Abstract
Thyrotropin receptor (TSHR) Ab’s of the stimulating variety are the cause of hyperthyroid Graves disease. MS-1 is a hamster mAb with TSHR-stimulating activity. To examine the in vivo biological activity of MS-1, mice were treated with purified MS-1 intraperitoneally and the thyroid response evaluated. MS-1 induced a dose-dependent increase in serum thyroxine (T4), with a maximum effect after 10 ∝g of MS-1 was administered. MS-1–secreting hybridoma cells were then transferred into the peritoneum of nude mice to study chronic thyroid stimulation. Serum MS-1 levels detected after 2 weeks were approximately 10–50 ∝g/ml, and the serum TSH was suppressed in all animals. Serum triiodothyronine levels were elevated, but only in animals with low serum MS-1 concentrations. In addition, there was a negative correlation between serum T4 and the serum MS-1 concentrations. These in vivo studies suggested a partial TSHR inactivation induced by excessive stimulation by MS-1. We confirmed this inactivation by demonstrating MS-1 modulation of TSHR function in vitro as evidenced by downregulation and desensitization of the TSHR at concentrations of MS-1 achieved in the in vivo studies. Thus, inactivation of the TSHR by stimulating TSHR autoantibodies (TSHR-Ab’s) in Graves disease patients may provide a functional explanation for the poor correlation between thyroid function and serum TSHR-Ab concentrations.
Authors
Takao Ando, Rauf Latif, Terry F. Davies
Abstract
The leukocyte integrin αMβ2/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-αMβ2 interaction in vivo, gene-targeted mice were generated in which the αMβ2-binding motif within the fibrinogen γ chain (N390RLSIGE396) was converted to a series of alanine residues. Mice carrying the Fibγ390–396A allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support αMβ2-mediated adhesion of primary neutrophils, macrophages, and αMβ2-expressing cell lines. The elimination of the αMβ2-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for αMβ2, integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.
Authors
Matthew J. Flick, XinLi Du, David P. Witte, Markéta Jiroušková, Dmitry A. Soloviev, Steven J. Busuttil, Edward F. Plow, Jay L. Degen
Abstract
Although arterial bifurcations are frequent sites for obstructive atherosclerotic lesions, the optimal approach to these lesions remains unresolved. Benchtop models of arterial bifurcations were analyzed for flow disturbances known to correlate with vascular disease. These models possess an adaptable geometry capable of simulating the course of arterial disease and the effects of arterial interventions. Chronic in vivo studies evaluated the effect of flow disturbances on the pattern of neointimal hyperplasia. Acute in vivo studies helped propose a mechanism that bridges the early mechanical stimulus and the late tissue effect. Side-branch (SB) dilation adversely affected flow patterns in the main branch (MB) and, as a result, the long-term MB patency of stents implanted in pig arteries. Critical to this effect is chronic MB remodeling that seems to compensate for an occluded SB. Acute leukocyte recruitment was directly influenced by the changes in flow patterns, suggesting a link between flow disturbance on the one hand and leukocyte recruitment and intimal hyperplasia on the other. It is often impossible to simultaneously maximize the total cross-sectional area of both branches and to minimize flow disturbance in the MB. The apparent trade-off between these two clinically desirable goals may explain many of the common failure modes of bifurcation stenting.
