Jeffrey A. Whitsett
Michael R. Knowles, Richard C. Boucher
Mesangial cells maintain normal glomerular function by mediating ECM remodeling and immune complex disposal. We have recently identified megsin, a novel member of the serine protease inhibitor (serpin) superfamily predominantly expressed in the mesangium. While our previous studies suggested a role for megsin in the pathogenesis of human glomerular diseases, its exact biological significance remained unknown. Here we produced two lines of megsin transgenic mice. Overexpression of megsin led to progressive mesangial matrix expansion and an increase in the number of mesangial cells. These glomerular lesions were accompanied by an augmented immune complex deposition, together with Ig’s and complement. Binding and functional assays in vitro identified plasmin as one biological substrate of megsin and confirmed its activity as a proteinase inhibitor. Transgenic animals exhibiting nephritis as a result of treatment with anti–glomerular basement membrane antiserum showed significantly more persistent expansion of the mesangial ECM than was seen in parental mice. Megsin therefore exerts a biologically relevant influence on mesangial function, and on the mesangial microenvironment, such that simple overexpression of this endogenous serpin engenders elementary mesangial lesions.
Toshio Miyata, Reiko Inagi, Masaomi Nangaku, Toshiyuki Imasawa, Masahiro Sato, Yuko Izuhara, Daisuke Suzuki, Atsusi Yoshino, Hiroshi Onogi, Minoru Kimura, Satoshi Sugiyama, Kiyoshi Kurokawa
Myostatin is a TGF-β family member that acts as a negative regulator of muscle growth. Mice lacking the myostatin gene (Mstn) have a widespread increase in skeletal muscle mass resulting from a combination of muscle fiber hypertrophy and hyperplasia. Here we show that Mstn-null mice have a significant reduction in fat accumulation with increasing age compared with wild-type littermates, even in the setting of normal food intake (relative to body weight), normal body temperature, and a slightly decreased resting metabolic rate. To investigate whether myostatin might be an effective target for suppressing the development of obesity in settings of abnormal fat accumulation, we analyzed the effect of the Mstn mutation in two genetic models of obesity, agouti lethal yellow (Ay) and obese (Lepob/ob). In each case, loss of Mstn led to a partial suppression of fat accumulation and of abnormal glucose metabolism. Our findings raise the possibility that pharmacological agents that block myostatin function may be useful not only for enhancing muscle growth, but also for slowing or preventing the development of obesity and type 2 diabetes.
Alexandra C. McPherron, Se-Jin Lee
The role of the renin-angiotensin system (RAS) in angiogenesis is little known. Here, we show that the angiotensin II (ATII) type 1 (AT1) receptor plays an important role in ischemia-induced angiogenesis. Well-developed collateral vessels and angiogenesis were observed in wild-type (WT) mice in response to hindlimb ischemia, whereas these responses were reduced in ATII type 1a receptor knockout (AT1a–/–) mice. Ischemia-induced angiogenesis was also impaired in WT mice treated with the AT1 receptor blocker TCV-116. These effects were not due to reduced systemic blood pressure (SBP), because hydralazine treatment preserved angiogenesis in WT mice although it reduced SBP to a level similar to that of AT1a–/– mice. Infiltration of inflammatory mononuclear cells (MNCs), including macrophages and T lymphocytes, was suppressed in the ischemic tissues of AT1a–/– mice compared with WT mice. Double immunofluorescence staining revealed that infiltrated macrophages and T lymphocytes expressed VEGF, and the expression of VEGF and monocyte chemoattractant protein-1 was also decreased in AT1a–/–. Finally, the impaired angiogenesis in AT1a–/– mice was rescued by intramuscular transplantation of MNCs obtained from WT mice, further indicating the importance of MNC infiltration in ischemia-induced angiogenesis. Thus, the ATII–AT1 receptor pathway promotes early angiogenesis by supporting inflammatory cell infiltration and angiogenic cytokine expression.
Ken-ichiro Sasaki, Toyoaki Murohara, Hisao Ikeda, Takeshi Sugaya, Toshifumi Shimada, Satoshi Shintani, Tsutomu Imaizumi
Agonistic αCD40 Ab’s have been shown to be potent immune adjuvants for both cell- and humoral-mediated immunity. While enhancing short-lived humoral immunity, the administration of a CD40 agonist during thymus-dependent immune responses ablates germinal center formation, prematurely terminates the humoral immune response, blocks the generation of B cell memory, and prevents the generation of long-lived bone marrow plasma cells. Interestingly, some of these effects of heightened CD40 engagement could be mimicked by enhancing the magnitude of antigen-specific T cell help. Taken together, these studies demonstrate that as the magnitude of CD40 signaling intensifies, the fate of antigen-reactive B cells can be dramatically altered. These are the first studies to describe the multifaceted function of CD40 in determining the fate of antigen-reactive B cells and provide novel insights into how CD40 agonists can short-circuit humoral immunity.
