Stephen H. Powis

Alleles and haplotypes of the MHC-encoded ABC transporters TAP1 and TAP2

TAP1 and TAP2 are two major histocompatibility complex (MHC) genes, located between HLA-DP and -DQ, whose products form a transporter molecule involved in endogenous antigen processing. Polymorphic residues have been described in both genes and, in this study, we have identified another polymorphic site within the adenosine triphosphate (ATP)-binding domain of TAP2. We have used the amplification refractoru mutation system (ARMS) polymerase chain reaction (PCR) to characterize TAP1 and TAP2 alleles and haplotypes in a reference panel of 115 homozygous typing cell lines. Of four possible TAP1 alleles, we observed three, and of eight possible TAP2 alleles, we observed five. Among 88 (homozygous typing cells) (HTCs) homozygous at HLA-DR, -DQ and TP, 80 were also homozygous at TAP1 and TAP2. Of 27 HTCs homozygous at HLA-DR and -DQ, but heterozygous at -DP, 14 were homozygous at TAP1 or TAP2 and 13 heterozygous, consistent with recombination taking place either side of the TAP loci. Of the fifteen possible combinations of TAP1 and TAP2 alleles, we observed eleven, each at a frequency similar to that predicted by individual allele frequencies. In this ethnically heterogeneous panel there is no indication that particular combinations of TAP1 and TAP2 have been maintained together.

Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors.

HLA‐F is a human non‐classical MHC molecule. Recombinant HLA‐F heavy chain was refolded with β2‐microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high‐affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA‐F. HLA‐F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA‐F could be immunoprecipitated from B cell lines and from HUT‐78, a T cell line. HLA‐F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA‐F tetramers stain peripheral blood monocytes and B cells. HLA‐F tetramer binding could be conferred on non‐binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA‐F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA‐F, suggest that HLA‐F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.

Inflammatory stress exacerbates lipid accumulation in hepatic cells and fatty livers of apolipoprotein E knockout mice.

The prevailing theory in non‐alcoholic fatty liver disease (NAFLD) is the “two‐hit” hypothesis. The first hit mainly consists of lipid accumulation, and the second is subsequent systemic inflammation. The current study was undertaken to investigate whether inflammatory stress exacerbates lipid accumulation in liver and its underlying mechanisms. We used interleukin‐1β (IL‐1β) and tumor necrosis factor alpha (TNF‐α) stimulation in human hepatoblastoma cell line (HepG2) cells and primary hepatocytes in vitro, and casein injection in apolipoprotein E knockout mice in vivo to induce inflammatory stress. The effects of inflammatory stress on cholesterol accumulation were examined by histochemical staining and a quantitative intracellular cholesterol assay. The gene and protein expressions of molecules involved in cholesterol trafficking were examined by real‐time polymerase chain reaction (PCR) and western blot. Cytokine production in the plasma of apolipoprotein E knockout mice was measured by enzyme‐linked immunosorbent assay. Our results showed that inflammatory stress increased cholesterol accumulation in hepatic cells and in the livers of apolipoprotein E knockout mice. Further analysis showed that inflammatory stress increased the expression of low‐density lipoprotein (LDL) receptor (LDLr), sterol regulatory element–binding protein (SREBP) cleavage activating protein (SCAP), and SREBP‐2. Confocal microscopy showed that IL‐1β increased the translocation of SCAP/SREBP‐2 complex from endoplasmic reticulum (ER) to Golgi in HepG2 cells, thereby activating LDLr gene transcription. IL‐1β, TNF‐α, and systemic inflammation induced by casein injection also inhibited expression of adenosine triphosphate–binding cassette transporter A1 (ABCA1), peroxisome proliferator‐activated receptor‐α (PPAR‐α), and liver X receptor‐α (LXRα). This inhibitory effect may cause cholesterol efflux reduction. Conclusion: Inflammatory stress up‐regulates LDLr‐mediated cholesterol influx and down‐regulates ABCA1‐mediated cholesterol efflux in vivo and in vitro. This may exacerbate the progression of NAFLD by disrupting cholesterol trafficking control, especially during the second hit phase of liver damage. (HEPATOLOGY 2008.)