Authors
Yoram Richter, Adam Groothuis, Philip Seifert, Elazer R. Edelman
Abstract
Activation of PKCβII is associated with the response to ischemia/reperfusion (I/R), though its role, either pathogenic or protective, has not been determined. In a murine model of single-lung I/R, evidence linking PKCβ to maladaptive responses is shown in the following studies. Homozygous PKCβ-null mice and WT mice fed the PKCβ inhibitor ruboxistaurin subjected to I/R displayed increased survival compared with controls. In PKCβ-null mice, phosphorylation of extracellular signal–regulated protein kinase-1 and -2 (ERK1/2), JNK, and p38 MAPK was suppressed in I/R. Expression of the immediate early gene, early growth response-1 (Egr-1), and its downstream target genes was significantly increased in WT mice in I/R, particularly in mononuclear phagocytes (MPs), whereas this expression was attenuated in PKCβ-null mice or WT mice fed ruboxistaurin. In vitro, hypoxia/reoxygenation-mediated induction of Egr-1 in MPs was suppressed by inhibition of PKCβ, ERK1/2, and JNK, but not by inhibition of p38 MAPK. These findings elucidate key roles for PKCβII activation in I/R by coordinated activation of MAPKs (ERK1/2, JNK) and Egr-1.
Authors
Tomoyuki Fujita, Tomohiro Asai, Martin Andrassy, David M. Stern, David J. Pinsky, Yu Shan Zou, Morihito Okada, Yoshifumi Naka, Ann Marie Schmidt, Shi-Fang Yan
Abstract
Administration of an agonistic anti-CD28 mAb paradoxically inhibits donor T cell expansion and prevents graft-versus-host disease (GVHD) in mice. Here we examined the mechanism of anti-CD28–mediated immunosuppression and found that anti-CD28 mAb activated, rather than blocked, CD28-mediated signaling in vivo. Anti-CD28 treatment prevented GVHD by selectively depleting alloantigen-activated donor T cells through apoptosis but spared the T cells that did not recognize recipient alloantigens. Overexpression of Bcl-xL did not protect T cells from depletion and did not affect GVHD prevention after anti-CD28 treatment. Depletion of activated T cells mediated through CD28 did not depend on the expression of death receptors Fas and TNF receptors type I and II, but both the depletion of activated T cells and the suppressive effect of anti-CD28 mAb on GVHD lethality required donor-derived IFN-γ production. This study demonstrates that agonistic Ab’s specific for the CD28 costimulatory molecule may be used as novel therapeutic agents to abrogate pathogenic T cell responses by selective depletion of activated T cells.
Authors
Xue-Zhong Yu, Michael H. Albert, Paul J. Martin, Claudio Anasetti
Abstract
OCH, a sphingosine-truncated analog of α-galactosylceramide (αGC), is a potential therapeutic reagent for a variety of Th1-mediated autoimmune diseases through its selective induction of Th2 cytokines from natural killer T (NKT) cells. We demonstrate here that the NKT cell production of IFN-γ is more susceptible to the sphingosine length of glycolipid ligand than that of IL-4 and that the length of the sphingosine chain determines the duration of NKT cell stimulation by CD1d-associated glycolipids. Furthermore, IFN-γ production by NKT cells requires longer T cell receptor stimulation than is required for IL-4 production by NKT cells stimulated either with immobilized mAb to CD3 or with immobilized “αGC-loaded” CD1d molecules. Interestingly, transcription of IFN-γ but not that of IL-4 was sensitive to cycloheximide treatment, indicating the intrinsic involvement of de novo protein synthesis for IFN-γ production by NKT cells. Finally, we determined c-Rel was preferentially transcribed in αGC-stimulated but not in OCH-stimulated NKT cells and was essential for IFN-γ production by activated NKT cells. Given the dominant immune regulation by the remarkable cytokine production of ligand-stimulated NKT cells in vivo, in comparison with that of (antigen-specific) T cells or NK cells, the current study confirms OCH as a likely therapeutic reagent for use against Th1-mediated autoimmune diseases and provides a novel clue for the design of drugs targeting NKT cells.
Authors
Shinji Oki, Asako Chiba, Takashi Yamamura, Sachiko Miyake
Abstract
While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation.
