Gout-associated uric acid crystals activate the NALP3 inflammasome

Development of the acute and chronic inflammatory responses known as gout and pseudogout are associated with the deposition of monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals, respectively, in joints and periarticular tissues. Although MSU crystals were first identified as the aetiological agent of gout in the eighteenth century and more recently as a 'danger signal' released from dying cells, little is known about the molecular mechanisms underlying MSU- or CPPD-induced inflammation. Here we show that MSU and CPPD engage the caspase-1-activating NALP3 (also called cryopyrin) inflammasome, resulting in the production of active interleukin (IL)-1beta and IL-18. Macrophages from mice deficient in various components of the inflammasome such as caspase-1, ASC and NALP3 are defective in crystal-induced IL-1beta activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystal-induced peritonitis in inflammasome-deficient mice or mice deficient in the IL-1beta receptor (IL-1R). These findings provide insight into the molecular processes underlying the inflammatory conditions of gout and pseudogout, and further support a pivotal role of the inflammasome in several autoinflammatory diseases.     

Structural and evolutionary analysis of HLA-D-region products

The major histocompatibility complex (MHC)--HLA in man and H-2 in mouse--encodes two classes of cell-surface antigens involved in the immune response. The amino acid sequences have been determined for a number of these molecules. Class I antigens, typified by the HLA-ABC antigens, are composed of a 43,000-molecular weight (MW) glycosylated transmembrane polypeptide with three external domains (alpha 1, alpha 2 and alpha 3), of which the one nearest the membrane (alpha 3) is associated with a 12,000-MW nonglycosylated polypeptide, beta 2-microglobulin. The HLA-D-region or class II antigens, DR, DC and SB, are composed of two glycosylated transmembrane polypeptides, of MWs 34,000 (alpha-chain) and 28,000 (beta-chain). Both chains have two external domains which presumably associate with each other, alpha 2, beta 2 being membrane proximal and alpha 1, beta 1 N-terminal and membrane distal. All four membrane-proximal domains (class I alpha 3, beta 2-microglobulin, class II alpha 2 and beta 2) have amino acid sequences that show significant similarities with immunoglobulin constant-region domains. This, together with the similarly placed internal disulphide bonds, suggests they might have an immunoglobulin-like structure (Fig. 1). We have now used computer graphics techniques to predict a detailed three-dimensional structure for the membrane-proximal domains of the class II antigens (alpha 2 and beta 2) based on the known coordinates of immunoglobulin constant domains (Fig. 2). The transmembrane regions of class II antigens have been modelled as two alpha-helices packed together. The proposed structure accounts for conservation of amino acids and leads to evolutionary predictions.     

A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family

Recent studies have demonstrated that CD3 is expressed on a subset of thymocytes with a CD4-CD8- (double negative) phenotype. At least some of these cells bear the CD3-associated gamma delta T-cell receptor (TCR gamma delta). Here we describe a second subset of double negative thymocytes which expresses CD3-associated alpha beta receptors (TCR alpha beta). Surprisingly, these cells express predominantly the products of a single V beta gene family (V beta 8). These CD4-CD8-, TCR alpha beta+ cells appear relatively late in ontogeny (between birth and day 5 of life) and thus are unlikely to be the precursors to the TCR alpha beta-bearing cells (CD4+CD8- and CD4-CD8+) already present at birth. They can be selectively expanded in vitro by stimulation with a monoclonal antibody to V beta 8 (F23.1) in the presence of interleukin I (IL-1). We propose that this cell type is a unique T-cell population distinguishable from typical TCR alpha beta+ T cells by its CD4-CD8- phenotype and a restricted TCR V beta repertoire. Analysis of the unique phenotype of these cells suggests that they may represent the normal counterpart of the defective CD4-CD8- T cells found in the lpr autoimmune mouse.     

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.     

Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.     

Regulation of T-cell receptor gamma-chain RNA expression in murine Thy-1+ dendritic epidermal cells

The epidermis of normal mice contains two distinct populations of dendritic cells derived from the bone marrow, Ia+ Langerhans cells and Ia- cells that express the Thy-1 alloantigen. The Thy-1-bearing dendritic epidermal cells (Thy-1+ dEC) have a surface phenotype similar to that of very early T-lineage cells, produce IL-2-like growth factors and exhibit cytotoxicity which is not restricted by the major histocompatibility complex (MHC). The relationship of Thy-1+ dEC to the T-cell lineage is unclear. Most T lymphocytes bear a receptor for antigen composed of an alpha chain and a beta chain associated with a nonpolymorphic complex termed CD3 (T3). A minor population carries a receptor in which CD3 is associated with a gamma/delta complex. We have analysed clones of Thy-1+ dEC for rearrangement and expression of the genes for the alpha-, beta- and gamma-chains of the T-cell receptor (TCR). They do not express alpha or beta but do carry a gamma/delta complex. Activation of the cells with Con A is associated with a rapid decrease in the steady-state level of gamma-chain RNA. Because Thy-1+ dEC resemble early stage T lymphocytes, down-regulation of TCR expression may reflect a necessary event during T cell differentiation.     

