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1.
Nat Immunol ; 12(12): 1184-93, 2011 Oct 23.
Article in English | MEDLINE | ID: mdl-22019834

ABSTRACT

Dendritic cells (DCs), which are known to support immune activation during infection, may also regulate immune homeostasis in resting animals. Here we show that mice lacking the ubiquitin-editing molecule A20 specifically in DCs spontaneously showed DC activation and population expansion of activated T cells. Analysis of DC-specific epistasis in compound mice lacking both A20 and the signaling adaptor MyD88 specifically in DCs showed that A20 restricted both MyD88-independent signals, which drive activation of DCs and T cells, and MyD88-dependent signals, which drive population expansion of T cells. In addition, mice lacking A20 specifically in DCs spontaneously developed lymphocyte-dependent colitis, seronegative ankylosing arthritis and enthesitis, conditions stereotypical of human inflammatory bowel disease (IBD). Our findings indicate that DCs need A20 to preserve immune quiescence and suggest that A20-dependent DC functions may underlie IBD and IBD-associated arthritides.


Subject(s)
Colitis/immunology , DNA-Binding Proteins/genetics , Dendritic Cells/immunology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Spondylitis, Ankylosing/immunology , Ubiquitin-Protein Ligases/genetics , Animals , Colitis/pathology , Colitis/prevention & control , Crohn Disease/genetics , Cysteine Endopeptidases , DNA-Binding Proteins/metabolism , Dendritic Cells/metabolism , Genetic Predisposition to Disease , Homeostasis/immunology , Humans , Lymphatic Diseases/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Differentiation Factor 88/metabolism , Nuclear Proteins/genetics , Polymorphism, Single Nucleotide , Signal Transduction , Splenomegaly/genetics , Spondylitis, Ankylosing/pathology , Spondylitis, Ankylosing/prevention & control , T-Lymphocytes/immunology , Tumor Necrosis Factor alpha-Induced Protein 3 , Ubiquitin-Protein Ligases/metabolism
2.
Proc Natl Acad Sci U S A ; 107(8): 3505-10, 2010 Feb 23.
Article in English | MEDLINE | ID: mdl-20133674

ABSTRACT

Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.


Subject(s)
Antigens, CD/metabolism , Apoferritins/metabolism , B-Lymphocytes/metabolism , Receptors, Transferrin/metabolism , T-Lymphocytes/metabolism , Antigens, CD/genetics , Cell Line , Cloning, Molecular , Endosomes/metabolism , Humans , Lysosomes/metabolism , Protein Binding , Receptors, Transferrin/genetics , Transferrin/metabolism
3.
J Immunol ; 183(5): 3150-9, 2009 Sep 01.
Article in English | MEDLINE | ID: mdl-19667083

ABSTRACT

Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host's immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity approximately 200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.


Subject(s)
Complement Activating Enzymes/antagonists & inhibitors , Complement Activating Enzymes/metabolism , Variola virus/immunology , Viral Matrix Proteins/antagonists & inhibitors , Viral Matrix Proteins/physiology , Amino Acid Motifs/genetics , Amino Acid Motifs/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Binding Sites/genetics , Binding Sites/immunology , Binding Sites, Antibody , CHO Cells , Complement Activating Enzymes/genetics , Complement C3b/metabolism , Cricetinae , Cricetulus , Glycosaminoglycans/antagonists & inhibitors , Glycosaminoglycans/metabolism , Heparin/metabolism , Humans , Hybridomas , Mice , Molecular Sequence Data , Point Mutation , Variola virus/genetics , Variola virus/pathogenicity , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Virulence Factors/antagonists & inhibitors , Virulence Factors/genetics , Virulence Factors/physiology
4.
Br J Haematol ; 143(3): 336-48, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18691170

ABSTRACT

Both atypical haemolytic uraemic syndrome (aHUS) and the HELLP syndrome (haemolytic anaemia, elevated liver enzymes, and low platelets) are thrombotic microangiopathies characterized by microvascular endothelial activation, cell injury and thrombosis. aHUS is a disease of complement dysregulation, specifically a gain of function of the alternative pathway, due to mutations in complement regulatory proteins and activating components. Recently, the same complement mutation identified in multiple patients with aHUS was found in a patient with the HELLP syndrome. The pathogeneses of both diseases are reviewed focusing on the role of the complement system and how its dysfunction could result in a thrombotic microangiopathy in the kidney in the case of aHUS and in the liver in the case of the HELLP syndrome.