Authors
Birgit Liliensiek, Markus A. Weigand, Angelika Bierhaus, Werner Nicklas, Michael Kasper, Stefan Hofer, Jens Plachky, Herman-Josef Gröne, Florian C. Kurschus, Ann Marie Schmidt, Shi Du Yan, Eike Martin, Erwin Schleicher, David M. Stern, Günter J. Hämmerling, Peter P. Nawroth, Bernd Arnold
Abstract
The IL-12Rβ2 gene is expressed in human mature B cell subsets but not in transformed B cell lines. Silencing of this gene may be advantageous to neoplastic B cells. Our objective was to investigate the mechanism(s) and the functional consequence(s) of IL-12Rβ2 gene silencing in primary B cell tumors and transformed B cell lines. Purified tumor cells from 41 patients with different chronic B cell lymphoproliferative disorders, representing the counterparts of the major mature human B cell subsets, tested negative for IL-12Rβ2 gene expression. Hypermethylation of a CpG island in the noncoding exon 1 was associated with silencing of this gene in malignant B cells. Treatment with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine restored IL-12Rβ2 mRNA expression in primary neoplastic B cells that underwent apoptosis following exposure to human recombinant IL-12 (hrIL-12). hrIL-12 inhibited proliferation and increased the apoptotic rate of IL-12Rβ2–transfected B cell lines in vitro. Finally, hrIL-12 strongly reduced the tumorigenicity of IL-12Rβ2–transfected Burkitt lymphoma RAJI cells in SCID-NOD mice through antiproliferative and proapoptotic effects, coupled with neoangiogenesis inhibition related to human IFN-γ–independent induction of hMig/CXCL9. The IL-12Rβ2 gene acts as tumor suppressor in chronic B cell malignancies, and IL-12 exerts direct antitumor effects on IL-12Rβ2–expressing neoplastic B cells.
Authors
Irma Airoldi, Emma Di Carlo, Barbara Banelli, Lidia Moserle, Claudia Cocco, Annalisa Pezzolo, Carlo Sorrentino, Edoardo Rossi, Massimo Romani, Alberto Amadori, Vito Pistoia
Abstract
Kidney podocytes and their slit diaphragms form the final barrier to urinary protein loss. This explains why podocyte injury is typically associated with nephrotic syndrome. The present study uncovered an unanticipated novel role for costimulatory molecule B7-1 in podocytes as an inducible modifier of glomerular permselectivity. B7-1 in podocytes was found in genetic, drug-induced, immune-mediated, and bacterial toxin–induced experimental kidney diseases with nephrotic syndrome. The clinical significance of our results is underscored by the observation that podocyte expression of B7-1 correlated with the severity of human lupus nephritis. In vivo, exposure to low-dose LPS rapidly upregulates B7-1 in podocytes of WT and SCID mice, leading to nephrotic-range proteinuria. Mice lacking B7-1 are protected from LPS-induced nephrotic syndrome, suggesting a link between podocyte B7-1 expression and proteinuria. LPS signaling through toll-like receptor-4 reorganized the podocyte actin cytoskeleton in vitro, and activation of B7-1 in cultured podocytes led to reorganization of vital slit diaphragm proteins. In summary, upregulation of B7-1 in podocytes may contribute to the pathogenesis of proteinuria by disrupting the glomerular filter and provides a novel molecular target to tackle proteinuric kidney diseases. Our findings suggest a novel function for B7-1 in danger signaling by nonimmune cells.
Authors
Jochen Reiser, Gero von Gersdorff, Martin Loos, Jun Oh, Katsuhiko Asanuma, Laura Giardino, Maria Pia Rastaldi, Novella Calvaresi, Haruko Watanabe, Karin Schwarz, Christian Faul, Matthias Kretzler, Anne Davidson, Hikaru Sugimoto, Raghu Kalluri, Arlene H. Sharpe, Jordan A. Kreidberg, Peter Mundel
Abstract
E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.
Authors
Ainhoa Iglesias, Matilde Murga, Usua Laresgoiti, Anouchka Skoudy, Irantzu Bernales, Asier Fullaondo, Bernardino Moreno, José Lloreta, Seth J. Field, Francisco X. Real, Ana M. Zubiaga
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