Loren D. Erickson, Brigit G. Durell, Laura A. Vogel, Brian P. O’Connor, Marilia Cascalho, Teruhito Yasui, Hitoshi Kikutani, Randolph J. Noelle
The CC chemokine receptor 3 (CCR3) is expressed by eosinophils, mast cells, and Th2 cells. We used CCR3–/– mice to assess the role of CCR3 in a murine model of allergic skin inflammation induced by repeated epicutaneous sensitization with ovalbumin (OVA), and characterized by eosinophil skin infiltration, local expression of Th2 cytokines, and airway hyperresponsiveness (AHR) to inhaled antigen. Eosinophils and the eosinophil product major basic protein were absent from the skin of sham and OVA-sensitized CCR3–/– mice. Mast cell numbers and expression of IL-4 mRNA were normal in skin of CCR3–/– mice, suggesting that CCR3 is not important for infiltration of the skin by mast cells and Th2 cells. CCR3–/– mice produced normal levels of OVA-specific IgE, and their splenocytes secreted normal amounts of IL-4 and IL-5 following in vitro stimulation with OVA, indicating effective generation of systemic Th2 helper responses. Recruitment of eosinophils to lung parenchyma and bronchoalveolar lavage (BAL) fluid was severely impaired in CCR3–/– mice, which failed to develop AHR to methacholine following antigen inhalation. These results suggest that CCR3 plays an essential role in eosinophil recruitment to the skin and the lung and in the development of AHR.
Weilie Ma, Paul J. Bryce, Alison A. Humbles, Dhafer Laouini, Ali Yalcindag, Harri Alenius, Daniel S. Friend, Hans C. Oettgen, Craig Gerard, Raif S. Geha
To investigate the role of insulin signaling on postnatal cardiac development, physiology, and cardiac metabolism, we generated mice with a cardiomyocyte-selective insulin receptor knockout (CIRKO) using cre/loxP recombination. Hearts of CIRKO mice were reduced in size by 20–30% due to reduced cardiomyocyte size and had persistent expression of the fetal β-myosin heavy chain isoform. In CIRKO hearts, glucose transporter 1 (GLUT1) expression was reduced by about 50%, but there was a twofold increase in GLUT4 expression as well as increased rates of cardiac glucose uptake in vivo and increased glycolysis in isolated working hearts. Fatty acid oxidation rates were diminished as a result of reduced expression of enzymes that catalyze mitochondrial β-oxidation. Although basal rates of glucose oxidation were reduced, insulin unexpectedly stimulated glucose oxidation and glycogenolysis in CIRKO hearts. Cardiac performance in vivo and in isolated hearts was mildly impaired. Thus, insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids. Insulin may also modulate cardiac myocyte metabolism through paracrine mechanisms by activating insulin receptors in other cell types within the heart.
Darrell D. Belke, Sandrine Betuing, Martin J. Tuttle, Christophe Graveleau, Martin E. Young, Mark Pham, Dongfang Zhang, Robert C. Cooksey, Donald A. McClain, Sheldon E. Litwin, Heinrich Taegtmeyer, David Severson, C. Ronald Kahn, E. Dale Abel
Glatiramer acetate (GA; Copaxone) is a random copolymer of glutamic acid, lysine, alanine, and tyrosine that is used therapeutically in patients with multiple sclerosis (MS). To investigate the mechanism of the drug’s immunomodulatory effect, we used immunophenotypic approaches to characterize the precise nature of GA-induced T cell responses. We demonstrate here that healthy individuals and untreated MS patients exhibit prominent T cell proliferative responses to GA. However, these responses are different in distinct subsets of T cells. Whereas GA-induced CD4+ T cell responses are comparable in healthy individuals and MS patients, CD8+ T cell responses are significantly lower in untreated MS patients. Treatment with GA results in upregulation of these CD8+ responses with restoration to levels observed in healthy individuals. Both CD4+ and CD8+ GA-specific responses are HLA-restricted. GA therapy also induces a change in the cytokine profile of GA-specific CD4+ and CD8+ T cells. This study provides the first direct immunophenotypic evidence, to our knowledge, of GA-specific CD8+ T cell responses and their upregulation during the course of therapy, which may suggest a role for these responses in the immunomodulatory effects of the drug.
Nitin J. Karandikar, Michael P. Crawford, Xiao Yan, Robert B. Ratts, Jason M. Brenchley, David R. Ambrozak, Amy E. Lovett-Racke, Elliot M. Frohman, Peter Stastny, Daniel C. Douek, Richard A. Koup, Michael K. Racke
Treatment of advanced, poorly immunogenic tumors in animal models, considered the closest simulation available thus far for conditions observed in cancer patients, remains a major challenge for cancer immunotherapy. We reported previously that established tumors in mice receiving an agonistic mAb to the T cell costimulatory molecule 4-1BB (CD137) regress due to enhanced tumor antigen–specific cytotoxic T lymphocyte responses. In this study, we demonstrate that several poorly immunogenic tumors, including C3 tumor, TC-1 lung carcinoma, and B16-F10 melanoma, once established as solid tumors or metastases, are refractory to treatment by anti–4-1BB mAb. We provide evidence that immunological ignorance, rather than anergy or deletion, of tumor antigen–specific CTLs during the progressive growth of tumors prevents costimulation by anti–4-1BB mAb. Breaking CTL ignorance by immunization with a tumor antigen–derived peptide, although insufficient to stimulate a curative CTL response, is necessary for anti–4-1BB mAb to induce a CTL response leading to the regression of established tumors. Our results suggest a new approach for immunotherapy of human cancers.