Subject(s)
HELLP Syndrome/etiology , Hemolytic-Uremic Syndrome/etiology , Blood Coagulation/immunology , Complement Pathway, Alternative/genetics , Female , HELLP Syndrome/genetics , HELLP Syndrome/immunology , Hemolytic-Uremic Syndrome/genetics , Hemolytic-Uremic Syndrome/immunology , Humans , Mutation , Pregnancy , Prognosis
5.
Blood ; 111(2): 624-32, 2008 Jan 15.
Article in English | MEDLINE | ID: mdl-17914026

ABSTRACT

The hemolytic uremic syndrome (HUS) is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies demonstrate that heterozygous mutations of membrane cofactor protein (MCP;CD46) predispose to atypical HUS (aHUS), which is not associated with exposure to Shiga toxin (Stx). Among the initial 25 MCP mutations in patients with aHUS were 2, R69W and A304V, that were expressed normally and for which no dysfunction was found. The R69W mutation is in complement control protein module 2, while A304V is in the hydrophobic transmembrane domain. In addition to 3 patients with aHUS, the A304V mutation was identified in 1 patient each with fatal Stx-HUS, the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and glomerulonephritis with C3 deposits. A major goal was to assess if these putative mutations lead to defective complement regulation. Permanent cell lines expressing the mutated proteins were complement "challenged," and membrane control of C3 fragment deposition was monitored. Both the R69W and A304V MCP mutations were deficient in their ability to control the alternative pathway of complement activation on a cell surface, illustrating the importance of modeling transmembrane proteins in situ.


Subject(s)
Amino Acid Substitution , Complement Activation/genetics , Glomerulonephritis/genetics , HELLP Syndrome/genetics , Hemolytic-Uremic Syndrome/genetics , Membrane Cofactor Protein/genetics , Mutation, Missense , Complement C3/genetics , Complement C3/metabolism , Female , Genetic Predisposition to Disease , Glomerulonephritis/metabolism , Glomerulonephritis/pathology , HELLP Syndrome/metabolism , HELLP Syndrome/pathology , Hemolysis/genetics , Hemolytic-Uremic Syndrome/metabolism , Hemolytic-Uremic Syndrome/pathology , Heterozygote , Humans , Liver/enzymology , Liver/pathology , Male , Membrane Cofactor Protein/metabolism , Models, Molecular , Pregnancy , Protein Structure, Tertiary/genetics , Shiga Toxin
6.
Vaccine ; 26 Suppl 8: I22-7, 2008 Dec 30.
Article in English | MEDLINE | ID: mdl-19388160

ABSTRACT

Nearly half of the proteins in the complement system serve in regulation. Control at the central step of C3 activation is provided by an orchestrated interplay of membrane and plasma regulators. A model system employing Chinese hamster ovary (CHO) cells transfected with human regulators was employed to assist in making functional comparisons. Also, in this experimental setup, the pathway and magnitude of complement activation can be varied while monitoring C4b/C3b deposition and cleavage as well as cytotoxicity. This review describes lessons learned and the application of this model for functionally characterizing mutations in regulators associated with atypical hemolytic uremic syndrome.


Subject(s)
Complement Activation , Complement C3/metabolism , Animals , CD55 Antigens/physiology , CHO Cells , Cricetinae , Cricetulus , Hemolytic-Uremic Syndrome/etiology , Hemolytic-Uremic Syndrome/genetics , Humans , Membrane Cofactor Protein/genetics , Membrane Cofactor Protein/physiology , Mutation
7.
Mol Immunol ; 44(1-3): 111-22, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16882452

ABSTRACT

The hemolytic uremic syndrome is characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. There are two general types. One occurs in epidemic form and is diarrheal associated (D+HUS). It has a good prognosis. The second is a rare form known as atypical (aHUS), which may be familial or sporadic, and has a poor prognosis. aHUS is increasingly recognized to be a disease of defective complement regulation, particularly cofactor activity. Mutations in membrane cofactor protein (MCP; CD46) that predispose to the development of aHUS were first identified in 2003. MCP is a membrane-bound complement regulator that acts as a cofactor for the factor I-mediated cleavage of C3b and C4b deposited on host cells. More than 20 different mutations in MCP have now been identified in patients with aHUS. Many of these mutants have been functionally characterized and have helped to define the pathogenic mechanisms leading to aHUS development. Over 75% of the reported mutations cause a reduction in MCP expression, due to homozygous, compound heterozygous or heterozygous mutations. This deficiency of MCP leads to inadequate control of complement activation on endothelial cells after an initiating injury. The remaining MCP mutants are expressed, but demonstrate reduced ligand (C3b/C4b) binding capacity and cofactor activity of MCP. MCP mutations in aHUS demonstrate incomplete penetrance, indicating that additional genetic and environmental factors are required to manifest disease. MCP mutants as a cause of aHUS have a favorable clinical outcome in comparison to patients with factor H (CFH) or factor I (IF) mutations. In 90% of the renal transplants performed in patients with MCP-HUS, there has been no recurrence of the primary disease, whilst >50% of factor I or factor H deficient patients have had a prompt recurrence. This highlights the importance of defining and characterizing the underlying genetic defects in patients with aHUS.