Identification of a novel psoriasis susceptibility locus at 1p and evidence of epistasis between PSORS1 and candidate loci

The pathogenesis of all forms of psoriasis remains obscure. Segregation analysis and twin studies together with ethnic differences in disease frequency all point to an underlying genetic susceptibility to psoriasis, which is both complex and likely to reflect the action of a number of genes. We performed a genome wide analysis using a total of 271 polymorphic autosomal markers on 284 sib relative pairs identified within 158 independent families. We detected evidence for linkage at 6p21 (PSORS1) with a non-parametric linkage score (NPL)=4.7, p=2 × 10-6 and at chromosome 1p (NPL=3.6, p=1.9 × 10-4) in all families studied. Significant excess (p=0.004) paternal allele sharing was detected for markers spanning the PSORS1locus. A further three regions reached NPL scores of 2 or greater, including a region at chromosome 7 (NPL 2.1), for which linkage for a number of autoimmune disorders has been reported. Partitioning of the data set according to allele sharing at 6p21 (PSORS1) favoured linkage to chromosomes 2p (NPL 2.09) and 14q (NPL 2.0), both regions implicated in previous independent genome scans, and suggests evidence for epistasis betweenPSORS1 and genes at other genomic locations. This study has provided linkage evidence in favour of a novel susceptibility locus for psoriasis and provides evidence of the complex mechanisms underlying the genetic predisposition to this common skin disease.

PPAR Agonists Protect Mesangial Cells from Interleukin 1β-Induced Intracellular Lipid Accumulation by Activating the ABCA1 Cholesterol Efflux Pathway

Previous studies have demonstrated that inflammatory cytokines such as interleukin-1beta (IL-1beta) promote lipid accumulation in human mesangial cells (HMC) by dysregulating the expression of lipoprotein receptors. Intracellular lipid accumulation is governed by both influx and efflux; therefore, the effect of IL-1beta on the efflux of lipid from HMC was investigated. IL-1beta was shown to inhibit (3)H-cholesterol efflux from HMC and increase total intracellular cholesterol concentration, probably as a result of reduced expression of the adenosine triphosphate (ATP) binding cassette A1 (ABCA1), a transporter protein involved in apolipoprotein-A1 (apo-A1)-mediated lipid efflux. To ascertain the molecular mechanisms involved, expression of peroxisome proliferator-activated receptors (PPAR) and liver X receptoralpha (LXRalpha) were examined. IL-1beta (5 ng/ml) reduced PPARalpha, PPARgamma, and LXRalpha mRNA expression. Activation of PPARgamma with the agonist prostaglandin J2 (10 micro M) and of PPARalpha with either bezafibrate (100 micro M) or Wy14643 (100 micro M) both increased LXRalpha and ABCA1 gene expression also and enhanced apoA1-mediated cholesterol efflux from lipid-loaded cells, even in the presence of IL-1beta. A natural ligand of LXRalpha, 25-hydroxycholesterol (25-OHC), had similar effects; when used together with PPAR agonists, an additive effect was observed, indicating co-operation between PPAR and LXRalpha in regulating ABCA1 gene expression. This was supported by the observation that overexpression of either PPARalpha or PPARgamma by transfection enhanced LXRalpha and ABCA1 gene induction by PPAR agonists. Taken together with previous data, it appears that, in addition to increasing lipid uptake, inflammatory cytokines promote intracellular lipid accumulation by inhibiting cholesterol efflux through the PPAR-LXRalpha-ABCA1 pathway. These results suggest potential mechanisms whereby inflammation may exacerbate lipid-mediated cellular injury in the glomerulus and in other tissues and indicate that PPAR agonists may have a protective effect.

Mechanisms of Dysregulation of Low-Density Lipoprotein Receptor Expression in Vascular Smooth Muscle Cells by Inflammatory Cytokines

Objective—Although inflammation is a recognized feature of atherosclerosis, the impact of inflammation on cellular cholesterol homeostasis is unclear. This study focuses on the molecular mechanisms by which inflammatory cytokines disrupt low-density lipoprotein (LDL) receptor regulation. Methods and Results—IL-1&bgr; enhanced transformation of vascular smooth muscle cells into foam cells by increasing uptake of unmodified LDL via LDL receptors and by enhancing cholesterol esterification as demonstrated by Oil Red O staining and direct assay of intracellular cholesterol concentrations. In the absence of IL-1&bgr;, a high concentration of LDL decreased LDL receptor promoter activity, mRNA synthesis and protein expression. However, IL-1&bgr; enhanced LDL receptor expression, overriding the suppression usually induced by a high concentration of LDL and inappropriately increasing LDL uptake. Exposure to IL-1&bgr; also caused overexpression of the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP), and enhanced its translocation from the endoplasmic reticulum to the Golgi, where it is known to cleave SREBP, thereby enhancing LDL receptor gene expression. Conclusions—These observations demonstrate that IL-1&bgr; disrupts cholesterol-mediated LDL receptor feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam cell formation. The implication of these findings is that inflammatory cytokines may contribute to intracellular LDL accumulation without previous modification of the lipoprotein.