Ryan A. Wilcox, Dallas B. Flies, Gefeng Zhu, Aaron J. Johnson, Koji Tamada, Andrei I. Chapoval, Scott E. Strome, Larry R. Pease, Lieping Chen
Cystic fibrosis is characterized by an early and sustained influx of inflammatory cells into the airways and by release of proteases. Resolution of inflammation is normally associated with the orderly removal of dying apoptotic inflammatory cells through cell recognition receptors, such as the phosphatidylserine receptor, CD36, and αv integrins. Accordingly, removal of apoptotic inflammatory cells may be impaired in persistent inflammatory responses such as that seen in cystic fibrosis airways. Examination of sputa from cystic fibrosis and non–cystic fibrosis bronchiectasis patients demonstrated an abundance of apoptotic cells, in excess of that seen in patients with chronic bronchitis. In vitro, cystic fibrosis and bronchiectasis airway fluid directly inhibited apoptotic cell removal by alveolar macrophages in a neutrophil elastase-dependent manner, suggesting that elastase may impair apoptotic cell clearance in vivo. Flow cytometry demonstrated that neutrophil elastase cleaved the phosphatidylserine receptor, but not CD36 or CD32 (FcγRII). Cleavage of the phosphatidylserine receptor by neutrophil elastase specifically disrupted phagocytosis of apoptotic cells, implying a potential mechanism for delayed apoptotic cell clearance in vivo. Therefore, defective airway clearance of apoptotic cells in cystic fibrosis and bronchiectasis may be due to elastase-mediated cleavage of phosphatidylserine receptor on phagocytes and may contribute to ongoing airway inflammation.
R. William Vandivier, Valerie A. Fadok, Peter R. Hoffmann, Donna L. Bratton, Churee Penvari, Kevin K. Brown, Joseph D. Brain, Frank J. Accurso, Peter M. Henson
There is increasing evidence that bacterial superantigens contribute to inflammation and T cell responses in psoriasis. Psoriatic inflammation entails a complex series of inductive and effector processes that require the regulated expression of various proinflammatory genes, many of which require NF-κB for maximal trans-activation. PS-519 is a potent and selective proteasome inhibitor based upon the naturally occurring compound lactacystin, which inhibits NF-κB activation by blocking the degradation of its inhibitory protein IκB. We report that proteasome inhibition by PS-519 reduces superantigen-mediated T cell–activation in vitro and in vivo. Proliferation was inhibited along with the expression of very early (CD69), early (CD25), and late T cell (HLA-DR) activation molecules. Moreover, expression of E-selectin ligands relevant to dermal T cell homing was reduced, as was E-selectin binding in vitro. Finally, PS-519 proved to be therapeutically effective in a SCID-hu xenogeneic psoriasis transplantation model. We conclude that inhibition of the proteasome, e.g., by PS-519, is a promising means to treat T cell–mediated disorders such as psoriasis.
Thomas M. Zollner, Maurizio Podda, Christine Pien, Peter J. Elliott, Roland Kaufmann, Wolf-Henning Boehncke
Primary HIV-1 infection causes extensive immune activation, during which CD4+ T cell activation supports massive HIV-1 production. We tested the safety and the immune-modulating effects of combining cyclosporin A (CsA) treatment with highly active antiretroviral therapy (HAART) during primary HIV-1 infection. Nine adults with primary HIV-1 infection were treated with CsA along with HAART. At week 8, all patients discontinued CsA but maintained HAART. Viral replication was suppressed to a comparable extent in the CsA + HAART cohort and in 29 control patients whose primary infection was treated with HAART alone. CsA restored normal CD4+ T cell levels, both in terms of percentage and absolute numbers. The increase in CD4+ T cells was apparent within a week and persisted throughout the study period. CsA was not detrimental to virus-specific CD8+ or CD4+ T cell responses. At week 48, the proportion of IFN-γ–secreting CD4+ and CD4+CCR7– T cells was significantly higher in the CsA + HAART cohort than in the HAART-alone cohort. In conclusion, rapid shutdown of T cell activation in the early phases of primary HIV-1 infection can have long-term beneficial effects and establish a more favorable immunologic set-point. Appropriate, immune-based therapeutic interventions may represent a valuable complement to HAART for treating HIV infection.
G. Paolo Rizzardi, Alexandre Harari, Brunella Capiluppi, Giuseppe Tambussi, Kim Ellefsen, Donatella Ciuffreda, Patrick Champagne, Pierre-Alexandre Bart, Jean-Philippe Chave, Adriano Lazzarin, Giuseppe Pantaleo