Tumour necrosis factor alpha stimulates resorption and inhibits synthesis of proteoglycan in cartilage

During inflammatory reactions, activated leukocytes are thought to produce a variety of small proteins (cytokines) that influence the behaviour of other cells (including other leukocytes). Of these substances, which include the interleukins, interferons and tumour necrosis factors (TNFs), interleukin-1 (IL-1) has been considered potentially a most important inflammatory mediator because of its wide range of effects. In vivo it is pyrogenic and promotes the acute phase response; in vitro it activates lymphocytes and stimulates resorption of cartilage and bone. Cartilage resorption is a major feature of inflammatory diseases such as rheumatoid arthritis, and IL-1 is the only cytokine hitherto known to promote it. TNFs are characterized by their effects on tumours and cytotoxicity to transformed cells, but share some actions with IL-1. I report here that recombinant human TNF alpha stimulates resorption and inhibits synthesis of proteoglycan in explants of cartilage. Its action is similar to and additive with IL-1, and it is a second macrophage-derived cytokine whose production in rheumatoid arthritis, or inflammation generally, could contribute to tissue destruction.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

A human recombinant haemoglobin designed for use as a blood substitute.

The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.     

Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors are members of a gene family of ligand-gated transmitter receptors that includes muscle nicotinic receptors, GABAA receptors and glycine receptors. Several lines of evidence indicate that neuronal nicotinic receptors can be made up of only two subunits, an alpha (alpha) subunit which binds ligand, and a non-alpha (n alpha) or beta (beta) subunit. The stoichiometry of each subunit in the functional receptor has been difficult to assess, however. Estimates of the molecular weight of neuronal nicotonic receptor macromolecules suggest that these receptors contain at least four subunits but probably not more than five. We have examined the subunit stoichiometry of the chick neuronal alpha 4/n alpha 1 receptor by first using site-directed mutagenesis to create subunits that confer different single channel properties on the receptor. Co-injection with wild-type and mutant subunits led to the appearance of receptors with wild-type, mutant and hybrid conductances. From the number of hybrid conductances, we could deduce the number of each subunit in the functional receptor.     

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.     

Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunoglobulin D.

The B-cell antigen receptor of the IgM and IgD class is a multimeric complex consisting of the membrane-bound form of the immunoglobulin molecule and two other proteins, Ig-alpha and Ig-beta. The Ig-alpha and Ig-beta proteins form a disulphide-linked alpha/beta heterodimer and are encoded by the mb-1 (ref 9, 10) and B29 genes, respectively. Surface expression of the membrane-bound IgM molecule requires assembly with the alpha/beta heterodimer. The IgD molecule, however, can be expressed on the cell surface in an alpha/beta-dependent and -independent form. We show here that in the alpha/beta-independent form the IgD molecule is anchored in the plasma membrane through a glycosyl-phosphatidylinositol linker. In the presence of the alpha/beta heterodimer, most of the otherwise glycosyl-phosphatidylinositol-linked IgD molecule is expressed on the cell surface as transmembrane proteins.     

The heavy chain of human B-cell alloantigen HLA-DS has a variable N-terminal region and a constant immunoglobulin-like region

The HLA-D region of the major histocompatibility complex (MHC) of man encodes polymorphic glycoproteins found predominantly on the cell surfaces of B cells and macrophages. These proteins mediate interactions, required for the induction of immune responses, among cells of the immune system and consequently are referred to as Ia (immune-response associated). Two families of Ia molecules, DR and DS (also known as DC), have been defined, the former analogous to the I-E (ref. 1) and the latter to the I-A molecules of the murine MHC. Both DR and DS molecules consist of two noncovalently associated polypeptide chains with molecular weights of 33,000 and 28,000, designated alpha and beta, respectively. The polymorphism of DR molecules is due to structural variation in the small subunit, DR beta, with the large subunit, DR alpha, being constant in structure. In contrast, both subunits DS alpha and DS beta are structurally variable when DS allotypes are compared. We have now isolated a cDNA clone from a DR7 cell line that contains the entire coding sequence for the DS alpha subunit and have compared its predicted amino acid sequence with that previously deduced from a DS alpha cDNA clone isolated from a DR4,w6 cell line. This comparison reveals that 10 of 11 amino acid differences are located within the alpha 1 (N-terminal) domain and that the alpha 2 or immunoglobulin-like domains are identical.     

The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor

The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.