Subject(s)
Hemolytic-Uremic Syndrome/etiology , Membrane Cofactor Protein/genetics , Mutation , Haplotypes , Hemolytic-Uremic Syndrome/classification , Hemolytic-Uremic Syndrome/genetics , Humans , Kidney Transplantation , Membrane Cofactor Protein/chemistry , Membrane Cofactor Protein/physiology
8.
Blood ; 108(4): 1267-79, 2006 Aug 15.
Article in English | MEDLINE | ID: mdl-16621965

ABSTRACT

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy with manifestations of hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies have shown that mutations in complement regulatory proteins predispose to non-Shiga toxin-associated HUS (non-Stx-HUS). We undertook genetic analysis on membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF) in 156 patients with non-Stx-HUS. Fourteen, 11, and 5 new mutational events were found in MCP, CFH, and IF, respectively. Mutation frequencies were 12.8%, 30.1%, and 4.5% for MCP, CFH, and IF, respectively. MCP mutations resulted in either reduced protein expression or impaired C3b binding capability. MCP-mutated patients had a better prognosis than CFH-mutated and nonmutated patients. In MCP-mutated patients, plasma treatment did not impact the outcome significantly: remission was achieved in around 90% of both plasma-treated and plasma-untreated acute episodes. Kidney transplantation outcome was favorable in patients with MCP mutations, whereas the outcome was poor in patients with CFH and IF mutations due to disease recurrence. This study documents that the presentation, the response to therapy, and the outcome of the disease are influenced by the genotype. Hopefully this will translate into improved management and therapy of patients and will provide the way to design tailored treatments.


Subject(s)
Complement Factor H/genetics , Complement Factor I/genetics , Hemolytic-Uremic Syndrome/genetics , Membrane Cofactor Protein/genetics , Mutation , Blood Component Transfusion , Complement C3b/genetics , Complement C3b/metabolism , Complement Factor H/biosynthesis , Complement Factor I/biosynthesis , Female , Gene Frequency/genetics , Genotype , Hemolytic-Uremic Syndrome/metabolism , Hemolytic-Uremic Syndrome/therapy , Humans , Kidney Transplantation , Male , Membrane Cofactor Protein/biosynthesis , Plasma , Protein Binding/genetics , Protein Biosynthesis/genetics , Recurrence , Shiga Toxin , Treatment Outcome
9.
Autoimmun Rev ; 4(8): 515-9, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16214088

ABSTRACT

Production of autoantibodies by B cells in systemic lupus erythematosus (SLE) can be interrupted via induction of regulatory and suppressor T cells. We have used the strategy of tolerizing lupus-prone (NZBxNZW)F(1) mice with an artificial peptide based on sequences common to several anti-double stranded (ds)DNA antibodies to induce regulatory and suppressor T cells that block production of anti-DNA antibodies and prolong their survival. At least one type of suppressor T cells (CD8+) and one type of regulatory T cell (CD4+ expressing the IL-2 receptor alpha chain CD25) are raised under this condition. While CD8+ suppressors (TS) require soluble factors to block help of T cells to B cells, regulatory CD4+CD25+ T cells (TR) curb the production of anti-DNA antibodies from B cells via cell contact through molecules that include membrane-bound TGFbeta and GITR. Moreover, CD8+ suppressors seem to act independently on antigen specificity, while TR act in an antigen-specific fashion. We hypothesize that the differences between these two lymphocyte subsets that share the common ability to dampen production of autoantibodies might underlie significant temporal and teleological advantages for optimal control of autoimmune reactivity.


Subject(s)
Antibodies, Antinuclear/biosynthesis , Antigens, Differentiation, T-Lymphocyte/analysis , B-Lymphocytes/immunology , Lupus Erythematosus, Systemic/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Humans , Lymphocyte Activation
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