyazawa Y, Tsutsui H, Mizuhara H, Fujiwara H, Kaneda K. Involvement of intrasinusoidal hemostasis in the development of concanavalin A-induced hepatic injury in mice. Hepatology 1998;27:497–506. [26] Bonder CS, Ajuebor MN, Zbytnuik LD, Kubes P, Swain MG. Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis. J Immunol 2004;172:45–53. [27] Kusters S, Gantner F, Kunstle G, Tiegs G. Interferon gamma plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A. Gastroenterology 1996;111:462–71. [28] Schumann J, Wolf D, Pahl A, Brune K, Papadopoulos T, van Rooijen N, et al. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am J Pathol 2000;157:1671–83. [29] Takeda K, Hayakawa Y, Van Kaer L, Matsuda H, Yagita H, Okumura K. Critical contribution of liver natural killer T cells to a murine model of hepatitis. Proc Natl Acad Sci USA 2000;97:5498–503. [30] Wong J, Johnston B, Lee SS, Bullard DC, Smith CW, Beaudet AL, et al. A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature. J Clin Invest 1997;99:2782–90. [31] Imhof BA, Aurrand-Lions M. Adhesion mechanisms regulating the migration of monocytes. Nat Rev Immunol 2004;4:432–44. [32] Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 1995;57:827–72. [33] Catalina MD, Estess P, Siegelman MH. Selective requirements for leukocyte adhesion molecules in models of acute and chronic cutaneous inflammation: participation of E- and P- but not L-selectin. Blood 1999;93:580–9. [34] Mohamadzadeh M, DeGrendele H, Arizpe H, Estess P, Siegelman M. Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion. J Clin Invest 1998;101:97–108. [35] Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 2007;7:678–89. [36] Hatada S, Ohta T, Shiratsuchi Y, Hatano M, Kobayashi Y. A novel accessory role of neutrophils in concanavalin A-induced hepatitis. Cell Immunol 2005;233:23–9.
Please cite this article in press as: Siegmund K, et al. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis. Immunol Lett (2013), http://dx.doi.org/10.1016/j.imlet.2013.06.005
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