TAP1 and TAP2 polymorphism in coeliac disease

Coeliac disease is strongly associated with HLA-DQ2, but it is possible that additional major histocompatibility complex genes also confer disease susceptibility. Encoded close to HLA-DQ are two genes, TAP1 and TAP2, whose products are believed to transport antigenic peptides from the cytoplasm into the endoplasmic reticulum. Comparison of 81 coeliac disease patients with caucasoid controls revealed an increased frequency of the alleles TAP1A and TAP2A in the patient population. However, no significant difference was found when patients were compared with HLA-DR and -DQ matched controls, indicating linkage disequilibrium between TAP1A, TAP2A, and HLA-DQ2. The TAP gene products do not have a major influence on susceptibility or resistance to coeliac disease in a Northern European Caucasoid population.

Limited polymorphism in HLA-DM does not involve the peptide binding groove

Class II major histocompatibility complex (MHC) molecules play a central role in the immune response by binding peptides and presenting them to helper T lymphocytes. Recently we described two novel genes, HLA-DMA and -DMB, which map in the human MHC between the HLA-DNA and HLA-DOB loci (Kelly et al. 1991). Although the α and β chains encoded by these genes were similar to those of other class II molecules, they were sufficiently diverse to suggest they may have arisen early in evolution before the duplications which gave rise to the main class II loci. It is likely that DMα and DMβ partner each other to form a class II molecule because, with the exception of HLA-DNA and HLA-DOB (Karlsson and Peterson 1992), the genes encoding the α and β chains of other class II molecules are physically closely linked

Major histocompatibility complex class I-related chain A allele mismatching, antibodies, and rejection in renal transplantation

Even when kidney allografts are well matched for human leukocyte antigen (HLA) and anti-HLA antibodies are not detected, graft rejection can still occur. There is evidence that some patients who lose their graft have antibodies specific for major histocompatibility complex (MHC) class I-related chain A (MICA) antigens. We investigated whether mismatching MICA alleles associates with MICA antibody production and graft rejection or dysfunction. MICA and HLA antibody screening in 442 recipients was performed, and specificities were confirmed in a subgroup of 227 recipients using single-antigen multiplex technology. For assignment of MICA antibody specificity, we used three independent assays. In addition, MICA alleles of 227 recipients and donors were determined by DNA sequencing. In all, 17 patients (7.5%) had MICA antibodies, and 13 patients (6%) developed MICA donor-specific antibodies (DSA). Multivariate analysis revealed MICA mismatching, as an independent significant factor associated with the presence of MICA antibodies (p = 0.009), and 14 mismatched MICA residues significantly correlated with MICA antibody production. MICA and HLA antibodies significantly associated with acute rejection (AR) and MICA DSA and HLA DSA correlated with decreased graft function by univariate and multivariate analysis. We conclude that mismatching for MICA epitopes in renal transplantation is a mechanism leading to production of MICA antibodies that associate with AR and graft dysfunction.

Hyperoxia improves the survival of intraportally transplanted syngeneic pancreatic islets.

Background. Hypoxia in the portal vein may compromise the survival of intraportally transplanted pancreatic islets. We therefore examined the effect of inspired oxygen on the outcome of islet transplantation. Methods. Blood glucose concentrations, glucose tolerance, and the size and number of surviving islets were measured in diabetic rats housed for 48 hr under hyperoxic (100% O2), hypoxic (11% O2), or normoxic (21%O2) conditions after intraportal transplantation of 350, 500, 700, or 1,000 syngeneic islets. Results. In normoxic diabetic rats, the smallest graft size to consistently restore normoglycemia was 1,000 islets. A graft size of 700 islets was effective in only three of nine animals, whereas 500 islets were ineffective in all eight animals undergoing transplantation. In contrast, in hyperoxically housed rats, graft sizes of 700 or 500 islets restored normoglycemia in eight of nine or five of eight animals, respectively. In those animals that became normoglycemic, the glucose tolerance of the hyperoxically treated rats receiving 700 islets was almost identical to that of normoxically housed animals receiving 1,000 islets. The average size of the islets 6 weeks after transplantation was the same in livers of hyperoxic and control rats. However, the total islet area and number of islets engrafted in hyperoxic rats was significantly increased when compared with livers from normoxic animals receiving the same graft size, so the area in hyperoxic rats receiving 700 islets was not significantly different from normoxic recipients of 1,000 islets. Conclusions. Hyperoxia posttransplantation increases the number of islets that survive the engraftment process and allows normalization of plasma glucose levels with a smaller number of transplanted islets.